Mills and Sternberg's Diagnostic Surgical Pathology [7 ed.] 9781975150754, 9781975150723, 1975150724

Table of contents :
Half Title Page
Title Page
Copyright Page
Dedication
Contributors
Preface to the Seventh Edition
Preface to the First Edition
Contents
VOLUME 1
SECTION I Skin, Soft Tissue, Bone, and Joints
1 Nonneoplastic Diseases of the Skin
2 Nonmelanocytic Cutaneous Tumors
3 Melanocytic Lesions
4 Muscle Biopsy in Neuromuscular Diseases
5 Soft Tissues
6 Joints
7 Nonneoplastic Diseases of Bones
8 Bone Tumors
SECTION II Breast
9 Breast
SECTION III Central Nervous System
10 Brain, Spinal Cord, and Meninges
SECTION IV Endocrine System
11 Neuroendocrine and Paracrine Systems
12 Pituitary and Sellar Region
13 Pathology of Thyroid and Parathyroid
14 Adrenal Glands
15 Paragangliomas
SECTION V Hematopoietic and Lymphatic Systems
16 Bone Marrow
17 Lymph Nodes
18 Spleen
SECTION VI Head and Neck
19 Jaws, Oral Cavity, and Oropharynx
20 Salivary Glands
21 Nose, Paranasal Sinuses, and Nasopharynx
22 Larynx
23 Ear and Temporal Bone
24 Eye and Ocular Adnexa
SECTION VII Intrathoracic Organs and Blood Vessels
25 Nonneoplastic Pulmonary Disease
26 Pulmonary Neoplasms
27 Pleura
28 Mediastinum
29 Heart
30 Blood Vessels
VOLUME 2
SECTION VIII Alimentary Canal and Associated Organs
31 Esophagus
32 Stomach
33 Nonneoplastic Intestinal Diseases
34 Intestinal Neoplasms
35 Pancreas
36 Nonneoplastic Liver Disease
37 Masses of the Liver
38 Gallbladder, Extrahepatic Biliary Tree, and Ampulla
39 Anus and Perianal Area
SECTION IX Urinary Tract and Male Genital System
40 Developmental Abnormalities of the Kidney
41 Adult Renal Diseases
42 Adult Renal Tumors
43 Renal Neoplasms of Childhood
44 Urothelial Tract: Renal Pelvis, Ureter, Urinary Bladder, and Urethra
45 Prostate and Seminal Vesicles
46 Nonneoplastic Diseases of the Testis
47 Testicular and Paratesticular Tumors
48 Penis
SECTION X Female Reproductive System and Peritoneum
49 Gestational Trophoblastic Disease
50 Placenta
51 Vulva and Vagina
52 Cervix
53 Uterine Corpus: Epithelial Tumors
54 Uterine Corpus: Mesenchymal Tumors
55 Ovarian Epithelial–Stromal Tumors
56 Sex Cord-Stromal, Steroid Cell, and Germ Cell Tumors of the Ovary
57 Miscellaneous Primary Tumors, Secondary Tumors, and Nonneoplastic Lesions of the Ovary
58 Fallopian Tube and Broad Ligament
59 Peritoneum
Index

Citation preview

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Healthcare professionals, and not the publisher, are solely responsible for the use of this work including all medical judgments and for any resulting diagnosis and treatments. Given continuous, rapid advances in medical science and health information, independent professional verification of medical diagnoses, indications, appropriate pharmaceutical selections and dosages, and treatment options should be made and healthcare professionals should consult a variety of sources. When prescribing medication, healthcare professionals are advised to consult the product information sheet (the manufacturer’s package insert) accompanying each drug to verify, among other things, conditions of use, warnings and side effects and identify any changes in dosage schedule or contraindications, particularly if the medication to be administered is new, infrequently used or has a narrow therapeutic range. To the maximum extent permitted under applicable law, no responsibility is assumed by the publisher for any injury and/or damage to persons or property, as a matter of products liability, negligence law or otherwise, or from any reference to or use by any person of this work. shop.lww.com

DEDICATION To Richard L. Kempson, maestro of surgical pathology and mentor to many; to my fellows and residents, who teach and inspire me; to my colleagues, who make me better; to my loving husband, Rick, who grounds me; and to Mira and Nate, who rock my world. Teri A. Longacre To my parents for instilling in me the value of education; to Henry Appelman, Stephen Geller, and John Yardley for mentoring me about life as well as pathology; to the residents, fellows, and colleagues who have made my career fun and enriching; and to my family: Adria, Hannah, Billy, Will, Sarah, David, and Mack, who keep me grounded and full of love. Joel K. Greenson To my wife Harmony Wu, who has remarkable insights about everything; to my residents, fellows, and colleagues, who always make “work” fun; and to Chris Fletcher, who introduced me to and helped me fall in love with surgical pathology. Jason L. Hornick To those who mentored and inspired me through my journey in academic medicine; to the fellows who keep me honest and excited to come to work; to my colleagues; to our patients who keep me humble and yearning to do better; to my wife Maria del Mar, children, and grandchildren who keep me grounded and without whom I would have never become who I am. Victor E. Reuter

CONTRIBUTORS

N. Volkan Adsay, MD Professor Chair of Pathology, Head of Surgical Sciences Department of Pathology KOC University-School of Medicine Istanbul, Turkey Hikmat Al-Ahmadie, MD Associate Attending Department of Pathology Memorial Sloan Kettering Cancer Center New York, New York Kimberly H. Allison, MD Professor of Pathology Department of Pathology Stanford University School of Medicine Vice Chair of Education, Director of Breast Pathology Department of Pathology Stanford Medical Center Stanford, California Pedram Argani, MD Professor Pathology and Oncology Johns Hopkins University Deputy Director of Surgical Pathology Department of Pathology The Johns Hopkins Hospital Baltimore, Maryland Sylvia L. Asa, MD, PhD Professor Department of Pathology

Case Western Reserve University Consultant in Endocrine Pathology Department of Pathology University Hospitals Cleveland Medical Center Cleveland, Ohio Leomar Y. Ballester, MD, PhD Assistant Professor Department of Pathology and Laboratory Medicine Department of Neurosurgery The University of Texas Health Science Center at Houston Houston, Texas Zubair W. Baloch, MD, PhD Professor of Pathology and Laboratory Medicine Department of Pathology and Laboratory Medicine University of Pennsylvania, Perelman School of Medicine Philadelphia, Pennsylvania Jose E. Barreto, MD Pathologist Instituto de Patologia e Investigacion Asunción, Paraguay Olca Basturk, MD Associate Professor Department of Pathology Memorial Sloan Kettering Cancer Center (MSKCC) New York, New York Gregory R. Bean, MD, PhD Assistant Professor of Pathology Department of Pathology Stanford University School of Medicine Assistant Professor of Pathology Department of Pathology Stanford University Medical Center Stanford, California J. Bruce Beckwith, MD Retired Missoula, Montana

Daniel M. Berney, MB B Chir FRCPath Consultant Histopathologist and Hon Professor of GU Pathology Barts Cancer Institute Queen Mary University of London London, United Kingdom Gerald J. Berry, MD Professor of Pathology Department of Pathology Stanford University Director of Anatomic Pathology Director of Cardiac and Pulmonary Pathology Department of Pathology Stanford University Medical Center Stanford, California Justin A. Bishop, MD Professor and Jane B. and Edwin P. Jenevein M.D. Distinguished Chair Department of Pathology UT Southwestern Medical Center Chief of Anatomic Pathology Department of Pathology Clements University Hospital Dallas, Texas S. Fiona Bonar, MB, FRCPA Adjunct Professor Department of Pathology School of Medicine University of Notre Dame Darlinghurst, New South Wales, Australia Pathologist Department of Anatomical Pathology Douglass Hanly Moir Pathology, Sonic Healthcare Macquarie Park, New South Wales, Australia Judith V. M. G. Bovée, MD, PhD Professor Department of Pathology Leiden University Consultant Pathologist Department of Pathology

Leiden University Medical Center Leiden, The Netherlands Sergey V. Brodsky, MD, PhD Professor Department of Pathology The Ohio State University Columbus, Ohio Jerome S. Burke, MD Adjunct Clinical Professor Department of Pathology Stanford University Medical Center Stanford, California Consultant Pathologist Department of Pathology Alta Bates Summit Medical Center Berkeley, California Miguel N. Burnier Jr., MD, PhD, FRCSC Professor, Department of Pathology, Ophthalmology, Oncology, Medicine, Surgery Director, The MUHC-McGill University Ocular Pathology Laboratory McGill University Montreal, Quebec, Canada Alberto Cavazza, MD Director Surgical Pathology Azienda USL/IRCCS Reggio Emilia, Italy Vivek Charu, MD, PhD Assistant Professor Department of Pathology Stanford University School of Medicine Stanford, California Sarah Chiang, MD Associate Attending Department of Pathology Memorial Sloan Kettering Cancer Center

New York, New York E. Karen Choi, MD Assistant Professor Department of Pathology University of Michigan Ann Arbor, Michigan Antonio L. Cubilla, MD Emeritus Professor Department of Pathology Universidad Nacional de Asuncion San Lorenzo, Paraguay Director Department of Pathology Instituto de Patologia e Investigacion Asunción, Paraguay Thomas J. Cummings, MD Professor Department of Pathology Duke University Medical Center Durham, North Carolina Ronald A. DeLellis, MD Consultant in Pathology, Rhode Island and The Miriam Hospitals Providence, Rhode Island Professor of Pathology and Laboratory Medicine (Emeritus) Warren Alpert Medical School of Brown University Providence, Rhode Island Jonathan I. Epstein, MD Professor of Pathology, Urology, and Oncology Reinhard Professor of Urological Pathology Johns Hopkins Medicine Director of Surgical Pathology Johns Hopkins Hospital Baltimore, Maryland Carol F. Farver, MD Professor, Thoracic Pathology Director, Division of Education Programs

Director, Thoracic Pathology Department of Pathology Michigan Medicine University of Michigan Ann Arbor, Michigan Ann K. Folkins, MD Assistant Professor Department of Pathology Stanford School of Medicine Director of Surgical Pathology Department of Pathology Stanford Health Care Stanford, California Talia L. Fuchs, MD Staff Specialist Department of Anatomical Pathology Royal North Shore Hospital Sydney, New South Wales, Australia Gregory N. Fuller, MD, PhD Professor Departments of Anatomic Pathology and Neuroradiology The University of Texas MD Anderson Cancer Center Deputy Chair and Chief Neuropathologist Department of Anatomic Pathology The University of Texas MD Anderson Cancer Center Houston, Texas C. Blake Gilks, MD Professor Emeritus Department of Pathology and Laboratory Medicine University of British Columbia Consultant Pathologist Department of Pathology and Laboratory Medicine Vancouver General Hospital Vancouver, British Columbia, Canada Anthony J. Gill, MD, FRCPA, AM Professor of Surgical Pathology Specialty of Clinical Pathology

University of Sydney Sydney, New South Wales, Australia Senior Staff Specialist Department of Anatomical Pathology Royal North Shore Hospital St Leonards, New South Wales, Australia Ryan M. Gill, MD, PhD Professor and Director of Surgical Pathology Department of Pathology University of California, San Francisco Anatomic Pathology Laboratory Director Department of Pathology UCSF Helen Diller Medical Center at Parnassus Heights San Francisco, California John R. Goldblum, MD Professor of Pathology Cleveland Clinic Lerner College of Medicine at CWRU Chairman Department of Pathology Cleveland Clinic Cleveland, Ohio Joel K. Greenson, MD Professor of Pathology Department of Pathology University of Michigan Medical School University of Michigan Health System Ann Arbor, Michigan Alejandro A. Gru, MD Associate Professor of Pathology and Dermatology University of Virginia Charlottesville, Virginia Alexandra M. Harrington, MD, MLS(ASCP)CM Professor Director of Hematopathology Department of Pathology Medical College of Wisconsin Milwaukee, Wisconsin

Juan C. Hernandez-Prera, MD Assistant Professor Department of Oncologic Sciences University of South Florida Assistant Member Department of Pathology Moffitt Cancer Center Tampa, Florida Jason L. Hornick, MD, PhD Director of Surgical Pathology and Immunohistochemistry Department of Pathology Brigham and Women’s Hospital Professor of Pathology Harvard Medical School Boston, Massachusetts Matthew T. Howard, MD Associate Professor Department of Laboratory Medicine and Pathology Mayo Clinic Rochester, Minnesota Brooke E. Howitt, MD Assistant Professor Department of Pathology Stanford University Stanford, California Associate Pathologist Department of Pathology Stanford Healthcare Stanford, California Pei Hui, MD, PhD Professor of Pathology Department of Pathology Yale School of Medicine Director of Gynecologic Pathology Service Surgical Pathology Yale-New Haven Hospital New Haven, Connecticut

Julie A. Irving, MD, FRCPC Clinical Associate Professor Department of Pathology University of British Columbia Vancouver, British Columbia, Canada Anatomical Pathologist Department of Laboratory Medicine, Pathology, and Medical Genetics Royal Jubilee Hospital Victoria, British Columbia, Canada Sanjay Kakar, MD Professor Department of Pathology University of California, San Francisco Chief, Gastrointestinal and Hepatobiliary Pathology Service Department of Pathology University of California San Francisco Medical Center San Francisco, California Chia-Sui Kao, MD Assistant Professor Department of Pathology Stanford University School of Medicine Stanford, California Director of Genitourinary Pathology Department of Pathology Stanford Healthcare Stanford, California Pawini Khanna, MD Pathologist Department of Pathology AdventHealth Orlando, Florida Christina S. Kong, MD Professor and Vice Chair for Clinical Affairs Department of Pathology Stanford University Chief of Pathology Department of Pathology Stanford Health Care

Stanford, California Steven H. Kroft, MD Professor and Chair Department of Pathology Medical College of Wisconsin Milwaukee, Wisconsin Melinda F. Lerwill, MD Assistant Professor Department of Pathology Harvard Medical School Director, Breast Pathology Service Associate Pathologist Department of Pathology Massachusetts General Hospital Boston, Massachusetts Virginia A. Livolsi, MD Professor Department of Pathology and Laboratory Medicine University of Pennsylvania Staff Pathologist Department of Pathology and Laboratory Medicine Hospital of the University of Pennsylvania Philadelphia, Pennsylvania Teri A. Longacre, MD Professor of Pathology Director Gynecologic Pathology Stanford Health Care Stanford, California M. Beatriz S. Lopes, MD, PhD Professor of Pathology and Neurological Surgery Department of Pathology University of Virginia School of Medicine Director of Neuropathology and Autopsy Department of Pathology University of Virginia Health System Charlottesville, Virginia

Fiona M. Maclean, MBBS Clinical Associate Professor Department of Clinical Medicine Macquarie University Sydney, New South Wales, Australia Pathologist Department of Anatomical Pathology Douglass Hanly Moir Pathology, Sonic Healthcare Macquarie Park, New South Wales, Australia William R. Macon, MD Professor Mayo Medical School Consultant Department of Laboratory Medicine and Pathology Mayo Clinic Rochester, Minnesota Cristina Magi-Galluzzi, MD, PhD Professor Department of Pathology University of Alabama at Birmingham Division Director of Anatomic Pathology Department of Pathology University of Alabama at Birmingham Birmingham, Alabama Shamlal Mangray, MBBS Chief Department of Pathology & Laboratory Medicine Nationwide Children’s Hospital Columbus, Ohio Charles C. Marboe, MD Professor of Pathology and Cell Biology at CUIMC Department of Pathology and Cell Biology Columbia University Vagelos College of Physicians and Surgeons New York, New York Anne M. Mills, MD Assistant Professor Department of Pathology

University of Virginia University of Virginia Health System Charlottesville, Virginia Stacey E. Mills, MD Emeritus Professor Department of Pathology University of Virginia Charlottesville, Virginia Jeffrey L. Myers, MD A. James French Professor of Diagnostic Pathology Vice Chair for Clinical Affairs and Quality Director, Michigan Medicine Laboratories (MLabs) Department of Pathology Michigan Medicine, University of Michigan Ann Arbor, Michigan Tibor Nádasdy, MD Professor of Pathology Department of Pathology The Ohio State University Director, Renal Pathology Department of Pathology The Ohio State University Wexner Medical Center Columbus, Ohio George J. Netto, MD Professor and Chair of Pathology Robert and Ruth Anderson Endowed Chair in Pathology UAB: The University of Alabama at Birmingham Birmingham, Alabama Alexandre N. Odashiro, MD, PhD Pathologist Department of Anatomical Pathology Charles LeMoyne Hospital Greenfield Park, Quebec, Canada Robert S. Ohgami, MD, PhD Professor and Chief of Hematopathology Department of Pathology

University of California, San Francisco San Francisco, California Esther Oliva, MD Professor Harvard Medical School Pathologist Department of Pathology Massachusetts General Hospital Boston, Massachusetts Scott R. Owens, MD Professor Department of Pathology University of Michigan Director, Division of Quality and Health Improvement Department of Pathology Michigan Medicine, University of Michigan Ann Arbor, Michigan Douglas C. Parker, MD, DDS Associate Professor Department of Pathology and Dermatology Emory University School of Medicine Dermatopathologist Department of Pathology Emory University Hospital Atlanta, Georgia Christopher G. Przybycin, MD Associate Professor Department of Pathology Cleveland Clinic Lerner College of Medicine Staff Pathologist Department of Pathology Cleveland Clinic Cleveland, Ohio Shyam Sampath Raghavan, MD Assistant Professor Department of Pathology University of Virginia

Charlottesville, Virginia Karen Rech, MD Associate Professor Department of Laboratory Medicine and Pathology Mayo Clinic Rochester, Minnesota Victor E. Reuter, MD Vice Chairman Department of Pathology Member, Memorial Sloan Kettering Cancer Center Professor Department of Pathology and Laboratory Medicine Weill Medical College of Cornell University New York, New York Drucilla J. Roberts, MD Associate Professor of Pathology Department of Pathology Harvard Medical School Pathologist Department of Pathology Massachusetts General Hospital Boston, Massachusetts Anja C. Roden, MD Professor of Pathology Department of Laboratory Medicine and Pathology Mayo Clinic Rochester Rochester, Minnesota Maria Romero, MD Chief of Anatomical Pathology CVPath Institute Gaithersburg, Maryland Giulio Rossi, MD Director/Chief Department of Pathology Hospital Santa Maria delle Croci Ravenna, Italy

Diego Fernando Sanchez, MD Pathologist and Bioinformatician Instituto de Patologia e Investigacion Asuncion, Paraguay Yu Sato, MD Research Fellow CVPath Institute Gaithersburg, Maryland Anjali A. Satoskar, MD Professor of Pathology Department of Pathology The Ohio State University Associate Director, Renal Pathology Department of Pathology The Ohio State University Wexner Medical Center Columbus, Ohio Jeanne Shen, MD Assistant Professor Department of Pathology Stanford University School of Medicine Stanford, California Ie-Ming Shih, MD, PhD Richard TeLinde Distinguished Professor Department of Gynecology and Obstetrics Johns Hopkins University School of Medicine Attending Pathologist Departments of Gynecology and Obstetrics and Pathology The Johns Hopkins Hospital Baltimore, Maryland Aatur D. Singhi, MD, PhD Associate Professor Department of Pathology University of Pittsburgh Pittsburgh, Pennsylvania Edward B. Stelow, MD Professor

Department of Pathology University of Virginia Charlottesville, Virginia Lester D. R. Thompson, MD Consultant Pathologist Head and Neck Pathology Consultations Woodland Hills, California Satish K. Tickoo, MD Attending Pathologist Department of Pathology Memorial Sloan Kettering Cancer Center New York, New York Michael Torbenson, MD Professor Department of Laboratory Medicine and Anatomic Pathology Mayo Clinic Rochester, Minnesota Elsa F. Velazquez, MD Dermatopathologist Department of Dermatology and Pathology Massachusetts General Hospital (MGH, MGPO) Newton, Massachusetts Renu Virmani, MD President CVPath Institute Gaithersburg, Maryland Hannes Vogel, MD Professor Department of Pathology Stanford University School of Medicine Director of Neuropathology Department of Pathology Stanford Health Care Palo Alto, California Bruce M. Wenig, MD

Professor Department of Oncologic Sciences University of South Florida Chairman and Senior Member Department of Pathology Moffitt Cancer Center Tampa, Florida Maria Westerhoff, MD Professor Department of Pathology University of Michigan Pathologist Department of Pathology Michigan Medicine, University of Michigan Ann Arbor, Michigan Evgeny Yakirevich, MD, DSc Professor Department of Pathology and Laboratory Medicine Warren Alpert Medical School of Brown University Pathologist Department of Pathology Rhode Island Hospital Providence, Rhode Island Rhonda K. Yantiss, MD Professor Department of Pathology and Laboratory Medicine Weill Cornell Medicine Attending Physician Department of Pathology and Laboratory Medicine New York Presbyterian Hospital New York, New York Robert H. Young, MD, FRCPATH Pathologist, Massachusetts General Hospital Robert E. Scully Professor of Pathology Harvard Medical School James Homer Wright Pathology Laboratories Massachusetts General Hospital Boston, Massachusetts

Richard J. Zarbo, MD, DMD Clinical Professor Department of Pathology Wayne State University School of Medicine System Chairman and Kathleen D. Ward Endowed Chair in Molecular Biology Pathology and Laboratory Medicine System Clinical Department Henry Ford Health System Detroit, Michigan Xuefeng Zhang, MD, PhD Associate Professor Department of Pathology Cleveland Clinic Lerner College of Medicine Staff Department of Pathology Cleveland Clinic Cleveland, Ohio

P R E FA C E TO T H E S E V E N T H EDITION

The seventh edition of Sternberg’s Diagnostic Surgical Pathology continues the effort of prior authors and editors to bring thoughtful diagnostic assistance to surgical pathologists at all levels of training and experience. Whenever possible, real-life diagnostic problems and pitfalls are emphasized. The preface to the first edition, reprinted on the next page, sets this tone right from the inception, and we have worked to preserve it. As with prior editions, the seventh edition brings considerable changes in authorship and, most importantly, content. The 6 years since the publication of the sixth edition have seen major advances in surgical pathology. The authors and editors have worked hard to incorporate this new material, including new molecular and immunohistochemical markers for diagnosis and prognosis of neoplasia, improved classification systems for diagnosis and prognosis, the role of pathology in new diagnostic and therapeutic techniques, and the recognition of new entities and variants. Where appropriate, updated World Health Organization terminology has been employed for tumor diagnosis. Surgical pathology is a visual specialty, and we continue to strive for the best illustrations, all of which have been color balanced to bring uniformity to the color illustrations in the text. Reference lists have been considerably updated, and, where possible, older references have been eliminated to save space. The editors of the seventh edition wish to especially thank Stacey E. Mills for his good stewardship as lead editor for the prior three editions. His vision and hard work continue to be reflected in this edition. The editors also wish to thank the contributing authors, past and present, a veritable “who’s who” in surgical pathology for establishing prior editions of this text as a

leader in the field and for making the seventh edition the best ever. Finally, we would like to thank the staff of Wolters Kluwer for their unfaltering, enthusiastic support of our text. Teri A. Longacre Joel K. Greenson Jason L. Hornick Victor E. Reuter

P R E FA C E TO T H E F I R S T EDITION

We speak of the loneliness of the long-distance runner, but there may be no one lonelier than a surgical pathologist working solo. Those working in large hospitals have the luxury of being able to consult ad lib with one or more pathologists about a given case, and may even have an associate who is a specialist in the area of difficulty. Easy access to consultation is a prerequisite for accurate diagnosis and, accordingly, for optimal patient care. It is especially critical in those instances when the busy pathologist has a low level of diagnostic doubt, but this is tempered by the need to sign out the case without consultation because of the press of time. Very difficult cases, those readily recognizable as problem cases, are in a sense less troublesome, as the need for a diagnostic consultation is selfevident. Therefore, knowing when and what one doesn’t know is of singular importance. A pathology reference library is the other information source for the working pathologist. Textbook consultation and human consultation go hand in hand. In this text, we have attempted to emphasize differential diagnosis of the surgical specimen and to keep to a minimum discussion of the natural history of disease, treatment, and autopsy findings. Although no textbook can take the place of a face-to-face discussion of a diagnostic problem (especially over a multiheaded microscope) between two or more pathologists, we have asked our authors to provide the reader with their reasoning in approaching differential evaluation of a biopsy specimen, thereby giving the flavor of a personal consultation. Moreover, the authors for the various chapters have been chosen based not only upon their recognized knowledge of the specific area but also upon their skill in

written communication. Since surgical pathologic diagnosis is a visual exercise, the book is generously illustrated with color and black-and-white photographs. In addition, the chapter authors have been liberal in their use of references, thereby enhancing the value of their presentations for the reader who wishes additional information. The section editors have worked closely with the chapter authors to ensure that the objectives of the text are met; namely, that it is a treatise on the diagnosis of conditions that confront the surgical pathologist. In summary, the goal of the editors is that this book will be a working companion, and thereby be accorded a place adjacent to the microscope of the reader. Stephen S. Sternberg Donald A. Antonioli Darryl Carter Joseph C. Eggleston Stacey E. Mills Harold A. Oberman

CONTENTS

Contributors Preface to the Seventh Edition Preface to the First Edition

VOLUME

1

SECTION I Skin, Soft Tissue, Bone, and Joints 1

Nonneoplastic Diseases of the Skin Douglas C. Parker

2

Nonmelanocytic Cutaneous Tumors Alejandro A. Gru and Shyam Sampath Raghavan

3

Melanocytic Lesions Shyam Sampath Raghavan and Alejandro A. Gru

4

Muscle Biopsy in Neuromuscular Diseases Hannes Vogel

5

Soft Tissues Jason L. Hornick

6

Joints Fiona M. Maclean and S. Fiona Bonar

7

Nonneoplastic Diseases of Bones Fiona M. Maclean and S. Fiona Bonar

8

Bone Tumors Judith V.M.G. Bovée

SECTION II Breast 9

Breast Gregory R. Bean and Kimberly H. Allison

SECTION III Central Nervous System 10

Brain, Spinal Cord, and Meninges Leomar Y. Ballester and Gregory N. Fuller

SECTION IV Endocrine System 11

Neuroendocrine and Paracrine Systems Sylvia L. Asa

12

Pituitary and Sellar Region M. Beatriz S. Lopes

13

Pathology of Thyroid and Parathyroid Zubair W. Baloch and Virginia A. Livolsi

14

Adrenal Glands Shamlal Mangray, Evgeny Yakirevich, and Ronald A. DeLellis

15

Paragangliomas Talia L. Fuchs and Anthony J. Gill

SECTION V

Hematopoietic and Lymphatic Systems 16

Bone Marrow Steven H. Kroft and Alexandra M. Harrington

17

Lymph Nodes Robert S. Ohgami, William R. Macon, Matthew T. Howard, and Karen Rech

18

Spleen Jerome S. Burke

SECTION VI Head and Neck 19

Jaws, Oral Cavity, and Oropharynx Richard J. Zarbo and Bruce M. Wenig

20

Salivary Glands Justin A. Bishop and Stacey E. Mills

21

Nose, Paranasal Sinuses, and Nasopharynx Justin A. Bishop and Stacey E. Mills

22

Larynx Edward B. Stelow

23

Ear and Temporal Bone Juan C. Hernandez-Prera and Bruce M. Wenig

24

Eye and Ocular Adnexa Alexandre N. Odashiro, Thomas J. Cummings, and Miguel N. Burnier Jr.

SECTION VII Intrathoracic Organs and Blood Vessels 25

Nonneoplastic Pulmonary Disease

Carol F. Farver and Jeffrey L. Myers

26

Pulmonary Neoplasms Giulio Rossi and Alberto Cavazza

27

Pleura Edward B. Stelow

28

Mediastinum Anja C. Roden

29

Heart Gerald J. Berry and Charles C. Marboe

30

Blood Vessels Renu Virmani, Maria Romero, and Yu Sato

VOLUME

2

SECTION VIII Alimentary Canal and Associated Organs 31

Esophagus Xuefeng Zhang and John R. Goldblum

32

Stomach Teri A. Longacre and Jeanne Shen

33

Nonneoplastic Intestinal Diseases Shyam Sampath Raghavan and Scott R. Owens

34

Intestinal Neoplasms Rhonda K. Yantiss

35

Pancreas Lester D.R. Thompson, Olca Basturk, N. Volkan Adsay, and Aatur D. Singhi

36

Nonneoplastic Liver Disease

Ryan M. Gill and Sanjay Kakar

37

Masses of the Liver Michael Torbenson

38

Gallbladder, Extrahepatic Biliary Tree, and Ampulla N. Volkan Adsay and Aatur D. Singhi

39

Anus and Perianal Area E. Karen Choi and Maria Westerhoff

SECTION IX Urinary Tract and Male Genital System 40

Developmental Abnormalities of the Kidney Vivek Charu and Pawini Khanna

41

Adult Renal Diseases Anjali A. Satoskar, Sergey V. Brodsky, and Tibor Nádasdy

42

Adult Renal Tumors Satish K. Tickoo and Victor E. Reuter

43

Renal Neoplasms of Childhood Pedram Argani and J. Bruce Beckwith

44

Urothelial Tract: Renal Pelvis, Ureter, Urinary Bladder, and Urethra Hikmat Al-Ahmadie and Victor E. Reuter

45

Prostate and Seminal Vesicles Jonathan I. Epstein and George J. Netto

46

Nonneoplastic Diseases of the Testis Christopher G. Przybycin and Cristina Magi-Galluzzi

47

Testicular and Paratesticular Tumors Chia-Sui Kao and Daniel M. Berney

48

Penis Elsa F. Velazquez, Diego Fernando Sanchez, Jose E. Barreto, and Antonio L. Cubilla

SECTION X Female Reproductive System and Peritoneum 49

Gestational Trophoblastic Disease Pei Hui and Ie-Ming Shih

50

Placenta Ann K. Folkins and Drucilla J. Roberts

51

Vulva and Vagina Teri A. Longacre and Brooke E. Howitt

52

Cervix Christina S. Kong

53

Uterine Corpus: Epithelial Tumors Anne M. Mills and Teri A. Longacre

54

Uterine Corpus: Mesenchymal Tumors Teri A. Longacre and Anne M. Mills

55

Ovarian Epithelial–Stromal Tumors C. Blake Gilks and Robert H. Young

56

Sex Cord-Stromal, Steroid Cell, and Germ Cell Tumors of the Ovary Esther Oliva and Robert H. Young

57

Miscellaneous Primary Tumors, Secondary Tumors, and Nonneoplastic Lesions of the Ovary Melinda F. Lerwill and Robert H. Young

58

Fallopian Tube and Broad Ligament Sarah Chiang and Robert H. Young

59

Peritoneum Julie A. Irving and Robert H. Young

Index

SECTION

I

Skin, Soft Tissue, Bone, and Joints

1

Nonneoplastic Diseases of the Skin Douglas C. Parker

INTRODUCTION Nonneoplastic or inflammatory skin diseases encompass a wide array of pathologic processes ranging from autoimmune diseases to infectious diseases to diseases of unknown etiology. In contrast to neoplastic dermatopathology, the histopathology of inflammatory skin diseases frequently does not exhibit a one-to-one correlation with a single diagnosis and requires correlation with the clinical presentation for a definitive diagnosis. In many instances, the dermatologist is neither looking for nor requires a specific histopathologic diagnosis. For instance, if the clinical differential diagnosis is between atopic dermatitis and psoriasis, the diagnosis of spongiotic dermatitis conveys the essential information to the clinician and guides appropriate therapy. That said, the advent of disease-specific biologic therapies targeting mediators of inflammation makes a definitive diagnosis more critical in some diseases, such as psoriasis. Although the diagnosis of many inflammatory skin diseases requires correlation with the clinical features, there are critical diagnoses, such as toxic epidermal necrolysis (TEN) and staphylococcal scalded skin syndrome, that the surgical pathologist may be asked to differentiate. The most accurate interpretation of the histopathology of inflammatory skin disease is accomplished if the pathologist is cognizant of the clinical differential diagnosis as well as the

histopathologic differential diagnosis. The pathologist must insist that an accurate clinical differential diagnosis or impression be submitted in addition to other data such as the age and sex of the patient and the anatomic site of the biopsy. Although dermatopathology specimens should be interpreted objectively, the final interpretation should always be correlated with the clinical findings. In this chapter, we have divided nonneoplastic skin diseases into various groups based on histopathologic patterns of inflammation (Tables 1.1 and 1.2). This approach is popular because it furnishes a basis for structured learning of these diseases without a prior knowledge of clinical dermatology. Like all classifications, this approach is not perfect, and it falls short at times because of the incredible complexity of the pathologic processes. Few diseases fit exclusively into only one category. Perhaps the best way to use this morphologic approach is to use the metaphor of a framework and superimposed templates. Think of each pattern as the framework and the specific histopathologic features of each disease as a template. Mentally superimposing the template then results in a modification of the original pattern. For example, in the diagnosis of lichenoid drug reaction, the pattern of lichenoid interface dermatitis is the framework. Superimposing a template of parakeratosis, eosinophils, and plasma cells over the framework leads to the diagnosis of lichenoid drug eruption. Of course, one must learn to recognize the basic patterns for this system to work effectively. TABLE 1.1 Definitions of Dermatopathology Terms Term

Definition

Acantholysis

Disruption of intercellular junctions between epidermal keratinocytes, resulting in loss of cohesion and rounding up of the affected cells

Acanthosis

An increase in the thickness of the stratum spinosum

Bulla

An intraepidermal or subepidermal blister >0.5 cm. Intraepidermal bullae may be secondary to either

spongiosis or acantholysis. Subepidermal bullae can result from disruption of basement membrane components, interface alteration, or dermal edema. Colloid bodies

Oval to round apoptotic keratinocytes typically found immediately above or below the epidermal basement membrane in interface dermatitis. These are also referred to as Civatte bodies.

Dyskeratosis

Abnormal, premature keratinization of keratinocytes. Dyskeratotic keratinocytes have brightly eosinophilic cytoplasm.

Epidermolysis

A distinctive alteration of the granular layer characterized by perinuclear clear spaces, enlarged and irregular keratohyalin granules, and an increase in the thickness of the granular layer. Acantholysis and epidermolysis are not synonyms; they are different pathologic processes.

Erosion

Partial-thickness loss of the epidermis

Exocytosis

The presence of inflammatory cells within the epidermis in association with spongiosis

Hydropic degeneration

See “Vacuolar epidermal interface alteration.”

Hyperkeratosis

An increase in the thickness of the stratum corneum. Hyperkeratosis may be either orthokeratotic or parakeratotic. Orthokeratotic hyperkeratosis is an exaggeration of the normal pattern of keratinization (i.e., no nuclei are seen in the stratum corneum). In parakeratotic hyperkeratosis, nuclei are retained in the stratum corneum.

Leukocytoclasis

Karyorrhexis and destruction of neutrophils. It frequently occurs in the setting of neutrophilic vasculitis (i.e., leukocytoclastic vasculitis).

Lichenoid epidermal interface alteration

Destruction of the basal keratinocytes resulting in “remodeling” of the basement membrane zone and associated dyskeratotic keratinocytes. A

bandlike lymphocytic inflammatory infiltrate is usually present. Orthokeratosis

Normal pattern of stratum corneum. Increased in hyperkeratosis

Papillomatosis

Abnormal elongation of the papillary dermis

Parakeratosis

Retention of nuclei in the epidermal stratum corneum

Pseudoepitheliomatous hyperplasia

Acanthosis and hyperplasia of the epidermis in a pattern that mimics squamous cell carcinoma. Epithelioma is an archaic term for carcinoma.

Pustule

A subcorneal, intraepidermal, or subepidermal vesicle or bulla filled with neutrophils

Scale crust

Parakeratotic debris, degenerated inflammatory cells, and fibrinous exudate on the surface of the epidermis

Spongiosis

Epidermal intercellular edema

Ulcer

Loss of the entire thickness of the epidermis. The dermis and subcutis may or may not be involved, depending on the depth of the ulcer.

Vacuolar epidermal interface alteration

Destruction of the basal keratinocytes characterized by the presence of intracytoplasmic vacuoles and dyskeratotic keratinocytes. A sparse to mild lymphocytic inflammatory infiltrate is usually present.

Vesicle

A small blister 0.5 cm

Crust

Fibrinopurulent exudate

Lichenification

Thickened, rough skin with accentuated skin markings. Lichenification, thickening of the skin from chronic rubbing or scratching, is not synonymous with lichenoid.

Macule

A flat lesion with change in skin color 1.0 cm

Papule

A solid elevation of the skin surface 1.0 cm

Plaque

A large, flat-topped papule >1.0 cm

Scale

Flakes of exfoliated epidermis

Vesicle

A small, fluid-filled blister 90%). As opposed to cutaneous follicle center lymphoma, clusters of plasmacytoid dendritic cells that are positive for CD123 are present in most cases (381,382). Dendritic cell meshworks (evaluated by CD21, CD23, CD35, or D2-40) are expanded and disrupted. Rearrangements of immunoglobulin heavy and light chain genes are frequently reported (380). Morphologically, there can be a resemblance to reactive lymphoid proliferations (LCC) in that MZL can feature a top-heavy infiltrate, reactive follicles, and occasional eosinophils. However, the findings of diffuse marginal zone cells, sheets of plasma cells (some with Dutcher bodies), and a B-to-T-cell ratio of 3:1 or greater favor the diagnosis of MZL, and immunohistochemical and gene rearrangement studies can be decisive. There can also be a resemblance between MZL and chronic lymphocytic leukemia, but unlike the latter, MZL typically does not express CD5 or CD43 (383). Primary cutaneous MZL is considered to be a low-grade lymphoma with an excellent prognosis. More recently, two subtypes of cutaneous MZL have been described: the most common class-switched form, with frequent plasma cells, IgG4+, and rich T-cell background; and the non–class-switched variant, which contains a background rich in B cells and a higher likelihood of local recurrence and systemic disease) (384). Recent studies have also shown cases of cutaneous MZL with associated epidermotropism and (385) presence of Epstein-Barr virus (EBV)-positive cells (386).

FIGURE 2.96 Cutaneous marginal zone lymphoma. A superficial and deep, perivascular and periadnexal lymphocytic infiltrate is present.

FIGURE 2.97 Cutaneous marginal zone lymphoma. The infiltrate is composed of small cells with the presence of atrophic germinal centers.

FIGURE 2.98 Cutaneous marginal zone lymphoma. Numerous kappa-positive plasma cells are seen by in situ hybridization.

FIGURE 2.99 Cutaneous marginal zone lymphoma. A lesser number of lambda-positive cells are seen. Plasma cells are kappa restricted.

Primary Cutaneous Follicle Center Lymphoma (PCFCL) Primary Cutaneous Follicle Center lymphoma (PCFCL) accounts for approximately 40% of all cutaneous B-cell lymphomas (387). Cutaneous dissemination in the setting of systemic follicular lymphoma is rare, and, as such, 60% of cutaneous follicular lymphomas are primary lesions. As is true for many lymphomas, PCFCL presents as a violaceous plaque or nodule, quite often in the head and neck region. Microscopically, the dermis is occupied by neoplastic follicles that contain cells with centrocytic, centroblastic, and indeterminate characteristics (Figs. 2.100 and 2.101). The lesions can have a follicular, follicular and diffuse, or predominantly diffuse growth in the dermis, and extend to the subcutis. The epidermis is spared. Between these follicles are small cells with cleaved nuclear contours. The internal portions of these follicles lack the characteristics of normal, organized germinal centers and their expected tingible body macrophages, apoptosis, and frequent mitotic activity. The neoplastic follicular cells usually have the following immunohistochemical profile: CD20+, CD79+, CD45RA+, CD10+ (Fig. 2.102), BCL-2-(Fig. 2.103), bcl-6+, CD5−, CD43−, CD23−, and cyclin D1− (388). Lesions with diffuse growth are frequently CD10-. BCL-2 immunohistochemical expression is present in about 25% of cases and is associated with the IGH-BCL2 translocation (389). As such, the presence of this translocation cannot be used to determine the origin on the disease. Rearrangement of immunoglobulin heavy and light chain genes is expected. The Ki67 in the abnormal follicles is typically low (90%). Demonstrable infection is not always proof that a follicular lesion of the skin is benign, because rare examples of PCFCL have been linked to infection with hepatitis C virus and other agents (390). MZL can also bear a morphologic resemblance to PCFCL; immunohistochemistry can be

useful as MZL lacks CD10 and bcl-6 expression. Primary cutaneous follicular lymphomas remain confined to the skin for prolonged periods, and, despite recurrences, involvement of extracutaneous sites is unusual (387). Adequate staging procedures (physical exam, CT, and PET-CT) are required in cases with BCL-2 coexpression to exclude the possibility of systemic FL. A variant of PCFCL with a spindle cell appearance, Crosti lymphoma, can sometimes be confused with mesenchymal lesions and show a predilection for the trunk (391,392). At a molecular level, 1p36 deletions have been recently reported in PCFCL, in addition to TNFRSF14 mutations (393,394).

FIGURE 2.100 Primary cutaneous follicle center lymphoma. Neoplastic follicles can be identified. The internal portions of these follicles lack the features of normal, organized germinal centers.

FIGURE 2.101 Primary cutaneous follicle center lymphoma. This abnormal follicle shows a predominance of large centroblasts.

FIGURE 2.102 Primary cutaneous follicle center lymphoma. The nodules are positive for CD10.

FIGURE 2.103 Primary cutaneous follicle center lymphoma. Nodules lack staining with BCL-2.

Diffuse Large B-Cell Lymphomas (DLBCL) As the name implies, this group of lymphomas are characterized by diffuse cutaneous infiltration by large neoplastic B cells. DLBCL can arise de novo or rarely by transformation from either MZL or follicular lymphoma (395). Cutaneous lesions can arise primarily or by secondary spread from another site; they have clinical characteristics similar to those of other Bcell lymphomas and can arise on the head and neck, trunk, and legs. Variant forms include DLBCL, leg type (this variant arises in cutaneous sites other than the legs in 12% of cases—(396)), a tumor of older adults that tends to have a worse prognosis than those arising in other cutaneous sites (397), and DLBCL, not otherwise specified (for cases that are CD10+ or MUM1- but BCL-6+). Microscopic findings include diffuse dermal growth of medium to large atypical cells that have the characteristics of immunoblasts (Fig. 2.104). Nuclei can have several nucleoli and are sometimes multilobated. Anaplastic, spindled, signet ring, immunoblastic, and plasmablastic types can occur, in addition to the previously mentioned T-cell–rich variety (395,398,399). The usual immunohistochemical profile is as follows: CD20+, CD79a+, CD19+, CD22+, bcl-6+, Ki-67+, BCL-2+, MUM-1+, CD5−, and cyclin D1− (400,401).

However, approximately 10% of tumors express CD5. CD30 positivity is sometimes seen, and commonly in anaplastic types (402). Rare examples of DLBCL tumors may express the ALK protein (395). The differential diagnosis between DLBCL and PCFCL can be challenging, particularly in cases of PCFCL with a diffuse growth and abundance of centroblasts. IgM and IgD are typically positive in DLCBL, but negative in PCFCL (403,404). P63 is also positive in a large subset of DLBCL cases, but negative in PCFCL (405). At a molecular level, about 75% of cases show MYD88 mutations, which is associated with more aggressive behavior (406-408). Cases with MYC translocations have also been documented (409). By Hans algorithm, DLBCL leg type is characteristically a nongerminal center–type lymphoma. Cases with germinal center profile should be classified as DLCBL-NOS.

FIGURE 2.104 Diffuse large B-cell lymphoma. Diffuse dermal growth of medium to large atypical cells.

Angiocentric Lymphomas There are two important B-cell lymphomas with angiocentric features. The first, intravascular DLBCL, was once thought to be malignant angioendotheliomatosis until it was determined that the neoplastic cells are lymphoid rather than endothelial in origin (410). This lymphoma occurs

in older adults and is characterized by purpuric macules, papules, and nodules together with other signs and symptoms related to small vessel occlusion, including disseminated intravascular coagulation, hypertension, nephrotic syndrome, and dementia (411). There is a “western” form, with symptoms primarily referable to the organ involved (especially neurologic and cutaneous), and an Asian form that features multiorgan failure and hemophagocytosis (412). It is a particularly aggressive disease that is only poorly responsive to therapy, although a rare case confined to the skin can have a prolonged course. Microscopically, small cutaneous vessels contain atypical lymphoid cells (Fig. 2.105) that, in most instances, express B-cell lineage antigens; the typical immunohistochemical profile is CD20+, CD22+, CD79a+, CDCD23−, CD5+/−, CD23−, and cyclin D1−. Negative CD23 and cyclin D1 expression, when combined with CD5 positivity, argues against other B-cell lymphoproliferative lesions that can express CD5 (usually considered a T-cell antigen), such as chronic lymphocytic leukemia and mantle cell lymphoma (413). Light chain restriction and immunoglobulin heavy chain gene rearrangements are regularly demonstrated (414). However, a rare T-cell variant of this lymphoma has been reported, and in those cases, the neoplastic cells express CD3 and CD45RO and may show T-cell receptor gene rearrangements (415,416).

FIGURE 2.105 Intravascular large B-cell lymphoma. Atypical lymphoid cells are found within dilated cutaneous vessels. These express B-cell lineage antigens.

The other angiocentric B-cell lymphoma is best known as lymphomatoid granulomatosis (LG). In this condition, the lung is the principal site of involvement, but the skin is considered the most common extrapulmonary site (417). LG is a disease of adults and presents with cough, dyspnea, chest pain, and skin lesions that include papules, plaques, and nodules (418,419). Spontaneous regression or response to chemotherapy has been reported, but, generally, this disorder pursues an aggressive course. LG may result from proliferations of neoplastic B cells that have been transformed by EBV (420). Microscopically, there is an angiocentric dermal infiltrate that can feature vascular occlusion, necrosis, and fibrinoid changes within vessels. The infiltrate is often polymorphous and includes small lymphocytes, macrophages, and plasma cells (417,421). However, variable numbers of large cells (the actual neoplastic cells) are identified (Fig. 2.106). The larger cells can be identified as B cells and express CD20, CD79a, and sometimes CD30 (418,420). EBV RNA can be identified in lesions by in situ hybridization methods, but this has apparently been an easier task in the lung than in skin lesions (422). Grading of skin lesions is done using the number of EBV+ cells in the infiltrate; higher grades show similar clinical behavior to DLBCL. Rearrangements of the immunoglobulin heavy chain gene are found, but there are no rearrangements of the T-cell receptor gene. Therefore, it appears that LG is in reality a kind of T-cell–rich B-cell lymphoproliferative disorder (420). In most patients with LG in the lung, the skin shows a paraneoplastic panniculitis, similar to erythema nodosum. Such lesions are negative for EBV.

FIGURE 2.106 Lymphomatoid granulomatosis. The angiocentric infiltrate contains small lymphocytes (T cells) and larger, neoplastic B cells.

EBV-Positive Mucocutaneous Ulcer EBV-positive mucocutaneous ulcer (EBV-MCU) has been recently incorporated into the WHO classification as a lymphoproliferative disorder associated with immunosuppression and/or immune-senescence of age (423). Patients typically develop sharply demarcated ulcers around the mouth, and sometimes in the anal mucosa. Spontaneous resolution usually occurs in 25% of cases, and commonly associated with improvement of the immune function. Histopathology of the involved areas shows a mucosal ulcer with reactive epithelial changes mucosa (Fig. 2.107). The infiltrate at the base of the ulcer shows a mixture of acute inflammatory cells (eosinophils, neutrophils), histiocytes, immunoblasts, and cells that resemble Hodgkin lymphoma variants (Fig. 2.108). Ares of necrosis are present. The immunophenotype of EBVMCU resembles classic Hodgkin lymphoma with expression of CD30 (Fig 2.109), CD15, and variable CD45. Dim PAX-5 expression is present. As opposed to classic Hodgkin lymphoma, the atypical cells are positive for CD20. The neoplastic cells are invariably EBV positive (424-426).

FIGURE 2.107 EBV-positive mucocutaneous ulcer. A sharply demarcated ulcer is present in the biopsy.

FIGURE 2.108 EBV-positive mucocutaneous ulcer. Large immunoblastic cells and Hodgkin-like cells are present.

FIGURE 2.109 EBV-positive mucocutaneous ulcer. CD30 expression is noted in the atypical cells.

SELECTED T-CELL LYMPHOMAS Mycosis Fungoides and Related Disorders Despite the awkward term, first used by Alibert in 1806, mycosis fungoides (MF) has been retained because it at least serves to distinguish this unique lymphoma from other cutaneous T-cell lymphomas that have different clinical presentations, clinical courses, and (in some cases) immunohistochemical profiles. It is the most common primary cutaneous T-cell lymphoma and is derived from a mature, postthymic peripheral T cell (427,428). The incidence and mortality rates appear to be increasing, although it still represents less than 0.5% of all non-Hodgkin lymphomas (429). There is frequently a history of a preceding dermatitis, often suspected to be contact dermatitis. Early manifestations of the disease are termed parapsoriasis, which is a term applied to a group of disorders of which two, parapsoriasis en plaque and retiform (variegate) parapsoriasis, have a strong association with MF. Although parapsoriasis

has been historically considered a precursor of MF, at least by many dermatologists, the prevailing view now seems to be that it represents an early stage of the disease (430,431). MF typically presents in the form of an erythematous, scaly eruption of long duration, often manifesting as patches or plaques found over the trunk. Eventually, these lesions progress to more generalized, infiltrated plaques or tumors that may ulcerate. Generalized erythroderma can be another clinical presentation, sometimes with lymphadenopathy and circulating atypical lymphocytes, which are features of Sézary syndrome (see following section on Sézary syndrome). MF can also begin with cutaneous tumors, without progressing through the prolonged superficial phase. This is known as the d’emblee variant, although it is likely that, with modern diagnostic methods, most of these cases could be shown to represent other varieties of cutaneous T-cell lymphoma, for example, subcutaneous panniculitic T-cell lymphoma (428). Unilesional MF does occur, although by its very nature, it can be difficult to diagnose; it should be distinguished from pagetoid reticulosis (Woringer-Kolopp disease), a limited form of mycosis fungoides that often responds well to local radiation therapy (432,433). Several other conditions are strongly linked with MF; these include follicular mucinosis (alopecia mucinosa) and syringolymphoid hyperplasia. Both can be associated with or evolve into MF (434,435), although many examples of alopecia mucinosa have a benign clinical course, and idiopathic examples of syringolymphoid hyperplasia have also been described. Besides pagetoid reticulosis, other clinicopathologic variants of MF include folliculotropic and granulomatous slack skin. The prognosis of MF depends on the extent of disease as reflected in the clinical stage. Those having only plaques and no systemic disease have an overall survival time of greater than 12 years (436), and individuals with limited plaque disease can have survival times approaching age-matched controls. However, patients with advanced cutaneous disease and lymph node and peripheral blood involvement have a median survival time of 5 years, and those with effacement of nodal architecture or visceral disease have a median survival time of 2.5 years (436). Transformation to large cell lymphoma is most often a harbinger of markedly diminished survival and is more commonly seen in tumor stages (437). The etiology of this lymphoma is still the subject of investigation. Previously, some considered MF to be a disease of “antigen

persistence,” with eventual breakdown of immune surveillance and evolution of a malignant clone of T cells (438). The current view is that this disease is a malignancy from the very beginning, held in check, at least initially, by host immunity (427). This concept is supported by the detection of T-cell receptor gene rearrangements early in the course of the disease. The possible role of human T lymphotropic virus (HTLV-1) or other viruses has also been the subject of investigative studies. Microscopically, early lesions that were formerly designated parapsoriasis show combinations of parakeratosis, acanthosis, and a mild superficial dermal perivascular or band-like infiltrate. Some lesions have the configuration of poikiloderma atrophicans vasculare: epidermal atrophy, vacuolar degeneration of the epidermal basilar layer, telangiectasia, and pigmentary incontinence (439,440). In patch-stage lesions, lymphocytes line up along the basilar layer and permeate the epidermis, forming small groups (couplets and triplets) and eventually larger intraepidermal or intrafollicular aggregates termed Pautrier microabscesses (Fig. 2.110). This typically occurs in the setting of minimal or absent spongiosis. Although cytologic atypia among lymphocytes is mild at first, as the disease progresses, there are increasing numbers of small- to medium-sized lymphocytes with hyperchromatic nuclei and irregular, or cerebriform, contours. Papillary dermal collagen becomes thickened and wiry, and atypical lymphocytes appear to permeate this thickened connective tissue (427,441). Admixtures with inflammatory cells, including reactive lymphocytes, plasma cells, eosinophils, or even granulomatous elements, can be seen at first, but as the disease progresses, inflammatory cells are difficult to detect. In plaque-stage lesions, lymphoid infiltrates become increasingly dense, creating a bandlike configuration in the superficial dermis and a patchy distribution of cells in the deeper dermis. An increasing percentage of intermediate and large lymphocytes can be seen, and often the lymphocytes within the epidermis show greater degrees of atypia than do those in the dermis (442). In tumor-stage lesions, large atypical cells with numerous mitoses predominate, and often the epidermal involvement diminishes. Transformation to large cell lymphoma can occur. Diagnostic criteria for this include the presence of at least 25% large cells that are greater than four times the size of an average lymphocyte. This is the only independent adverse prognostic factor in patients with advanced-stage disease (443)

(Fig. 2.111) (437). CD30 expression should not be used to define large cell transformation (444,445).

FIGURE 2.110 Mycosis fungoides. Pautrier microabscesses can be identified within the epidermis.

FIGURE 2.111 Transformed mycosis fungoides. Lymphocytes with large, markedly atypical nuclei can be identified. Many of these are CD30+.

The typical immunohistochemical profile is CD2+, CD3+, CD4+, CD5+, and TCRβ+ and CD8− (446). However, reactive CD8+ cells are often identified in the dermal infiltrate. CD7 “dropout” is a helpful feature of MF but is not always easy to discern. Most often, the numbers of CD7+ cells are diminished rather than absent, particularly when compared with their numbers in benign inflammatory diseases. Loss of CD7 in the epidermal component can be a helpful clue to the diagnosis of MF, and the combination of CD7 deficiency and T-cell receptor gene rearrangements is highly specific for the disease (447). A CD8+ lymphoproliferative disorder with resemblances to MF also occurs; some may refer to this as CD8+ MF (448). These lesions often show a more indolent clinical course. Pediatric forms of MF also have a hypopigmented appearance clinically, and are usually CD8+ (449). Approximately 10% to 15% of cases of MF show CD30 expression. Interlesional and intralesional variability of CD30 expression can be seen, a subject of major importance, as targeted CD30 therapy is one of the first lines of treatment in patients with advanced disease (450,451). The diagnosis of MF, particularly in its early stages, can be a major diagnostic challenge. A common problem is the distinction of MF from chronic, reactive inflammatory dermatoses. At present, there is no easy solution to this because the gold standard for early diagnosis of MF is still traditional light microscopy (441). Clues to the diagnosis include clustering of lymphocytes within the epidermis, particularly in the absence of spongiosis; lacunae around intraepidermal lymphocytes (“haloed cells”); singly distributed lymphocytes in the basilar epidermis along a broad front; and single lymphocytes permeating wiry papillary dermal collagen. There are several published guidelines to aid in the diagnosis of MF, employing grading systems with major and minor criteria (427). Major criteria are based on the density of the dermal infiltrate, prominence of epidermotropism, and degree of cytologic atypia; minor criteria include papillary dermal fibroplasia, degree of atypia of intraepidermal lymphocytes, and lack of inflammatory elements. Sézary Syndrome This form of cutaneous T-cell lymphoma consists of a triad of erythroderma, lymphadenopathy, and atypical circulating T cells in the

peripheral blood. Some consider it the “leukemic” variant of MF (452). It is mainly a disease of adults. Severe pruritus, palmoplantar keratoderma, and nail dystrophy sometimes accompany the process, and, on occasion, there can be an apparent eruption of numerous seborrheic keratoses. There are at least 1000 atypical cells (Sézary cells) per cubic millimeter of blood (Fig. 2.112) (428). Some believe that the diagnosis should require the demonstration of a clonal T-cell population in the peripheral blood (453). This syndrome typically has an aggressive clinical course, and the 5-year survival rates range from 10% to 33%. Treatments have included topical and systemic chemotherapy, monoclonal antibodies, retinoids, cyclosporine, and extracorporeal photopheresis. Recent information indicates that malignant cells in Sézary syndrome express lymph node homing molecules CCR7 and L-selectin and are also positive for CD27 and CCR4, all features of central memory T cells, whereas those of MF are CCR7−, L-selectin−, and CD27− but CCR4+, characteristics of resident effector memory T cells. The findings suggest that these are separate lymphomas derived from distinctive T-cell subsets (454).

FIGURE 2.112 Sezary syndrome. Circulating malignant cells in the peripheral blood area seen with cerebriform nuclei.

On biopsy, lesions of Sézary syndrome often feature significant parakeratosis and acanthosis, sometimes of psoriasiform proportions (455). There may be a band-like subepidermal lymphocytic infiltrate containing cells with cerebriform nuclei. Epidermotropism and lining up of lymphocytes along the basilar layer can be observed (456), but these changes may be less pronounced than in patch and plaque stages of typical examples of MF (Fig. 2.113) (455). Furthermore, the microscopic changes of nonspecific dermatitis can be seen in up to one-third of cases, making diagnosis a challenging prospect. At times, several biopsies may be necessary before a definitive diagnosis can be rendered. Lymph node infiltration by Sézary cells can also be identified. The usual immunoprofile (not including the T-cell subsets mentioned earlier) is similar to that of MF, so the lymphoid cells usually mark as helper T cells (CD4+) (457). CD7 dropout can occur, but some CD7+ cases are also encountered. T-cell receptor gene rearrangements are regularly found (453,458). There appears to be little or no correlation between histopathologic pattern or degree of lymph node involvement and prognosis. However, PAS-positive cytoplasmic inclusions, large circulating Sézary cells, and large cell transformation within cutaneous infiltrates are associated with diminished survival (457).

FIGURE 2.113 Sézary syndrome. Malignant infiltrate mostly centered in the dermis.

CD30 Positive Lymphoproliferative Disorders, Including Primary Cutaneous Anaplastic Large Cell Lymphoma and Lymphomatoid Papulosis These conditions have as a common thread the cutaneous infiltration of anaplastic CD30+ cells, hence the term CD30-positive lymphoproliferative disorders (CD30+ LPD). CD30+LPD are associated with MF in 20% to 25% of cases. Primary cutaneous anaplastic large cell lymphoma (ALCL) often presents as a single violaceous nodule or multiple grouped nodules confined to one body site (459,460). Microscopically, there is a dense dermal infiltrate of large, pleomorphic or immunoblastic cells that may extend into the subcutis (Fig. 2.114). Many of the cells display abundant, pale-to-eosinophilic cytoplasm and irregularly shaped or multiple nuclei containing one or more nucleoli (461). Mitoses are frequent. There is a tendency for the neoplastic cells to grow in cohesive aggregates, which can raise the possibility of malignant melanoma or poorly differentiated squamous cell carcinoma. Accompanying inflammatory cell types can include neutrophils or eosinophils (462). A large proportion of the atypical cells are CD30+ (>75% of the tumor cells) (460). There is also evidence of T-cell lineage because these cells also express CD2, CD3, CD4, and CD45RO. They also express cytotoxic markers (TIA-1, granzyme B). Loss of certain pan–T-cell antigens is sometimes encountered. Unlike the systemic form of ALCL, primary cutaneous ALCL is negative for anaplastic lymphoma–related tyrosine kinase (ALK-1) in most instances (463). The vast majority of cases show a T-cell receptor gene rearrangement. Unlike systemic ALCL with cutaneous involvement, which responds poorly to chemotherapy and has an adverse prognosis, primary cutaneous ALCL is often an indolent disease; lesions may regress spontaneously (~25%), and tumor dissemination occurs only late in the clinical course, if at all (464). Approximately 20% to 25% of cases of cutaneous ALCL show a translocation involving the DUSP22/IRF4 gene. Such translocation is associated with epidermotropic cells, a more monotonous population of intermediate-sized lymphoma cells, and a characteristic dim expression of CD30 in the epidermis, and strong in the dermis (465-467). A subset of cases show a predominant intravascular pattern (468). As opposed to cases of systemic ALCL that show secondary skin dissemination, most primary cutaneous lesions are negative for EMA. Cases with TP63 rearrangements show a more aggressive clinical course (469).

FIGURE 2.114 Primary cutaneous anaplastic large cell lymphoma. There is a dense dermal infiltrate of markedly atypical cells, in this case accompanied by numerous neutrophils and eosinophils.

Lymphomatoid papulosis was first set apart as a distinct entity by Macaulay (470,471), who (along with others) recognized a self-healing eruption of papulonecrotic skin lesions that closely resembled the established cutaneous disease pityriasis lichenoides et varioliformis acuta (Mucha-Habermann disease) but, on biopsy, featured significant cytologic atypia. Lymphomatoid papulosis primarily affects adults, with a mean age at diagnosis of 43 years, although children can also have the disorder. Recurrent crops of papulonodules form over the trunk and extremities. These ulcerate and heal with varioliform scars (472). The disease can persist for years without other evidence of malignancy, but a proportion of individuals (10%- 20%) develop malignant lymphoma (473). The lymphomas that occur include MF, Hodgkin disease, and ALCL (415). Lymphomatoid papulosis can be controlled or improved with therapies such as methotrexate and psoralen with ultraviolet A light (PUVA). Longterm follow-up of these individuals is warranted because of the risk of malignant transformation. Microscopically, there is often a wedge-shaped dermal infiltrate, with the base of the wedge near the dermal-epidermal interface and the point of the wedge in the deeper dermis; the epidermis shows varying degrees of

spongiosis, exocytosis, and necrosis, sometimes with ulceration (Fig. 2.115). There are now considered to be five histopathologic types of the disease (474,475). Type A shows large, atypical cells containing large, vesicular nuclei and clumped chromatin (similar to those of ALCL), admixed with small lymphocytes, neutrophils, eosinophils, and macrophages. The image is somewhat reminiscent of Hodgkin disease, and in fact, some of the atypical cells can mimic the Reed-Sternberg cells of the latter condition. Type B lesions feature cells with enlarged, hyperchromatic, and convoluted nuclei resembling the cells of MF. Transition between the two forms does occur. The type C lesion features solid sheets of atypical cells resembling those of ALCL. Type D, described in 2010, shows marked epidermotropism composed of neoplastic cells expressing CD8 and CD30—a cytotoxic phenotype, simulating aggressive epidermotropic CD8+ T-cell lymphoma (476). A fifth type, type E, features ulcerated lesions with angioinvasive, angiodestructive infiltrates composed of small- to medium-sized atypical lymphocytes that express CD30 and (frequently) CD8 (477). Another subtype of LyP shows also the DUSP22/IRF4 translocation (478).

FIGURE 2.115 Lymphomatoid papulosis. In this example, the epidermis is infiltrated by atypical lymphoid cells. Similarly atypical cells, with large, irregularly shaped, hyperchromatic nuclei, are seen within the underlying dermis.

Immunohistochemically, the atypical cells of lymphomatoid papulosis are often CD4+ and CD8−, with the exceptions noted earlier (479), particularly in type D and type E disease. The anaplastic cells that predominate in type A and C lesions are CD30+ (460). The infiltrating cells bear activation and proliferation markers such as CD25 and Ki-67, and cytotoxic granule proteins are also expressed (480). T-cell receptor gene rearrangements can be detected in approximately half of the cases, most often in type B or mixed cases (481). The differential diagnosis includes pityriasis lichenoides et varioliformis acuta, which lacks significant cytologic atypia, and cutaneous involvement by Hodgkin disease. With regard to the latter differential, true Reed-Sternberg cells have the immunoprofile of CD30+, CD15+, and CD45R−, whereas the cells of lymphomatoid papulosis are CD30+, CD15−, and CD45R+ (482). Type C lymphomatoid papulosis may be impossible to differentiate from ALCL without clinical data. LEUKEMIA CUTIS Skin involvement in patients with leukemia can be categorized as specific leukemic infiltrates and nonspecific manifestations. The latter category will not be discussed here but would include reactive processes such as Sweet syndrome, drug eruptions, or the insect bite–like reactions that have been described in patients with leukemias and lymphomas (483). Specific cutaneous infiltrates have been reported with most leukemias, including acute myeloid leukemia, acute myelomonocytic leukemia, acute erythroleukemia, acute lymphoblastic leukemia, chronic myelogenous leukemia, chronic myelomonocytic leukemia, chronic lymphocytic leukemia, adult T-cell leukemia, T-cell prolymphocytic leukemia, and hairy cell leukemia (484). Cutaneous involvement is particularly common in monocytic and chronic lymphocytic leukemias (484,485). The lesions consist of papules and nodules that are often particularly monomorphous in their appearance; plaques, ulcers, and purpuric lesions also occur. Acute myeloid leukemia is sometimes accompanied by chloroma, a grayish-green tumor that results from the presence of high concentrations of myeloperoxidase in infiltrating myeloblasts (485). The appearance of cutaneous lesions is typically associated with a poor prognosis (486). Microscopic features include dense dermal infiltrates of atypical cells that permeate collagen bundles and are separated from the epidermis by a grenz zone (Fig. 2.116) (487). A perivascular or periadnexal distribution

of the cells is common (484). The specific type of leukemia can often be diagnosed by particular attention to cytologic detail, but this can be aided by histochemistry and immunohistochemistry. Thus, for example, cells of myeloid leukemia express lysozyme, chloroacetate esterase (in myelocytes and mature granulocytes), and myeloperoxidase; monocytic and myelomonocytic leukemias are positive for CD43 and CD68 (488); and acute lymphoblastic leukemia shows the profile of CD19+, CD20+ (sometimes), CD79a+, CD10+, and HLA-DR+. Diagnostic issues arise in cases when there is no other known clinical evidence of leukemia (aleukemia cutis), when there is a question of leukemia versus lymphoma, or when there are only a few leukemic cells obscured by an inflammatory infiltrate (e.g., some examples of Sweet syndrome) (487).

FIGURE 2.116 Leukemia cutis. There is a dense infiltrate of atypical cells that permeate dermal collagen and are separated from the epidermis by a grenz zone. This biopsy was obtained from a patient with myelomonocytic leukemia.

Regarding immunohistochemical staining, myeloid leukemia cutis is likely when CD3−/CD20− infiltrates are found to be CD43+/MPO+ or CD43+/MPO−/CD68+/CD56−/CD117− (489). Examples that are CD56+ can be distinguished from the lymphoma, blastic plasmacytoid dendritic cell neoplasm, by using stains for CD4, CD123, and CD56 that are positive in the latter disorder (CD4 may also be positive in myeloid leukemias with

monocytic differentiation). Those that prove to be CD117+ can be distinguished from mast cell sarcoma by staining for tryptase or MITF, each of which stains mast cells but not myeloid cells (489,490). S-100 positivity has been reported in rare examples of myeloid leukemia cutis, and, in one recent case, this was associated with cytophagic activity (491). Such a result could lead to an erroneous diagnosis of melanoma, Rosai-Dorfman disease, or phagocytic activity associated with a lymphoid neoplasm, thus emphasizing the need for use of immunohistochemical panels as well as clinicopathologic correlation.

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3

Melanocytic Lesions Shyam Sampath Raghavan ■ Alejandro A. Gru

The spectrum of benign and malignant melanocytic proliferations is both complex and fascinating. An awareness of the normal developmental biology of the human melanocyte provides diagnostic insight into the morphologic diversity of melanocytic disorders. An understanding of the spectrum of benign proliferative patterns is necessary for recognizing the aberrant histologic and cytologic features of malignant melanoma and precursor lesions. Melanocytes originate from neural crest precursor cells, a pluripotent cell that arises from the dorsal-most point of the neural tube (1). Neural crest cells also give rise to neurons, glial cells, and Schwann cells (1,2). After undergoing epithelial-to-mesenchymal transition (EMT), neural crest cells that are destined to give rise to melanocytes migrate to the dorsolateral pathway by expression of matrix metalloproteinases. These cells become melanoblasts, the precursors of melanocytes, and migrate, proliferate, and terminally differentiate en route to their eventual destinations in the epidermis, choroid, iris, and leptomeninges (1). Numerous signaling pathways and genes are crucial in the regulation of melanocyte migration and development, including the PAX3 (paired-box 3), SOX10 (sexdetermining region Y [SRY]-box 10), MITF, endothelin 3, and endothelin receptor B (EDNRB) (1). The WNT/β-catenin pathway is also essential because overexpression of β-catenin in zebrafish promotes melanoblast formation and reduces the formation of neurons and glia (3). Once they reach their destination, melanoblasts develop the dendritic morphology and the ultrastructural characteristics of mature melanocytes (4,5). Their

embryogenesis and their developmental relationship to other cells of the peripheral nervous system likely contribute to the wide morphologic spectrum of benign melanocytic lesions (5-7). An example of this phenomenon includes entities such as pigmented neurofibroma and malignant melanotic nerve sheath tumor (melanotic schwannoma), the association of leptomeningeal melanocytosis with giant congenital melanocytic nevi (CMN), and the commonly observed phenomenon of neurotization in acquired melanocytic nevi (8). Stromal-epithelial interactions during embryogenesis influence the development of distinct melanocyte subpopulations that differ in anatomic distribution and the potential to evolve into various types of melanocytic proliferations (9-15). These features underscore the heterogeneous nature of malignant melanoma with respect to the different site-dependent features. As melanocytes are quite biologically distinct from cells of epithelial origin, criteria used to distinguish benign from malignant melanocytic proliferations and to identify precursor and borderline lesions are quite different from their epithelial counterparts. For example, the migration of melanocytes across the basement membrane zone of the epidermis and into the papillary dermis is part of the “normal” development of many benign melanocytic lesions, including most of the acquired melanocytic nevi. In addition, upward or lateral migration of melanocytes within the epidermis may be seen in several benign lesions (16), including Spitz nevi, pigmented spindle cell nevus (17), and acral nevi (18). As such, the isolated histologic observation of “invasive” behavior does not necessarily imply malignancy in a melanocytic lesion. In summary, the nature of melanocytic lesions is a reflection of the remarkably complex embryologic processes by neural crest cells and melanoblasts, along with their interaction with a complex and intricate microenvironment. To some degree, melanocytes indefinitely maintain some of their more primitive characteristics (i.e., ability to migrate and secrete matrix metalloproteinases), which makes an assessment of their malignancy challenging. In this chapter, we provide a basic framework to approach common benign, intermediate, and malignant melanocytic lesions.

BENIGN MELANOCYTIC PROLIFERATIONS HISTOLOGIC AND CYTOLOGIC CHARACTERISTICS Most benign melanocytic lesions tend to demonstrate varying combinations of three proliferative patterns that are readily apparent under low-power examination. One of these patterns is lentiginous hyperplasia, which describes a pattern of crowded single-cell melanocytic growth along the dermal-epidermal junction, centered at the base and sides of the rete ridges (Fig. 3.1). This pattern is often seen in compound nevi and lentigines. Another pattern, nested proliferation, describes the clonal growth of melanocytes, forming numerous nests within the epidermis or along the dermal-epidermal junction (Fig. 3.2). Lastly, pagetoid spread describes a pattern of single-cell scatter throughout the full thickness of the epidermis (Fig. 3.3). Although pagetoid spread is often associated with melanoma, it can be seen in some benign entities such as nevi of special site and Spitz nevi, typically occurring in the center of the lesion.

FIGURE 3.1 Lentiginous melanocytic hyperplasia, benign. The melanocytes are moderately increased in number and align the basal layer.

FIGURE 3.2 Nested melanocytic proliferation, junctional nevus. Cytologically bland melanocytes proliferate in nests within the epidermis.

FIGURE 3.3 Pagetoid spread. Melanocytes demonstrate upward migration into the epidermis. This example was taken from a superficial spreading melanoma.

The cytologic characteristics of benign melanocytes or nevus cells are variable. As such, comparing their size and nuclear features to those of adjacent normal keratinocytes or endothelial cells is often helpful. The nucleus of a normal melanocyte residing within the basal layer of the epidermis is typically somewhat smaller and slightly more hyperchromatic than that of a neighboring keratinocyte (Fig. 3.4) (19). A normal melanocyte will have a uniform chromatin pattern and will lack prominent nucleoli. The nuclear contour often appears polygonal or indented, and the cell cytoplasm appears clear as a result of artifactual retraction.

FIGURE 3.4 Normal melanocyte. The nuclei display characteristic polygonal, hyperchromatic features and appear smaller than the adjacent keratinocyte nuclei. The cytoplasm is collapsed, forming a thin eosinophilic rim about the nucleus. Dendritic processes are evident in one melanocyte as eosinophilic processes extending to the intercellular space of adjacent keratinocytes.

A wide spectrum of atypical nuclear changes may be seen in benign melanocytic proliferations, including mild hyperchromasia, subtle nuclear enlargement, and variably prominent nucleoli. These changes are generally reactive, degenerative, or senescent/involutional in nature, rather than malignant. Benign melanocytes rarely have significant hyperchromasia, irregular clumped chromatin, coarse nuclear membranes, or significant mitotic activity; these are all worrisome findings for melanoma. These cytologic findings can be used to stratify where a melanocytic proliferation sits on the benign-malignant axis. It should be noted, however, that certain lesions (Spitz nevi) and regions (i.e., genital and acral) are permitted more cytologic atypia than others. Symmetry is one of the hallmarks of a benign melanocytic proliferation and is readily apparent at low magnification. In a symmetric compound nevus, the junctional (epidermal) component extends to a width comparable to the underlying dermal component.

When the junctional component extends far beyond the underlying dermal component, the lesion is said to demonstrate a “shoulder,” which is a marker of aberrant development (20). Such apparent histologic asymmetry may reflect the asynchronous migration of the junctional melanocyte population into the papillary dermis or an accelerated junctional melanocytic proliferation in what otherwise was a normally evolving lesion. While uniform symmetry alone is not a definitive marker for benignity, asymmetry should certainly encourage the pathologist to inspect the lesion more carefully. Dermal maturation is the process by which melanocytes become smaller and more cytologically banal with increasing dermal depth. This occurs in benign intradermal or compound melanocytic nevi, and its absence can be a distinguishing characteristic in melanoma (with some exceptions in cases of pseudomaturation of the so-called nevoid melanoma). In benign nevi, superficial melanocytes tend to be larger with more abundant cytoplasm, whereas deeper melanocytes become smaller and sometimes more spindled, with delicate-appearing nuclei. Mitotic figures should never be found in the deep aspect of benign proliferations. Associated with maturation is involution, the symmetric replacement of dermal nevomelanocytes by normal dermal connective tissue. A wide spectrum of histologic patterns of involution may be seen in benign melanocytic lesions, including schwannian differentiation (so-called “neurotization”), lipomatous degeneration, and inflammatory regression. A variety of cytologic patterns of involution may also be observed, including giant cell transformation, the presence of bizarre cells, and balloon cell formation. In all cases, however, such histologic patterns of senescence should appear uniform and symmetric. LENTIGO SIMPLEX The common lentigo or lentigo simplex is a benign pigmented lesion characterized by accumulation of melanin pigment in basilar keratinocytes and melanocytic hyperplasia (a normal ratio of keratinocytes and melanocytes is ~10-20:1). Lentigines are acquired

lesions that arise during childhood and appear as small, uniformly dark brown macules. They may occur anywhere on the skin, but generally show a predilection for acral sites. Microscopic examination reveals lentiginous melanocytic hyperplasia that is usually distributed along the tips of rete ridges (Fig. 3.5). The adjacent keratinocytes are usually hyperpigmented, and a slight degree of epidermal rete hyperplasia may be present. The histopathologic features are distinguished from those of acquired melanocytic nevi by the absence of nested melanocytes (21).

FIGURE 3.5 Lentigo simplex. Dense melanin deposition in basilar keratinocytes is observed primarily along rete ridges. Lentiginous melanocytic hyperplasia may be obscured by the pigment.

Unlike acquired melanocytic nevi and solar lentigo, lentigo simplex lesions are not associated with mutations of BRAF (V600E), PIK3CA, or FGFR3 (22-24).

SOLAR LENTIGO The solar or actinic lentigo is a pigmented lesion resulting from chronic sun exposure. There is marked variation in microscopic findings among these lesions (Fig. 3.6). Solar lentigines appear as multiple, often poorly circumscribed areas of macular hyperpigmentation that may exceed 1 cm in diameter. Histologically, solar lentigines demonstrate hyperpigmentation of basilar keratinocytes and lentiginous melanocytic hyperplasia with a background of solar damage/elastosis. Occasionally, there will be marked secondary epidermal rete hyperplasia, sometimes simulating the appearance of a reticulated seborrheic keratosis. It should be noted that in small biopsies of large lesions on sun-damaged skin, caution is advised in rendering an outright diagnosis of solar lentigo. The observation of cytologically benign changes within a limited tissue sample does not necessarily rule out the presence of adjacent histopathologic atypia.

FIGURE 3.6 Solar lentigo. An irregular pattern of increased melanin pigment within keratinocytes is seen in the epidermis. Solar elastosis, telangiectasia, and

slight chronic inflammation are seen in the underlying dermis.

A variant of solar lentigo is the so-called ink spot or reticulated lentigo, which can often resemble melanoma clinically. Histologically, this variant shows lentiginous melanocytic hyperplasia and narrow segments of prominent basilar pigmentation that can focally extend to the stratum spinosum and stratum corneum (25). Another variant of solar lentigo/lentigo simplex is the mucosal (labial) melanotic macule, which is present on the mucosa of the lip and genital sites. Recent studies have shown that solar lentigo and seborrheic keratosis share common mutations in FGFR3, PIK3CA, and RAS (23,24), potentially indicating that these lesions lie on a spectrum of evolution. Interestingly, these same mutations have been detected in a proportion of benign lichenoid keratosis lesions, also suggesting that lichenoid keratosis represents a common pattern of regression of solar lentigo and seborrheic keratosis (26). ACQUIRED MELANOCYTIC NEVUS The acquired melanocytic nevus is the most common melanocytic tumor in humans and typically appears on the skin or mucosa as small brown macules and papules (27). Ultraviolet (UV) exposure, particularly intermittent exposure in childhood, is associated with the formation of these nevi (28). Common sites of occurrence include the head and neck, sun-exposed trunk, and extremities. Molecular studies have shown that most acquired melanocytic nevi arise from activating mutations in BRAF (85%) and NRAS (29,30), which are distinct from the molecular drivers involved in other benign nevi such as blue nevi or Spitz nevi (see later discussion). Acquired nevi first develop in childhood and increase over the first three decades of life and then decrease with age; over an entire lifetime, a person may develop hundreds of nevi (27,31). As such, a new melanocytic lesion in an elderly patient should be approached with caution. Similarly, the junctional component of melanocytic nevi tends to diminish with age; hence, proliferations with a prominent junctional component in older patients should be examined carefully. Histologically, three

major subtypes of acquired melanocytic nevi exist, representing the stages in the developmental progression of benign nevi: junctional, compound, and intradermal. The junctional nevus is the earliest stage of an intraepidermal melanocytic proliferation. Nevomelanocytes are dispersed in multiple discrete nests along the dermal-epidermal junction, although lentiginous melanocytic hyperplasia may also be present. Junctional nevi appear clinically as small, slightly raised, and deeply pigmented lesions. The compound melanocytic nevus includes both a junctional component and the infiltration of the dermis by nevus cells distributed singly and in nests. Clinically, such lesions appear elevated or dome shaped, and they are less intensely pigmented than the junctional nevi. The intradermal nevus no longer displays a junctional component, and nevomelanocytes are confined to the dermis. These lesions are often associated with varying degrees of senescent/involutional change, such as neurotization. Clinically, they appear flesh colored or lightly pigmented, and they are dome shaped or pedunculated. As discussed earlier, the dermal components of acquired nevi should mature, meaning that nevomelanocytes should become smaller, less pigmented, and more cytologically bland with increasing dermal depth. The designations junctional, intradermal, and compound do not necessarily refer to discrete melanocytic entities but rather to the histologically characteristic stages in the natural progression of the common acquired melanocytic nevus. Certain cytologic features are characteristic of stages in the evolution of melanocytic nevi (Fig. 3.7). The intraepidermal nevus cell, referred to as the epithelioid melanocyte or type A nevus cell, contains a round-to-oval nucleus that is slightly smaller than the nuclei of the adjacent keratinocytes (Fig. 3.8). The nucleus contains finely dispersed chromatin that is similar to that of neuroendocrine cells and, occasionally, a single inconspicuous nucleolus. The cytoplasm is prominent and often contains moderately coarse melanin granules.

FIGURE 3.7 Acquired compound nevus. The nevus cells are present in the epidermis and papillary dermis in nests with minimal involvement of the superficial reticular dermis.

FIGURE 3.8 Type A nevus cells, compound nevus. Clusters of epithelioid nevus cells with well-demarcated cell boundaries, abundant eosinophilic cytoplasm, and occasional intranuclear inclusions. This type of melanocyte is typically present in the intraepidermal nests and superficial papillary dermis.

The lymphocyte-like or type B nevus cell is part of the dermal component of compound nevi (Fig. 3.9). The nucleus is small and round, and it contains uniformly dispersed chromatin with no apparent nucleoli. Scant, nonpigmented cytoplasm is evident.

FIGURE 3.9 Type B nevus cells, compound nevus. Epithelioid and monomorphous melanocytes with granular chromatin and inconspicuous nucleoli.

The neural or type C nevus cell is often present at the base of melanocytic lesions (Fig. 3.10). This cell is typically spindle shaped, and it contains a somewhat smaller oval nucleus with a banal chromatin pattern. These fusiform cells come to rest at, or singly infiltrate, the superficial reticular dermal collagen bundles.

FIGURE 3.10 Type C nevus cells, compound nevus. Fusiform and spindle melanocytes with scant cytoplasm singly infiltrate the superficial reticular dermis at the base of the lesion.

Different patterns of epidermal hyperplasia may be seen in association with melanocytic nevi (32,33). Acanthotic epidermal proliferation with pseudo–horn cyst formation may clinically and histologically mimic a seborrheic keratosis. Extensive reticulated hyperplasia is common in some pedunculated dermal nevi. An inflammatory host cell response is a common finding within acquired nevi. It can be caused by external trauma, for example, from excoriation, shaving, or plucking hairs. A prominent inflammatory response can also be seen in a “halo nevus,” in which lymphocytes infiltrate and destroy melanocytes (34-36) (Fig. 3.11). Clinically, this results in an enlarging peripheral rim of hypopigmentation around a common acquired nevus (37,38). It should not be confused with the inflammatory host response seen in dysplastic or malignant melanocytic proliferations (see the following texts).

FIGURE 3.11 Halo nevus. A dense lymphocytic infiltrate obscures residual junctional and dermal nests of nevus cells.

Long-term clinical observation of benign nevi reveals that most lesions undergo gradual involution (39,40). Junctional melanocytes regress, and residual dermal nevus cells are ultimately replaced by fibrous stroma. Melanocytic nevi can display histologic and cytologic variations that reflect senescence/involution, including neurotization or schwannian differentiation, in which the formation of neural structures in loose connective tissue often simulates a neurofibroma (Fig. 3.12); lipomatous degeneration, with infiltration of dermal nevi by fat cells; and osseous metaplasia. It should be noted that such histologic and cytologic patterns may also occur in melanoma and should, therefore, never be used as the sole criterion for discriminating between benign and malignant melanocytic tumors.

FIGURE 3.12 Dermal nevus. Extensive neurotization or schwannian differentiation of dermal nevus cells is present. Nests of dermal nevus cells may resemble peripheral neural structures, such as Wagner-Meissner corpuscles.

Most common acquired melanocytic nevi and lentigines display symmetry, uniform pigment distribution, and smooth boundaries with adjacent skin. These lesions are biopsied when there are visual features of asymmetry, uneven pigment distribution, and irregular or jagged borders, as these are features that may indicate evolving melanoma. In many cases, an atypical-appearing pigmented lesion proves to be microscopically benign. A “changing mole” is the most common clinical cause for biopsy of pigmented lesions. In an otherwise previously uniform symmetric nevus, concerning changes typically include an increase in size, the formation of irregular borders with adjacent normal skin, and a peripheral change in color. Although such clinical changes can be characteristic of dysplastic nevi, a substantial percentage of these lesions display entirely benign cytologic features. Microscopically, the principal correlate is a so-called “shoulder” of lentiginous junctional melanocytic proliferation beyond the lateral border of the underlying dermal component. Careful cytologic assessment of the

peripheral lentiginous junctional component in addition to that of the more central domain is essential. The absence of significant nuclear changes must be confirmed. The biologic significance of peripheral changes and a “growing shoulder” within previously stable nevi is unclear. It is widely accepted that most common acquired nevi begin as junctional nevi. Subsequent dermal migration results in the establishment of a dermal component. The lentiginous junctional pattern of melanocytic hyperplasia is typically associated with an active, growing phase. One possibility is that, in many benign changing moles, radial junctional proliferation is reactivated. Because the clinical appearance of reactivated radial proliferation in a benign nevus may be indistinguishable from that of many dysplastic nevi and some superficial spreading melanomas (SSMs), biopsy and careful histologic evaluation are indicated. CONGENITAL MELANOCYTIC NEVUS CMN are nevi defined by their presence at birth or in the first few weeks of life (41). These lesions occur in approximately 1% of neonates (42). They are classified as small, medium, large, or giant, depending on their size and anatomic location. Small- and mediumsized nevi are common, although large and giant CMN are rare, occurring in 1 per 20,000 to 1 per 500,000 newborns (41). Melanoma risk in patients with small CMN is less than 1%, whereas in large/giant CMN, it is approximately 5% (41,43). Congenital nevi involving the craniosacral axis have the highest risk of neurocutaneous melanosis. In most cases, the history of a pigmented lesion present from birth or the existence of easily recognizable clinical features readily permits a clinical diagnosis. Clinically, CMN often display irregular surface contours, heterogeneous pigmentation, and are hair bearing. Most congenital nevi are not sampled because they display asymmetric features but because they have changed in appearance. Histologically, both compound and dermal patterns are seen. Some congenital nevi are microscopically indistinguishable from

benign acquired nevi. In the most histologically characteristic lesions, the distribution of nevus cells throughout the dermis is more extensive than that seen in acquired nevi, typically involving the lower two-thirds of the reticular dermis and surrounding adnexal structures (Fig. 3.13). The presence of nevus cells within cutaneous structures, including sebaceous lobules, arrector pili muscles, and the perineurium of peripheral nerves, is characteristic of congenital nevi and is not seen in acquired nevi (Figs. 3.14 and 3.15) (44,45). The cytologic features of CMN cells and their patterns of maturation and senescence differ little from those previously described for acquired nevi (46,47).

FIGURE 3.13 Congenital intradermal nevus. Nevus cells are deep within the reticular dermis and in close association with the appendages.

FIGURE 3.14 Congenital compound nevus. Nevus cells are infiltrating a sebaceous lobule.

FIGURE 3.15 Congenital compound nevus. Nevus cells are infiltrating a peripheral nerve twig.

The clinical differences between congenital nevi and common acquired nevi may reflect the distribution and density of nevomelanocytes within the underlying appendageal epithelium and arrector pili muscles (Figs. 3.13 to 3.15). Congenital nevi may appear as a localized collection of individual pigmented lesions in a nonrandom, usually linear array, likely representing formation along Blaschko lines (48). As with most acquired melanocytic nevi in which a change in size or contour is reported, the most common benign microscopic correlate observed is peripheral junctional lentiginous melanocytic hyperplasia. Another common abnormality that would prompt biopsy of a congenital nevus is the emergence of a so-called “proliferative nodule.” This is an expansile proliferation of epithelioid cells arising within a congenital nevus that can show benign or atypical cytologic features. Recent studies show that immunohistochemically, these lesions may represent a borderline category between nevus and melanoma (49), whereas genetic analysis shows that they have a different aberration pattern than melanomas and typically have gains of whole chromosomes (50). Most small congenital nevi will show a BRAF mutation; however, larger congenital nevi are more likely to show an NRAS mutation (41,49). It should be noted that some common acquired nevi can show similar histologic findings as CMN, such as infiltration around adnexa and involvement of arrector pili muscles. The etiology and pathogenesis is subject to debate. In our practice, these are termed nevi with congenital features. BALLOON CELL NEVUS This peculiar change can be found in 1.7% of all melanocytic proliferations (51,52) and can be seen in almost any type of melanocytic proliferation, including intranodal nevus cell nests (53). It can also be identified clinically via dermoscopy (54). Microscopically, balloon cell change manifests as melanocytes with clear-to-foamy cytoplasm containing a small benign-appearing hyperchromatic nucleus (Fig. 3.16). Melanocytic pigment can be conspicuous or

absent. If nuclear atypia is prominent and other features of malignancy are identified, the rare balloon cell variant of malignant melanoma should be considered (55). Early studies (56,57) have shown balloon cell change results from impaired melanin synthesis with subsequent ballooning cellular degeneration (58).

FIGURE 3.16 Balloon cell nevus. Nevus composed of clear-to-foamy melanocytes cytoplasm containing a small benign-appearing hyperchromatic nucleus.

NEVI OF SPECIAL SITES A percentage of nevi arising in particular anatomic sites have been recently recognized to show atypical histologic characteristics despite a benign clinical course. These sites include the ear, scalp, milk line (breast to genital areas), and acral skin. This particular group of lesions exhibits distinct and often worrisome cytologic and architectural features that separate them from other nevi and are the source of possible diagnostic pitfalls. The etiology of these peculiar cytologic and architectural features is unknown. Special site nevi of the ear and scalp are particularly challenging diagnostically, given their location on sun-exposed skin and relatively common occurrence. These nevi accounted for 10% of scalp nevi in one series (59) and up to 42% of periauricular lesions in another

series (60). Ear lesions tend to show poor lateral circumscription and a stromal reaction composed of lymphocytic inflammation and dermal fibroplasia (61). Large nests can be identified at the tips and sides of the rete, as well as in the inter-rete spaces (61,62). Pagetoid spread is typically not prominent. Scalp lesions can show large, confluent, and bizarre-shaped nests along the dermal-epidermal junction in a random distribution (61). Lentiginous growth, including down adnexal structures, along with pagetoid spread, can be conspicuous (61). Both types can show cytologic atypia, often with enlarged hyperchromatic nuclei with clumped chromatin. Nevi of the milk line, genital nevi, and nevi of flexural sites share several cytologic and architectural features. Nevi of the milk line comprise the axillary folds, the mammary area, the umbilicus, and the inguinal folds. Milk line and genital nevi (Fig. 3.17) share the “nested and discohesive” pattern, characterized by the presence of large, confluent nests at the dermal-epidermal junction, along with diminished melanocytic cohesion (61,62). Vulvar nests are often oval and lie along the rete ridges. Other distinctive changes of this group of lesions are stromal fibroplasia, which is usually coarser than in dysplastic nevi, and frequent lentiginous proliferation of the nevomelanocytes. Genital lesions, especially those occurring in the vulva, often show more prominent atypical features, such as moderate-to-severe cytologic atypia (epithelioid cells with prominent nucleoli) and occasional pagetoid spread. Also, the nests in these genital lesions are large, irregular, and frequently exhibit prominent pigmentation (63). Melanocytic nevi in genital skin in the setting of lichen sclerosus can also show worrisome features that can potentially mimic a melanoma diagnosis (64,65).

FIGURE 3.17 Compound nevus with features of nevi of special sites. Large intraepidermal and dermal melanocytic nests are associated with fusion of rete ridges and irregular pigmentation. Dyscohesion of nested nevomelanocytes is frequently observed. These features are commonly seen in lesions in the milk line and genital area.

Melanocytic proliferations occurring in acral skin (hands and feet) are typically more cellular and arranged in a lentiginous pattern as opposed to a nested pattern. Up to 85% of benign acral nevi show bridging between rete (61). In addition, acral nevi can show upward pagetoid spread; in cases where this is quite prominent, the term melanocytic acral nevus with intraepithelial ascent of cells (MANIAC) can be used (61). The melanocytic nests are often discohesive and irregular in size and shape, sometimes adopt a crescentic shape, and may coalesce (Fig. 3.18). Pagetoid spread is present in 36% to 61% of all nevi in the palm and soles (16,61). In the benign lesions, the areas of pagetoid ascent are restricted to the areas of nested proliferation and have benign morphology. In contrast, the pagetoid cells in acral lentiginous melanoma extend well beyond the nests in a haphazard array (66,67).

FIGURE 3.18 Acral nevus. Intraepidermal nests are irregularly disposed at the tips of the rete ridges but also occasionally between the rete ridges. Pagetoid spread is commonly seen, but it is always confined to the areas of nested proliferation.

It is important to keep in mind that all these lesions invariably show benign features, such as maturation and absence of dermal mitoses. These clues are helpful in differentiating these sometimes worrisome looking but benign lesions from dysplastic nevi and malignant melanoma. COMBINED NEVUS On occasion, elements of two different types of dermal or epidermal melanocytic proliferation may be present in the same pigmented lesion (68). These so-called combined nevi commonly include components of an acquired melanocytic nevus and the common blue nevus (69), though combinations with deep penetrating nevi, BAP-1– inactivated nevi, Spitz nevi, and balloon cell nevi have been described as well. It is important to distinguish combined nevi from

collision nevi, as well as a melanoma arising in association with a nevus. DEEP PENETRATING NEVUS Clinically, deep penetrating nevi can occur de novo as blue papules or nodules and can be mistaken for a blue nevus or melanoma. The most common presentation is within a preexisting acquired or congenital nevus in which the development of a focal bluish discoloration raises the possibility of malignant degeneration. This lesion can be mistaken for malignant melanoma in as many as 40% of cases (70). Histologically, this nevus consists of a symmetric wedge-shaped proliferation of epithelioid, lightly pigmented melanocytes extending into the deep dermis, and, sometimes, the subcutaneous fat (Fig. 3.19). When it occurs de novo, there is usually an intraepidermal/junctional component. When it occurs in a preexisting nevus, it usually does so as a focal transformation within the dermal component. The types of cells that comprise the deep penetrating nevus are most commonly the inverted type A nevus cells (epithelioid) but sometimes are also the spindled type B nevus cells. Mitoses are very rare and, when present, should lead to thorough sectioning to rule out malignant transformation (43).

FIGURE 3.19 Deep penetrating nevus. A proliferation of epithelioid melanocytes with abundant cytoplasm admixed with melanophages. These lesions will show nuclear expression of β-catenin.

Recent studies have characterized the underlying genetic drivers that ultimately lead to the formation of deep penetrating nevi. In addition to mutations in the MAP kinase (MAPK) pathway, deep penetrating nevi are driven by activating mutations in the Wnt/βcatenin pathway (71). This leads to nuclear localization of β-catenin within the nevomelanocytes. As such, immunohistochemistry for βcatenin can be useful diagnostically. LEF1, which is activated downstream of β-catenin, has also shown to be upregulated in these nevi and can similarly be used to aid in the diagnosis (72). NODAL MELANOCYTIC NEVI The finding of nests of benign melanocytes within lymph nodes was first reported in 1931 by Stewart and Copeland (73). Within the literature, the incidence of benign nodal nevi ranges from 1% to 22% (74). The incidence of nodal nevi in sentinel lymph nodes of patients

with cutaneous melanomas is 3.9%, which is higher when compared with the incidence in nonsentinel lymph nodes in melanoma patients (1.01%) (75). Despite the common prevalence, the origin of these melanocytes is unknown. The melanocytes in nodal nevi are small and bland and can be challenging to identify on standard hematoxylin and eosin stains (Fig. 3.20). They are often identified with immunostains, which are utilized routinely in evaluation of sentinel nodes in patients with melanoma. In the majority of cases, nodal nevi are intracapsular or intratrabecular, although cases of intraparenchymal nests have been reported (76).

FIGURE 3.20 Nodal blue nevus. There is a capsular proliferation of benign melanocytes with heavy pigmentation. Note the extraparenchymal location of the melanocytes.

Pathologists should be aware of the relatively high incidence of nodal nevi among sentinel lymph nodes in malignant melanoma in order to avoid erroneous interpretations. In addition to the intracapsular and intraseptal location of the nests, a distinguishing feature from metastatic melanoma is bland cytology, which is distinct from pleomorphic melanoma cells. Recent studies have shown PRAME (PReferentially expressed Antigen in MElanoma—see later discussion) to be a useful immunohistochemical tool for this

distinction (77). PRAME is positive in most melanoma types and is consistently negative in nodal nevi (77). PERSISTENT/RECURRENT MELANOCYTIC NEVUS Persistent/recurrent nevi are nevi that reappear after biopsy or excision and often present as a pigmented lesion at a scar. Usually, such a recurrence is seen within several months of the procedure and is clinically worrisome owing to irregular pigmentation and architecture (78-83). Typically, these lesions show a trizonal appearance with a lentiginous proliferation of melanocytes in the epidermis, broad fibrosis within the dermis consistent with a scar, and a nevoid proliferation underneath the fibrosis, in keeping with a residual nevus (Figs. 3.21) (81,84). The melanocytes can show prominent cytologic atypia, though it is less than what is typically seen in melanoma.

FIGURE 3.21 Recurrent melanocytic nevus. At low power, a linear fibrotic scar is seen in the superficial dermis between an atrophic epidermis devoid of adnexa and an underlying aggregate of residual dermal nevus cells.

REGRESSING MELANOCYTIC NEVUS The presence of a dense, superficial lymphoid cell infiltrate in an otherwise cytologically benign melanocytic nevus usually reflects inflammatory regression. The best-known example of such

regression is the halo nevus, in which inflammatory regression occurs in a symmetric centripetal manner (Fig. 3.11). Although the clinical appearance of a halo nevus is quite distinctive, the microscopic features may be no different from those observed in a changing pigmented lesion in which the progressive change in pigmentation is irregular or asymmetric (85).

SPITZ NEVUS AND VARIANTS SPITZ NEVUS Spitz nevi are benign melanocytic neoplasms composed of large epithelioid and/or spindled cells that were first described by Sophie Spitz in 1948 as “melanomas of childhood” (86). Despite the atypical and sometimes worrisome histologic features, these lesions are ultimately benign. In her initial case series of 13 children, all but one patient had an indolent clinical course. This type of nevus is usually observed in children and young adults, but has been reported in almost every age group. Common anatomic sites include the head and neck and extremities. Clinically, this nevus presents as a small, solitary, dome-shaped nodule. Because of a prominent vascular component in the tumor stroma and a relative lack of melanin pigmentation, it is frequently misdiagnosed clinically as a hemangioma or pyogenic granuloma. Spitz nevi are typically well-circumscribed and symmetric proliferations of epithelioid and/or spindled melanocytes. There are junctional, compound, and intradermal forms (Fig. 3.22) (87,88). Large nests are common and extend from the epidermis to the dermis in a wedge-shaped distribution. Within the epidermis, there is often “clefting” around the nests. Other features include the formation of “kamino bodies” (nodules of hyaline material that are accentuated by periodic acid-Schiff [PAS] stains) along with epidermal hyperplasia and central pagetoid spread (89,90). Cytologically, the melanocytes contain slightly enlarged nuclei with uniform nucleoli and variable degrees of pleomorphism. Mitotic

activity is low (6 mm) and share worrisome features with melanoma, such as irregular borders, asymmetry, and heterogeneous pigmentation. Taking into account a constellation of clinical, histologic, and molecular findings, current classification delineates three distinct groups within the “Spitz” category: Spitz nevi, atypical Spitz tumors, and Spitz melanoma (108). Histologically, atypical Spitz tumors are larger than classic Spitz nevi, are asymmetric, and can extend deep into the dermis or subcutaneous fat. They can show complete consumption or effacement of the epidermis with prominent pagetoid spread. Cytologic atypia is somewhat more severe than what is typically present in Spitz nevi. Mitoses are much more common and, in some cases, can be as frequent as 2 to 4 per mm2. For these lesions, sentinel lymph node biopsy (SLNB) is recommended. The reported range of positivity for SLNB is 26% to 50% (111-113). Spitz melanoma is a newly designated entity that combines malignant histologic and clinical findings with molecular drivers present in Spitz nevi (see prior discussion). Melanomas that have

features of Spitz neoplasms but lack classic Spitz drivers (i.e., contain a BRAF V600E mutation) are designated spitzoid melanomas. Spitz melanomas are often larger than 10 mm, asymmetric, poorly circumscribed, and lack maturation (108). Mitotic activity tends to be brisk (>6 per mm2) with deep figures and atypical forms. Marked pleomorphism is often present. These lesions are treated with wide excision and sentinel node biopsy. Although the previous criteria are often helpful for making a diagnosis, many cases are challenging to classify. For example, the authors have seen cases with bland cytologic features and good circumscription, but with robust mitotic activity. In such instances, the use of ancillary techniques such as FISH and comparative genomic hybridization (CGH) can be quite useful. Both of these tests represent forms of genomic copy number analysis (114). While this is discussed in more detail later, briefly, nevi tend to have stable copy number profiles, whereas melanomas have multiple copy number gains and losses. Regardless of these findings, clinicopathologic correlation is essential when evaluating these types of melanocytic neoplasms. BAP1-INACTIVATED MELANOCYTIC NEOPLASMS BAP1 is a tumor-suppressor gene located on chromosome 3 that is associated with BRCA1. Recent studies have shown germline mutations in BAP1 cause a familiar tumor syndrome characterized by an increased risk for melanocytic tumors (115). These tumors can also occur sporadically and are characterized by a proliferation of epithelioid melanocytes with abundant “glassy” cytoplasm and welldefined cytoplasmic borders (Fig. 3.28). Most lesions demonstrate a BRAF V600E mutation or an NRAS mutation, in addition to a BAP1– inactivating mutation and loss of the wild-type allele (27,114,115). Owing to their cytologic characteristics, they were previously categorized as atypical Spitz tumors, but it is now understood that they are not genetically related to Spitz nevi. They tend to be low grade and do not recur after excision (114,115). Distinction between a BAP1–inactivated nevus and melanoma can be very challenging.

FIGURE 3.28 BAP1–inactivated melanocytic tumor. (A) There is a proliferation of epithelioid melanocytes with well-defined cytoplasmic borders. (B)

Immunohistochemistry for BAP-1 shows loss of expression within this population.

DERMAL MELANOCYTOSES Certain acquired and congenital pigmented lesions are associated with proliferative disorders of dermal melanocytes (116,117). These dermal melanocytes are believed to be derived from ectopic nests of migratory neural crest cells (118) or nerve sheath (Schwann) cell precursors with the capacity for both nerve sheath and melanocytic differentiation (119). These lesions include the common and cellular blue nevus, the Mongolian spot, and the nevi of Ota and Ito. All of these neoplasms are characterized by a proliferation of delicate spindle cells with cytoplasmic melanin granules admixed with bundles of reticular dermal collagen and scattered melanophages. Clinically, the dermal melanocytoses display a blue or gray color that results from the absorption of long wavelengths of visible light by dermal melanin and reflection of the shorter blue wavelengths (Tyndall effect). MONGOLIAN SPOT The Mongolian spot is a gray, blue, or brown macule that is present over the lower back or buttocks (120). It is present at birth in most Asian neonates, and it usually regresses within several years. It is rather uncommon in Caucasians. Histologically, the lesion appears as a dermal proliferation of rare elongated melanocytes containing fine melanin pigment granules, admixed with occasional melanophages. The melanocytes are randomly scattered, usually in the lower two-thirds of the reticular dermis and subcutis. NEVUS OF OTA AND NEVUS OF ITO These uncommon pigmented lesions are acquired hamartomas of dermal melanocytes, usually occurring in the first or second decade of life. They are observed most frequently in Asians, but they may also occur in individuals of Hispanics, African, and Native American

descent. They appear clinically as macular areas of irregular bluegray pigmentation (121). The nevus of Ota occurs in periorbital and temporal skin in the distribution of the first and second branches of the trigeminal nerve (122). The nevus of Ito occurs over supraclavicular and scapular skin in the distribution of the lateral supraclavicular and brachial nerves (108). These lesions tend to persist and do not fade away with time. Histologically, these lesions appear to be distinct from the Mongolian spot. Low-power examination reveals deeply pigmented cells scattered sparsely, usually throughout the entire reticular dermis but sometimes limited only to the upper one-third. Fusiform dermal melanocytes with delicate melanin pigment granules are present with the melanophages, often in a perivascular distribution (Fig. 3.29). They show diffuse staining for HMB-45.

FIGURE 3.29 Nevus of Ota. The dermal melanocytes exhibit dendrites filled with fine melanin granules.

COMMON BLUE NEVUS The common blue nevus is a solitary, small (1 cm). Histologically, the lesion displays a well-circumscribed cellular mass of interweaving fascicles of predominantly nonpigmented or lightly pigmented spindle cells (Fig. 3.31) (108,117,129,130). Cellular atypia and mitotic activity are absent. Melanophages can be numerous and heavily pigmented. One characteristic form of the cellular blue nevus is that of a well-circumscribed dermal proliferation that adopts a bulbous configuration going deep into the subcutaneous tissue with a “dumbbell” shape. Another commonly encountered pattern is that of a lesion with a growth pattern parallel to the epidermis with extension deep into the fascial planes, thus designated “plaquelike cellular blue nevus.” Morphologic features can occasionally overlap with the common blue nevus; an arbitrary cutoff of 50% or more hypercellularity has been instituted by some authors to differentiate these entities (131).

FIGURE 3.31 Cellular blue nevus. Dense proliferation of spindled melanocytes admixed with hemosiderin-laden macrophages.

On occasion, peculiar areas of myxoid/liquefactive degeneration that is distinct from tumor cell necrosis can be seen. The presence of these areas could be attributed to an ischemic effect because these lesions tend to occur in pressure-prone areas (i.e., buttocks).

Cellular blue nevi can show focal perineural and even intralymphatic invasion; these findings should be carefully evaluated in the context of the lesion. The histologic differential diagnosis may include other cellular spindle cell tumors, such as a cellular dermatofibroma and, possibly, leiomyoma. A close examination reveals the presence of melanin-containing fusiform dermal melanocytes interspersed throughout the lesion. An important differential diagnosis is the socalled malignant blue nevus (melanoma arising in blue nevus), which relies on the presence of necrosis, infiltrative “sarcomatoid” growth pattern, cellular atypia, and pleomorphism with large epithelioid cells as well as a mitotic rate of greater than 2 per mm2 (129,132). However, this distinction is not always so straightforward, sometimes requiring the use of an “atypical” category. EPITHELIOID BLUE NEVUS (BLUE NEVUS WITH EPITHELIOID CELLS) Epithelioid blue nevi (EBN) describe a subset of blue nevi with a significant population of plump epithelioid cells with a “fried-egg” appearance (108). Recently, classification has separated these nevi from the so-called pigmented epithelioid melanocytoma (PEM), which is found in Carney complex (see following section) (108,133,134). EBN usually occur in the trunk and extremities of children and adults. Microscopically, the lesions are composed of a roughly dome-shaped dermal proliferation of epithelioid cells with abundant coarsely pigmented cytoplasm bearing large nuclei with prominent nucleoli. Some cells can show a perinuclear area that is spared of pigment. A variable amount of more conventional dendritic blue nevus cells as well as melanophages can be found admixed with the epithelioid cells. The presence of a junctional component usually indicates a combined nevus. ATYPICAL BLUE NEVUS Atypical blue nevus represents an intermediate category that lies between cellular blue nevi and the so-called malignant blue nevi (blue nevus–like melanoma). Fortunately, it is quite rare.

Histologically, there is a large nodule of tumor that effaces the dermis and destroys nearby adnexal structures. The melanocytes are spindled to epithelioid with variable pigmentation, and there are areas of increased mitotic activity with the possibility of tumor necrosis. Criteria have been proposed for this diagnosis, which include lesions with a cellular blue nevus morphology that exhibit size greater than 3 cm, increased cellularity, increased mitotic count, and areas of necrosis (135). Unfortunately, there is poor interobserver reproducibility, and studies with stronger clinicopathologic correlation and follow-up data are required to further delineate these lesions (136).

PIGMENTED EPITHELIOID MELANOCYTOMA PEM is an uncommon melanocytic neoplasm composed of epithelioid and spindled melanocytes with heavy pigmentation. These tumors share a resemblance to both “animal-type melanoma” (137), because of the presence of abundant melanin, and EBN seen in Carney complex, because of their epithelioid morphology. They have the ability to metastasize to regional lymph nodes, but otherwise demonstrate a benign clinical course. A recent study showed these tumors to follow an indolent course and contain mutations in the PRKAR1A gene (108,138). This led to the rather novel designation of PEM. These lesions can occur in patients with Carney complex (myxomas, malignant melanotic nerve sheath tumors, endocrine neoplasms) or sporadically. Clinically, the first sign of this lesion is a slowly enlarging blueblack plaque or nodule. The lesion occurs commonly on the scalp and extremities and occasionally on the trunk. Although the lesion may first appear as a punctuate discoloration, it can eventually reach the size of 10 to 12 cm. The lesions are usually not associated with other pigmented lesions or a history of melanoma. Histologically, PEM appears as a heavily pigmented, usually dermal-based lesion with a wedge-shaped configuration (Fig. 3.32). Extension to the subcutaneous fat has been identified. There is a

mixture of epithelioid and dendritic melanocytes admixed with melanophages (Fig. 3.33). The epithelioid cells can occasionally demonstrate marked pleomorphism or show multinucleation. Mitotic activity can be identified in some cases. Despite occasional sentinel node metastasis, these cases exhibit indolent behavior with very good prognosis (139).

FIGURE 3.32 Pigmented epithelioid melanocytoma. Numerous melanin-laden cells fill the dermis. Cytologically, all of the cells are heavily pigmented melanocytes with only a rare melanophage admixed.

FIGURE 3.33 Pigmented epithelioid melanocytoma. The densely pigmented dermal melanocytes have characteristic nuclei and display prominent blue nucleoli.

MOLECULAR ASPECTS OF BLUE NEVI AND DERMAL MELANOCYTOSES In addition to having distinct histologic findings, this group of melanocytic lesions differ from banal acquired nevi on a molecular basis as well. BRAF and RAS mutations, typical in common acquired nevi, are usually not present in this group of lesions (140). A majority of blue nevi (83%) show mutations in the heterotrimeric G-protein α subunit (GNAQ and GNA11) genes (108). Mutations in these genes (particularly GNA11) are also present in 46% of uveal melanomas (141,142), which may explain the higher incidence of uveal melanomas in individuals with nevus of Ota. PEMs typically have mutations in the protein kinase A regulatory subunit 1α (PRKAR1A, 17q22-24), which is involved in cyclic adenosine monophosphate signaling. This is correlated with loss of expression of PRKAR1A by immunohistochemistry (138).

DYSPLASTIC NEVUS Dysplastic nevi (also known as atypical nevi or Clark nevi) are a subset of acquired melanocytic nevi characterized by architectural disorder, cytologic atypia, and atypical clinical findings (108,143). They were initially described in patients with hereditary melanoma syndromes but were later identified both in patients with nonfamilial melanoma and in patients unaffected by melanoma (108). Dysplastic nevi are clinically distinct. They appear as either single or multiple slightly raised pigmented lesions that rarely occur before puberty. They are larger than common nevi, usually greater than 0.6 cm in diameter, and always have a flat (junctional) component. Their borders are irregular and often have heterogeneous pigmentation. They tend to occur on skin that is intermittently sun exposed, such as the back. For years, there has been controversy as to whether or not dysplastic nevi represent an intermediate between clearly benign and clearly malignant melanocytic proliferations (29,30,144). In various studies, there has been evidence of a precursor dysplastic nevus adjacent to a malignant melanoma, but this is identified in a minority of cases (29,108). Experts do agree that patients with numerous dysplastic nevi have a higher risk of developing de novo melanoma (108). Further, the presence of multiple dysplastic nevi may identify a person with dysplastic nevus syndrome (145,146). Individuals with this hereditary autosomal dominant disorder may develop hundreds of dysplastic nevi, among which one or more primary melanomas are likely to develop (147). Interestingly, the melanomas present in patients with dysplastic nevus syndrome appear to do so at earlier stages (a feature that is perhaps linked to the frequent clinical follow-up these patients get) and are usually managed by wide local excision methods. Histologically, dysplastic nevi are characterized by architectural disorder and cytologic atypia. In the banal common acquired nevus, the lesion is usually symmetric and junctional nests that are small and typically located at the tips of the rete ridges. If there is a dermal

component, the junctional component does not extend significantly past the underlying dermal proliferation (the so-called “shoulder”). Dysplastic nevi tend to break some or all of these patterns. Nests can be enlarged and located at the sides of the rete, or even in the inter-rete spaces (108,148). A characteristic finding is bridging, in which there is a connection between nests of adjacent rete ridges. Occasional single melanocytes in between the rete ridges (inter-rete spaces) can be identified. The overall nevus can be asymmetric with a prominent “shoulder” (Fig. 3.34). The superficial dermis may show a lymphocytic response with lamellar (concentric) fibroplasia (Fig. 3.35). While normal melanocytes are typically smaller than the size of a keratinocyte, the melanocytes in a dysplastic nevus can be enlarged with hyperchromatic nuclei, clumped chromatin, and variably prominent nucleoli (108,149,150). See Table 3.1 for further cytologic characterization.

FIGURE 3.34 Dysplastic nevus. There is mild cytologic atypia with architectural disorder, most notably in the form of bridging nests. There is a surrounding fibrosis. The dermal component shows maturation.

FIGURE 3.35 Dysplastic nevus. The proliferation of melanocytes exhibits variable and discontinuous atypia and variable distribution of nests. Note the drapelike fibrosis known as lamellar fibroplasia.

TABLE 3.1 Cytologic Characteristics of Dysplastic Nevi Grade

Nuclear size (relative to basal keratinocytes)

Chromatin

Nucleoli

Mild

×1

Mildly hyperchromatic

Absent

Moderate

×1-1.5

Moderately hyperchromatic

Small

Severe

>×1.5

Prominently hyperchromatic, coarse clumpy chromatin

Prominent

Dysplastic nevi share many of the same driver mutations as common acquired nevi, such as mutations in BRAF and NRAS (29). However, recent studies have shown that dysplastic nevi have a

variety of “secondary” mutations, such as loss of p16 (CDKN2A) and mutations in TP53 (29,108,148). However, there have been no studies to date that attribute a particular mutation, or set of mutations, to the morphologic characteristics seen in dysplastic nevi.

MALIGNANT MELANOMA BACKGROUND AND HISTOLOGIC CHARACTERISTICS The diagnosis of malignant melanoma is one of the most challenging aspects of dermatopathology. The neoplastic transformation of human melanocytes to melanoma may occur de novo, or via a precursor lesion, such as a common acquired nevus or a dysplastic nevus (151-156). Approximately 30% to 50% of melanomas contain histologic remnants of a benign nevus. A recent study examined the molecular characteristics of 37 primary melanomas along with their adjacent precursor lesions. Although the majority of benign lesions harbored BRAF or NRAS mutations, adjacent intermediate lesions or frank melanoma demonstrated the accumulation of additional mutations (TERT, p16/CDKN2A, TP53, PTEN), providing evidence of an evolutionary path from a benign precursor to frank melanoma (29). Clinically, melanomas can be identified using the “ABCDE” paradigm, which has been a cornerstone of early detection: Asymmetry, Border (irregular or poorly defined border), Color (variation in color), Diameter (>6 mm), and Evolving (appears different from the rest, or changing in size, shape, or color) (157). Other useful clinical approaches include dermoscopy and biopsy of nevi that appear to be the “ugly duckling.” The latter assumes that an individual’s nevi share common characteristics and that melanoma deviates from this typical pattern (157). In the same vein, the authors recommend a similar approach to evaluating worrisome lesions histologically, as discussed earlier (158). First, melanomas are larger than benign nevi and can be quite broad (at least >6 mm) (108,159). While benign common acquired

nevi are typically symmetric with a well-defined border, malignant melanomas show marked asymmetry, both in the size and distribution of nests, and also in the extension of a junctional component over the dermal component, also known as the “shoulder.” These “shoulders” can extend for many rete in a lentiginous pattern, likely contributing to the poorly defined border and irregular color variation. Certain subtypes of melanoma (discussed later) can show near confluence of melanocytes at the dermal-epidermal junction along with marked pagetoid spread. Other cases can show extension along dermal eccrine or follicular epithelium in continuity with the adjacent epidermal junctional proliferation. Cytologically, the melanocytes within a malignant melanoma can show varying degrees of nuclear and cytoplasmic atypia, which should be evaluated in the context of the architectural findings. For example, melanoma with mildly atypical cells may show lentiginous confluence, prominent pagetoid spread, and extensive adnexal extension. Moderately atypical cells feature coarse, irregular nuclear membranes with peripheral chromatin condensation. The cytoplasm of these cells contains finely distributed melanin pigment, and cell borders are evident. Severely atypical cells feature marked pleomorphism and hyperchromasia. Atypical mitotic figures, vacuolated nuclei with coarse peripheral chromatin clumping, and irregular eosinophilic nucleoli can be conspicuous. With regard to the dermal component, invasive melanomas do not show cytologic maturation and may have deep dermal mitoses. Senescent changes, including the formation of multinucleated nevus cells or balloon cells, are typically seen in benign nevi, but they may also occur in melanoma and premalignant lesions. Therefore, the presence of such findings should not be used to rule out a diagnosis of melanoma. Atypical melanocytic proliferations usually elicit a host inflammatory cell response. This can be seen in both dysplastic nevi and melanoma. The response is typically lymphocytic, asymmetrically infiltrates the lesion along its periphery, and leads to the accumulation of melanophages, dermal fibrosis, and

telangiectasia. The term “regression” is often used to describe this pattern when loss of melanocytes occurs. The presence of extensive regression in the absence of a pigmented lesion is always worrisome, and it may indicate the existence of a preceding melanoma or a severely atypical nevus. USEFUL IMMUNOHISTOCHEMICAL TECHNIQUES IN THE EVALUATION OF MELANOMA General melanocytic markers, such as SOX10, S100, Melan-A, tyrosinase, HMB-45, and MITF, can be used to distinguish melanoma from nonmelanocytic tumors (160-162). It should be noted that poorly differentiated melanoma or DM may lose expression of these markers (i.e., Melan-A). Immunohistochemistry for the tumor-suppressor p16 has long been utilized for decades in identifying melanoma, but the authors do not advocate for widespread use because it can be lost in benign nevi and retained in malignant melanoma (163). Ki67 and phosphohistone H3 (PHH-3, a marker that labels mitoses at the prophase stage) can be used to evaluate the dermal proliferation rate. A Ki67 proliferation index of more than 5% in the dermis is concerning for melanoma (164-169). PRAME is a new immunohistochemical stain that has been shown to be very useful in the diagnosis of melanoma (see later section for detailed description) (170,171). MALIGNANT MELANOMA: GROWTH PHASE The growth pattern of melanoma can be divided into two categories: (1) radial growth phase (RGP) and (2) vertical growth phase (VGP). The histologic recognition of RGP and VGP forms is important because RGP primary melanoma does not metastasize (172) and its removal is curative (172). The transition to VGP melanoma, however, heralds the development of metastatic behavior. In a patient with VGP melanoma, a residual risk for metastasis lingers despite the complete removal of the primary lesion. This risk can be estimated by evaluating other factors, including the depth of

penetration of the tumor, the presence or absence of an immune response, epidermal ulceration, and microscopic satellites (173). Primary Melanoma—Radial Growth Phase RGP melanoma is a slowly developing lesion that may exist for months to years before identification (168,169). The tumor appears clinically as a circumferentially enlarging pigmented plaque measuring at least 0.5 to 1 cm in diameter. The border contour is markedly irregular, and areas of intensely black pigmentation are mixed with hypopigmented and erythematous regions. Pigmented basal cell carcinomas and pigmented seborrheic keratoses may, on occasion, simulate this appearance. Microscopically, these tumors display varying degrees of melanocytic hyperplasia with severe cytologic atypia and extensive epidermal involvement (156). The cellular morphology and pattern of infiltration vary with the specific subtype of melanoma (see following section). RGP melanoma may be limited to the epidermis, in which case it is designated melanoma in situ, or it may be invasive, with superficial infiltration of the papillary dermis, at most extending to the superficial vascular plexus. Primary melanomas involving the reticular dermis or subcutaneous fat virtually always display VGP characteristics and thus are clinically ominous. The papillary dermal component of RGP melanomas consists of a mixture of atypical single cells and nests infiltrating a fibrovascular stroma (Fig. 3.36). The invasive nests are typically no larger than the nests within the epidermis or along the dermal-epidermal junction. The cell population is pleomorphic, and a lymphocytic host response is usually present at the base of the lesion. Mitotic figures are absent. Histologic evidence of a precursor lesion, such as a dysplastic or congenital nevus, is often found.

FIGURE 3.36 Malignant melanoma—radial growth phase. Markedly atypical melanocytes are dispersed singly and in small clusters throughout the epidermis and papillary dermis.

Primary Melanoma—Vertical Growth Phase A frequent clinical feature of VGP melanoma is the rapid formation of a discrete nodule, either within a plaque of RGP melanoma or on otherwise normal skin. The clinical entity of nodular melanoma corresponds to the formation of a VGP melanoma without evident overlying RGP (see following section). VGP melanomas are histologically identified by one or more expansile dermal nodules of malignant melanocytes (Fig. 3.37). VGP melanomas usually arise in a preexisting RGP melanoma, and the VGP component is typically larger than the epidermal or junctional nests comprising the RGP melanoma. The presence of mitotic activity within the dermis is now considered a sign that the melanoma is in VGP. A host immune response is present less often than in RGP.

FIGURE 3.37 Malignant melanoma—vertical growth phase. An expansile nodule of malignant melanocytes infiltrates the papillary dermis.

The recognition of VGP melanoma is usually straightforward, particularly in cases with deep dermal extension (Clark levels IV and V; see following text) and in most level III lesions. Approximately 10% of thin primary melanomas displaying VGP features ultimately metastasize, whereas thin primary melanomas in RGP are curable by surgical excision. MALIGNANT MELANOMA: SUBTYPES In 2018, the World Health Organization (WHO) recently proposed a new classification for cutaneous, mucosal, and uveal melanoma (108,173,174). This scheme classifies melanoma based on clinical, histologic, epidemiologic, and genomic characteristics, leading to the formation of nine distinct subsets/pathways within two larger subgroups: (1) melanomas associated with cumulative solar damage (CSD) and (2) those not consistently associated with CSD (108,174). These are discussed in the subsequent section.

LOW CUMULATIVE SOLAR DAMAGE MELANOMA/SUPERFICIAL SPREADING MELANOMA This is the most common form of melanoma in Western counties and occurs as a result of “intermittent” sun exposure, which leads to lowto-moderate cumulative sun exposure over an individual’s lifetime. In contrast to high CSD melanomas, the background dermis in these patients has minimal solar elastosis (108,174). Common locations for low CSD/SSM include the back, arms, and posterior legs/calf. Other risk factors include a family history of melanoma, large numbers of benign acquired melanocytic nevi, and one or more dysplastic nevi. SSM may arise in association with a preexisting common acquired, congenital, or dysplastic nevus. Histologically, the RGP of SSM is characterized by an intraepidermal proliferation of cytologically atypical melanocytes arranged at the dermal-epidermal junction with prominent pagetoid scatter (Fig. 3.38). SSM has a tendency to form nests within the epidermis, which can occur at various levels (108,174). When this melanoma enters the VGP, cytologic features of the VGP component influence the prognosis; patients with VGP lesions containing epithelioid melanoma cells fare worse than those with lesions composed predominantly of spindle cells (175).

FIGURE 3.38 Superficial spreading melanoma (low cumulative solar damage melanoma). Intraepidermal proliferation of atypical melanocytes with pronounced pagetoid spread.

Recent studies have shown BRAF V600E mutations to be the most common driver mutation in SSM (29,30,176,177). Interestingly, this is the same mutation that is found in most banal nevi. SSM typically accumulates additional mutations in genes, such as TERT, CDKN2A, and TP53. High Cumulative Solar Damage Melanoma/Lentigo Maligna Melanoma Lentigo maligna melanoma (LMM) tends to occur in the heavily sunexposed population, including, but not limited to, outdoor workers and frequent sun tanners, and characteristically in the head and neck region (174). Clinically, it appears as a slow-growing, poorly circumscribed macular area of hyperpigmentation that evolves over time and may grow to several centimeters in diameter. They may get mistaken for solar lentigines or flat pigmented seborrheic or actinic keratoses. They most often display only radial growth characteristics and can be curable with excision. Foci of vertical growth may occur in long-standing lesions (178-180). Histologically, the RGP of LMM consists of a prominent proliferation of markedly atypical melanocytes at the dermalepidermal junction. Although focal areas of pagetoid spread may be apparent, prominent nesting is not typical. In the background, there is often marked actinic damage (solar elastosis), basilar keratinocytic hyperpigmentation, and epidermal atrophy (Fig. 3.39) (108,181,182). Extension of malignant melanocytes along the junctional areas of follicular and eccrine epithelium is a common feature. Single-cell infiltration of the papillary dermis may be observed in RGP lesions. Lesions with VGP formation may appear indistinguishable from the VGP of superficial spreading or nodular melanoma, displaying both epithelioid and spindle cell morphologies (183). Desmoplastic and neurotropic invasive patterns are often associated with VGP formation in LMM (184).

FIGURE 3.39 Lentigo maligna melanoma. In an atrophic epidermis, there is a predominantly basilar proliferation of single and nested malignant melanocytes with extension to the external root sheath of the follicle. Note the multinucleated giant cells and pagetoid spread.

The most common driver mutations are in NF1, NRAS, and KIT (174,185). BRAF V600K mutations have also been identified. These tumors have a very high mutation burden with a prominent UV signature (174). Desmoplastic Melanoma DM represents a unique variant of melanoma that occurs on skin with high CSD. The difference between DM and ordinary melanoma is supported not only by a different clinical and microscopic appearance but also by differences in immunohistochemical profile, gene expression profile, and biologic behavior. Clinically, DM is often amelanotic and mimics a scar, fibroma, or basal cell carcinoma. It has a predilection for sun-exposed areas, especially in the head and neck regions, but it can also appear in the acral and mucosal regions. It presents more often at a later age than ordinary

melanoma and occurs more commonly in males. In clinically pigmented lesions, there is usually an accompanying melanocytic component, which may be a banal nevus, melanocytic hyperplasia, or lentigo maligna (186). Microscopically, DM is represented by an infiltrating proliferation of atypical and hyperchromatic spindle cells surrounded by a desmoplastic stroma with scattered foci of inflammation and lymphoid aggregates (Fig. 3.40) (174). The stroma contains abundant dense collagen and scattered fibroblasts. There may be an overlying junctional melanocytic proliferation, most commonly an LMM.

FIGURE 3.40 Desmoplastic melanoma. There is a proliferation of mildly atypical spindle cells within the dermis. There are associated lymphoid aggregates, which are commonly seen in desmoplastic melanoma. Note the overlying lentigo maligna melanoma.

Immunohistochemically, they are consistently positive for S-100 and SOX10, focally positive for MART-1, and negative or rarely focally positive for HMB-45. Under electron microscopy, they show

granules consistent with premelanosomes. Another frequent finding in these lesions is the presence of extensive perineural and intraneural involvement around the infiltrating edge of the lesion, sometimes discontiguous to the main tumor. Therefore, it is important to emphasize the need for careful evaluation of the resected specimen to avoid recurrences. DM can be considered a “pure DM,” in which 90% or more of the lesion is desmoplastic, or a “combined” DM, in which less than 90% of the lesion is represented by desmoplasia and the remaining component consist of the epithelioid cells typical in VGP melanoma (187). The importance of this classification is that it appears to have prognostic significance, with the pure variant having a better prognosis with a lower frequency of regional lymph node involvement (187-189). Similar to the high CSD LMM, DM has a high mutational burden with a strong UV signature. Inactivating mutations of NF1, promoter mutations of NFKBIE, and other activating mutations of the MAPK pathway have been observed (108,174). Spitz Melanoma Spitz melanoma was discussed earlier in this chapter. It consists of a low CSD form of melanoma with a particular set of mutations that are shared with Spitz nevi, including activating mutations of HRAS and rearrangements of receptor tyrosine kinases (ROS1, ALK, MET, NTRK1, NTRK3, RET, MERTK) or serine/threonine kinases in the MAPK pathway (BRAF, RAF1, MAP3K8) (27,95-99). For additional details, see section “Atypical Spitz Tumors and Spitz Melanoma.” Acral Lentiginous Melanoma Acral lentiginous melanoma refers to melanoma occurring on the glabrous (non–hair bearing) aspects of the hands and feet, which include plantar, palmar, subungual, and periungual areas (174). It is the most frequent type of melanoma in people with increased pigmentation as they are not as susceptible to the CSD subtypes of melanoma; this includes individuals of African and Asian descent (174). It accounts for approximately 10% of cases of melanoma in

individuals of Caucasian descent. Clinically, the lesions initially appear as densely pigmented macules with irregular borders. Because a thickened, hyperkeratotic epidermis overlies the primary lesion, the clinical appearance may be less ominous than that of other melanomas. The biologic course of acral lentiginous melanoma probably does not differ significantly from the course of low CSD/SSM; however, acral melanomas are often relatively advanced at the time of diagnosis (190-192). The RGP form of acral lentiginous melanoma displays a lentiginous proliferation of cytologically atypical spindled and dendritic melanocytes, often with accompanying retiform epidermal hyperplasia (Fig. 3.41). Pagetoid spread is characteristic, as is extension down eccrine epithelium. The VGP form usually consists of spindle cells, and it is often accompanied by epidermal ulceration.

FIGURE 3.41 Acral lentiginous melanoma. Acral skin is involved by a lentiginous and pagetoid proliferation of spindled and epithelioid melanocytes with invasive of the underlying dermis.

Acral melanomas typically have a low burden of point mutations and a high incidence of copy number variations (174). Alterations in CCND1 and KIT are common, and one study showed KIT mutations in 36% of cases (185). Less common events include mutations in BRAF, NRAS, and kinase fusions. Nodular Melanoma Although most melanomas evolve over months to years in a plaquelike growth of primary RGP melanoma before vertical growth develops, the rapid onset of an invasive nodule of primary VGP melanoma may occur, giving rise to the clinical entity known as “nodular melanoma” (Fig. 3.42). Nodular melanoma was initially described as one of the types of melanoma in the original classification, but today, we know that many of the “nodular” melanomas in reality correspond to one of the previously described types of melanoma where a VGP occurs rapidly. This is supported by the fact that nodular melanomas can share clinical and molecular features with superficial spreading and lentiginous variants of melanoma.

FIGURE 3.42 Nodular melanoma. (A) Large expansile nodule of malignant melanocytes with overlying ulceration. (B) Large nodules of melanoma fill and expand the dermis.

Other Subtypes of Melanoma

Mucosal Melanoma. This entity is defined as a melanoma occurring on a mucous membrane, which includes oral, nasal, or genital sites (108). The RGP presents with a lentiginous pattern of growth of single atypical melanocytes (Fig. 3.43). There may be limited areas of nesting or pagetoid spread. These tend to have a low somatic mutational burden. KIT and NRAS mutations have been described. BRAF point mutations are uncommon (174).

FIGURE 3.43 Mucosal melanoma. A large dermal-based nodule of melanoma is present underlying a mucosal surface.

Melanoma Arising in a Congenital Nevus. Although rare, melanomas can arise in a congenital nevus, usually the “giant” subtype. There can be a junctional component that is similar to LMM and SSM, or a pure dermal component. These melanomas have driver mutations in NRAS and accumulate additional mutations (i.e., TERT and CDKN2A) (174). Melanoma Arising in Blue Nevus. This is a very rare melanoma that most often occurs in the fifth decade of life, with a male predilection (174). It most commonly occurs in a background of a cellular blue nevus. The malignant component shows a tumorigenic proliferation of large markedly atypical cells with necrosis and/or

ulceration. Similar to blue nevi, the driver mutation is typically in the GNAQ or GNA11 genes. These melanomas will often show abundant copy number alterations. PROGNOSTIC FACTORS IN MALIGNANT MELANOMA When reporting malignant melanoma, the pathologist must document a series of morphologic parameters with the goal of forecasting the course of disease and guiding the clinician toward an appropriate therapeutic approach. Some of these prognostic factors are included in the American Joint Committee on Cancer (AJCC) staging system, which was revised and updated in 2017 (eighth edition) (173). With regard to the pathologic stage, Breslow thickness (193) and ulceration are the most important factors of the tumornode-metastasis (TNM) staging system. Mitotic activity, although always noted, does not affect the pTNM stage. At a bare minimum, Breslow depth and ulceration should always be reported. In addition to these important characteristics, the authors also advocate for including other important details in the pathology report, which are discussed later. Tumor Thickness Tumor thickness represents the primary determinant of the T classification for staging of melanoma. The lesions are classified as T1 through T4 depending on the measured tumor thickness, with designation as follows: T1: less than 1 mm; T2: 1 to 2 mm; T3: 2 to 4 mm; and T4: greater than 4 mm. T1 is further subcategorized into T1a (C, m.3460G>A) Neurogenic weakness w/ataxia and retinitis pigmentosa/Leigh syndrome (m.8993T>G, m.8993T>C) Transfer RNA (tRNA) gene point mutations

Mitochondrial encephalomyopathy, lactic acidosis, and stroke (MELAS) (tRNALeu(UUR)) Myoclonic epilepsy with ragged red fibers (MERRFs) (tRNALys) Severe infantile myopathy with COX deficiency (tRNAGlu) Chronic progressive external ophthalmoplegia (m.3243A>G, m.4274T>C) Myopathy (m.14709T>C, m.12320A>G) Cardiomyopathy (m.3243A>G, m.4269A>G) Diabetes and deafness (m.3243A>G, m.12258C>A) Encephalomyopathy (m.1606G>A, m.10010T>C) Nonsyndromic sensorineural deafness (m.7445A>G) Items in parentheses ( ) indicate genes involved. The diagnosis of the mitochondrial myopathies can be elusive, and genetic studies are often not the answer. There is often poor correlation between genotype and phenotype. There may be an overlapping of features between syndromes. In many cases, the genetic defect cannot be identified. Respiratory chain enzyme assays may be informative in defining a pattern of enzyme deficiency but does not provide a genetic basis. Finally, a single molecular abnormality may be found in more than one phenotype, and some syndromes are associated with several genetic defects. For instance, Leigh syndrome, progressive external ophthalmoplegia (PEO), and others have been associated with abnormalities in multiple genes. Although a specific diagnosis is not rendered based on the muscle biopsy, it is helpful in approaching these cases to be familiar with the most well-recognized syndromes associated with mitochondrial myopathy. Kearns-Sayre. This syndrome consists of a triad of onset before age 20 years, pigmentary retinopathy, and PEO. Other common features are heart block, cerebellar ataxia, and very short stature. Although a number of defects in oxidative phosphorylation are reported, many cases have a defect in the cytochrome oxidase

(COX) system that results from a variety of large-scale deletions in the mtDNA (192). The deletions are large (up to 5 kb), and they include several genes. A frequent mutation is a deletion of 4977 base pairs that has been called the common deletion. The muscle biopsy shows RRFs and COX-deficient fibers. Mitochondrial Encephalopathy, Lactic Acidosis, and StrokeLike Episodes. The MELAS syndrome is caused by a point mutation in the tRNALeu(UUR) mitochondrial gene, often with an A → G mutation in base pair 3243. The genetic defect is often associated with an abnormality in complex I of the respiratory chain. MELAS occurs in children, resulting in muscle weakness, seizures, strokes in atypical vascular distributions, dementia, and lactic acidosis (193). Computed tomography (CT) may show calcifications in the basal ganglia. Abnormal mitochondria are seen in the smooth muscle elements of blood vessels during ultrastructural examination (194). RRFs stain positively for COX activity. Myoclonic Epilepsy With Ragged Red Fibers. A point mutation (A8344G) in the mitochondrial tRNALys gene is responsible for the MERRF syndrome (195). This maternally inherited syndrome is associated with a defect in complex IV of the respiratory chain. The clinical manifestations, which begin in childhood, include myoclonus, ataxia, and dementia. In muscle biopsy specimens stained histochemically, ragged fibers are positive for SDH but negative for COX. Leigh Syndrome (Subacute Necrotizing Encephalomyelopathy). This is a multisystem disease that usually manifests during the first year of life. Multiple genetic defects are responsible for the same phenotype, which includes failure to thrive, hypotonia, brain stem dysfunction, and ataxia. Muscle pathology is usually either normal or may show COX deficiency without RRFs (196).

Mitochondrial Neurogastrointestinal Encephalomyopathy. Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is an autosomal recessive disorder that is most often associated with mutations of the thymidine phosphorylase (TYMP) gene. Fewer cases have been reported with mutations of RRM2B. It occurs in childhood and late teens, presenting with gastrointestinal dysmotility, cachexia, ptosis, ophthalmoplegia, neuropathy, leukoencephalopathy, and lactic acidosis. Biopsy of muscle shows COX-deficient RRFs and neurogenic atrophy (197,198). Neuropathy, Ataxia, and Retinitis Pigmentosa. Neuropathy, ataxia, and retinitis pigmentosa (NARP) is a disease of young adults, in which the name describes the main features, and may also be accompanied by muscle weakness and dementia. The MT-ATP6 gene is mutated in this syndrome. The muscle biopsy is often unremarkable but may show neurogenic atrophy. Mitochondrial Recessive Ataxia Syndrome. In addition to ataxia, this syndrome of childhood and young adults may manifest sensory neuropathy, PEO, myoclonus, and myopathy. It is linked to mutations in the polymerase gamma (POLG1) gene, which is also implicated in Alpers-Huttenlocher syndrome, other neonatal or infantile-onset encephalopathies, sensory ataxic neuropathy, spinocerebellar ataxia, and seizures (199,200). Muscle biopsies are frequently normal but may show mtDNA depletion (196). In the workup of mitochondrial disease, an underlying condition must be excluded. The secondary mitochondrial disorders with COXdeficient fibers include IBM, DM, lipid storage disease, and dystrophic myopathies, chiefly the dysferlinopathies (201). Mitochondrial myopathy has also been described as a consequence of drug therapy, such as reverse transcriptase inhibitors for HIV infections. Myoadenylate Deaminase Deficiency Adenylate deaminase, or adenosine monophosphate deaminase (AMPD), is an enzyme found in skeletal muscle; a deficiency may

lead to exercise intolerance in some patients, who experience aches and muscular cramps without significant muscle weakness on neurologic examination (202,203). The gene coding for deaminase in muscle (AMPD1) is situated on chromosome 1p21. Myoadenylate deaminase deficiency can be detected with enzyme histochemistry, although absent activity may be noted as an incidental finding in muscle biopsies from asymptomatic patients. NEUROPATHIC DISEASES Neuromuscular Junction Disorders Both immune-mediated and congenital disorders of the neuromuscular junction have been described (204,205). Although the clinical presentations are quite heterogeneous, fatigable weakness is a common theme, especially involving the extraocular muscles. Ptosis is quite common. Electromyogram (EMG), rather than muscle biopsy, is most important in establishing the diagnosis. In most routine biopsies of limb muscles, an investigation of the endplates is not practical because encountering these structures is serendipitous at best. Directed biopsies of intercostal muscles can reliably obtain tissue for specialized immunohistochemical and ultrastructural investigation of neuromuscular junctions (4). Autoimmune Myasthenias. Myasthenia gravis, which is more frequent in young adult women, is characterized by the insidious onset of easy fatigability in the extraocular and facial muscles (204). Patients with myasthenia gravis synthesize antibodies to the postsynaptic membrane of the motor endplate, thus blocking neuromuscular transmission and damaging the endplate region. Autoantibodies to the acetylcholine receptor (AChR) or musclespecific kinase (MuSK) are most common. The clinical diagnosis depends on a positive response to anticholinesterase agents and a decrement in motor action potentials during repetitive stimulation. If a muscle biopsy is obtained, the specimen is often normal, or it may be characterized by type 2 fiber atrophy in a minority of cases.

Ultrastructurally, the junctional folds of the endplates are simplified, and the synaptic clefts are widened (206). Congenital Myasthenic Syndromes. In this group of rare genetic disorders, the safety margin of neuromuscular transmission is compromised (205). Many genes-encoding proteins of the nerve terminals, extracellular matrix, or endplates are known to cause myasthenic syndromes. Repetitive stimulation and single-fiber EMG are key diagnostic tests. Muscle biopsy findings are mostly nonspecific, but type 1 fiber predominance, type 2 fiber atrophy, numerous tubular aggregates, or ultrastructural pathology of the neuromuscular junction may be clues to the diagnosis. Denervating Diseases Overview. Neurogenic atrophy of muscle results from genetic or acquired disease of the lower motor neurons or their axons. They may account for nearly one-third of diagnoses in muscle biopsies. The etiology of the denervating condition cannot be accurately determined by examining a muscle biopsy specimen because the histopathologic changes in the affected muscle are very similar in all denervating diseases, except in infants. In early denervation, the selective atrophy of type 2 fibers is frequently the only abnormality, so the diagnosis of denervation depends on confirmatory clinical information. With continuing denervation, the proportions of atrophic type 1 and type 2 fibers become relatively equal. At first, denervated fibers are randomly scattered or small groups representing single motor units. Chronic denervation is characterized by atrophy of larger groups in addition to fiber-type grouping that typifies reinnervation (207). Target fibers may be seen in early or frequently in chronic neuropathic disease with reinnervation of muscle. A muscle biopsy from long-standing neuropathic disease may manifest a host of secondary myopathic changes, including myonecrosis, endomysial fibrosis, rimmed vacuoles, and internalized nuclei with fiber splitting. Eventually, the muscle will have end-stage histopathology. Although typically not necessary,

immunohistochemistry may be useful in identifying denervation. In normal fibers, neural cell adhesion molecule (N-CAM) staining can only be seen at the neuromuscular junctions, but denervated fibers demonstrate N-CAM staining of the entire sarcolemma. The diagnostic specificity of N-CAM immunostaining for denervation is compromised by its upregulation in regenerating fibers (208). Neuronal nitric oxide synthase (nNOS) is absent from denervated fibers. When fibers are then reinnervated, nNOS expression reappears at the sarcolemma (209). Anti-nestin immunohistochemistry is a more specific marker whereby expression is upregulated specifically in denervation (210). Neuropathic Disease in Infants. SMA is the most common neurogenic disease in young children (211). Approximately 95% of individuals are homozygous for a deletion of the survival motor neuron 1 gene, SMN1, located on chromosome 5q11.2-13.3. The age at onset, which ranges from birth to early childhood, dictates the natural history. In general, the earlier the onset is, the more unfavorable the prognosis. Infants (classified as SMAI) typically exhibit the floppy infant syndrome with severe weakness, a weak cry, poor suck, and respiratory difficulties, although cognitive function is normal. Fasciculations are difficult to appreciate, except in the tongue. Life expectancy of these infants, before the recent advent of genetic therapy, is less than 2 years. The muscle pathologic picture in SMA1 differs from that of other neurogenic atrophies. In the classic presentation, most fibers in each fascicle are abnormally small and rounded (Fig. 4.44). Other fibers, often grouped, are normal in diameter or hypertrophic. The massively enlarged fibers are predominantly type 1 in many cases. With progressively older onset of SMA due to increasing copy numbers of SMN2, the neuropathic features of the muscle biopsy are more likely to resemble the neurogenic atrophy described earlier. A heterogeneous group of additional genetic disorders (termed non-5q SMA) also cause neuropathic disease in childhood (212,213).

FIGURE 4.44 Infantile spinal muscular atrophy. Most of the fibers in these fascicles are abnormally small and rounded. Dramatically hypertrophied fibers are often grouped and of type 1 specificity (hematoxylin and eosin).

MISCELLANEOUS DISEASES MYOSIN HEAVY-CHAIN LOSS MYOPATHY (CRITICAL ILLNESS MYOPATHY) This entity occurs in acutely and severely ill patients who manifest profound generalized weakness, often affecting the respiratory muscles but sparing facial muscles. It is rapidly progressive, although reversible if it is recognized early and is properly treated. A related and sometimes concurrent condition is critical illness polyneuropathy. Myosin loss myopathy is potentiated by treatment with steroids and muscle relaxants by pharmacologic neuromuscular blockers (214). In muscle biopsies, there is atrophy and selective loss of myosin thick filaments in both fiber types (215). Myosin ATPase activity is typically reduced in the center of the affected

fibers at any pH. Antimyosin heavy-chain immunohistochemistry is less affected (216). Myofiber necrosis is an unusual finding. By electron microscopy, actin thin filaments, unassociated with myosin, form perpendicular arrays with Z discs (215). AGING Subtle myopathic change occurs in the elderly patient who is weak. Aging patients who have sarcopenia may not have other muscle disease, and their biopsies seem nonspecific. Normal aging patients have fewer fibers, and the fibers are smaller, but the atrophy is mild and highly dependent on activity (217). The atrophy is typically almost selective for type 2 fibers. Owing to alterations in filament expression and motor neuron innervation, an increase in type 1 fibers and a commensurate loss of type 2 fibers occur. MUSCLE HERNIAS Muscle hernias are unusual and may be confused with other masslike lesions such as lipomas. They consist of a small portion of the main muscle, which protrudes through the fascia, becoming more prominent with muscle contraction and producing pain or local discomfort at the site (218). They tend to occur in younger individuals who are physically active such as weight lifters or elite athletes or in the untrained after violent exercise to which they are unaccustomed. Underlying factors include muscle hypertrophy with an elevation in compartment pressure, stress on the fascia, and perhaps fenestrations in the fascia of some. The most common sites are the anterior tibial, biceps brachii, rectus abdominis, and gastrocnemius. The tissue may exhibit a variety of pathologic changes, often of a myopathic nature. MUSCLE HEMORRHAGE Muscle hemorrhage or hematoma may result from direct trauma or in the setting of critical illness, coagulation disorders, or cirrhosis, where it may appear to be “spontaneous” (219). Most common

locations are the quadriceps femoris, rectus sheath, and iliopsoas. Intensifying pain is a common reason for surgical evacuation. Persistent presence of muscle hemorrhage increases the risk of later myositis ossificans.

PITFALLS The submission of a specimen with insufficient clinical data may lead to a delay in the diagnosis or to an incorrect diagnosis. The clinical information guides the investigation of the biopsy specimen beyond the routine workup. Several special techniques are available, but from a practical and economic standpoint, not all of them can be used in every case. Some biopsies are done with the intended or eventual goal of genetic or enzymatic testing; thus, care should always be taken to preserve as much snap-frozen tissue as possible in that eventuality. It may be practical, for example, to forego entirely formalin fixation and paraffin embedding with small pediatric biopsies. The clinician sending the biopsy in this situation may simply want to know if the patient has myopathy and its features. Whenever the diagnosis and the pathologic findings do not match, communication with the contributor is a wise decision. The pathologist should proceed with circumspection with a biopsy from a patient without weakness. Some patients experience only intermittent weakness and are not weak at the time of the examination. Many of these patients complain of pain or cramps, suggesting a metabolic myopathy or a neurogenic process. In others, the weakness is of physiologic origin without a morphologic correlate, and a biopsy is contraindicated. An inadequate specimen is one of the most important reasons why a muscle biopsy is a noncontributory procedure. As discussed in the section “Collection and Preparation,” the biopsy should represent the disease. If symptoms are limited to proximal leg muscles, a biopsy of calf muscles is likely to be normal. A muscle in which there is an active disease process is more apt to contain diagnostic features, sometimes ideally revealed by ultrasound or MRI. Severely involved

muscle in which the disease process has been long standing and chronic often results in a biopsy with end-stage disease and nonspecific findings. Muscles subjected to prior trauma or affected by an unrelated disease process should be avoided for biopsy. Needle tracts produced during EMG studies or intramuscular injections of therapeutic medications are common types of traumatic injury. The changes in such muscles—fiber necrosis, regeneration, inflammation, and endomysial fibrosis—mimic those of certain neuromuscular diseases, and they can mislead the pathologist. The biopsy specimen should be taken from the belly of the muscle, away from tendinous insertions, the normal histologic features of which may be misinterpreted as pathologic. At the tendinous interface, muscle fibers typically vary more in size; the internal nuclei are numerous; and the fibers are separated by trabeculae of dense collagen as they attach to the tendon (Fig. 4.45). Similar but less pronounced features are found at the interface of muscles and fascia. The interfaces should not be confused with fibrosis.

FIGURE 4.45 Subfascial muscle. In this location and in myotendinous insertions, the muscle fibers normally vary in size, and they are often surrounded by fibrous tissue with numerous internally placed nuclei (hematoxylin and eosin).

Certain muscles are commonly biopsied, and the pathologist becomes familiar with their appearance. Not all muscles look alike. The pathologist should be careful when receiving an unfamiliar muscle. For example, the paraspinal muscles normally contain increased numbers of internal nuclei, and groups of fascicles are separated by abundant connective tissue that resembles fibrosis. In more distal leg muscles may show neuropathic changes from spinal disc disease or compression neuropathies that are unrelated to the disease for which the biopsy was obtained. Vacuolization of the fibers is a frequent consequence of improper freezing, transport, or storage of the specimen. These ice crystals must be distinguished from pathologic vacuoles seen in storage diseases. Ice crystals tend to be linear or assume noncircular shapes in contrast to pathologic vacuoles, which tend to be round. PAS and fat stains should be used to rule out lipid and glycogen storage whenever there is a question about the origin of the vacuoles. Specimens submitted in saline or kept too moist show fluid accumulation between the fibers that resembles edema. The fibers are also often vacuolated, disrupted, or “blown out” as though they exploded (Fig. 4.46). These changes, which may be mistaken for an acute myopathy with edema and fiber necrosis, occur in the absence of inflammation or phagocytosis that would be expected in a pathologic reaction. Such mishandling may result in a loss of enzyme staining and the false impression of an enzyme deficiency.

FIGURE 4.46 Muscle specimen submitted in saline. Fluid between fibers mimics edema. Several fibers are damaged and disrupted and appear blown out.

PERIPHERAL NERVE PATHOLOGY The diagnostic biopsy of a peripheral nerve along with a muscle biopsy may occur when a peripheral neuropathy is the prime consideration. As with muscle biopsies, the biopsy presupposes that a comprehensive physical and neurologic examination, laboratory studies, and electrodiagnostic testing (EMG and nerve conduction velocity [NCV]) have been performed. The sural nerve in the lateral ankle region, a purely sensory nerve, is the most traditionally biopsied, leaving the patient with only a minor sensory deficit. Also analogous to muscle biopsies, the nerve biopsy should only be performed by an experienced surgeon. A segment of at least 3 cm in length in adults and as long as possible in very young patients should be carefully dissected, without stretch or cautery, and provided for immediate processing. Portions are placed in formalin and glutaraldehyde (Fig. 4.47). Freezing of nerve biopsies is not

indicated because of their vulnerability to freeze artifact, the lack of any need for cryosections toward enzyme histochemistry, and very infrequent need for molecular testing.

FIGURE 4.47 Nerve biopsy, with suggested priorities for processing a specimen for paraffin sections and glutaraldehyde fixation for plastic sections, microdissection (“teasing”), and electron microscopy.

The formalin-fixed portion should be embedded longitudinally and, for cross sections, and stained by H&E, trichrome, Luxol fast blue for myelin, Bielschowsky silver impregnation for axons, and Congo red for amyloid in all adult nerve biopsies. Six H&E-stained levels are recommended because of the highly focal nature of some pathologic

changes, chiefly inflammation. The glutaraldehyde fixative should ideally be isotonic (suggested formula is 10 mL 8% glutaraldehyde + 10 mL 0.1 M cacodylate buffer at pH 7.4 +10 mL deionized water). Routine hypertonic glutaraldehyde used for liver or kidney biopsies imparts a kinking artifact to nerve biopsies, which interferes with obtaining ideally circular cross sections from the plastic resin– embedded tissue. The resin sections are far superior to paraffin sections (Fig. 4.48A,B) in allowing the microscopist the ability to discern the relative losses of large and small myelinated fibers (unmyelinated fibers may only be visualized by electron microscopy), and patchy versus diffuse fiber loss, diabetic microangiopathy, onion bulbs, and other evidence of demyelination and remyelination. Another technique that may be useful in distinguishing axonal from primary demyelinating neuropathies as well as the condition known as tomaculous neuropathy is nerve microdissection or “nerve teasing,” which is done with glutaraldehyde-fixed nerve that is then fixed in osmium to render the lipid-rich myelin sheaths dark.

FIGURE 4.48 Cross section of sural nerve biopsy stained for myelin, affording a rough approximation of myelinated fiber density (Luxol fast blue) (A). Cross section of glutaraldehyde-fixed, plastic-embedded nerve from the same case providing a greatly superior advantage in distinguishing large from small myelinated fibers (toluidine blue) (B).

Nerve biopsies may yield a frustrating lack of etiologic specificity in the final diagnosis, such as in many idiopathic axonal neuropathies. They are distinctly unhelpful in acute inflammatory polyradiculoneuropathy (Guillain-Barré syndrome), somewhat more

suggestive in chronic inflammatory polyradiculoneuropathy (CIDP), small fiber neuropathies, and diabetes mellitus; and most useful in conditions with distinctive pathology, including vasculitis, amyloidosis, sarcoidosis, leprosy, and various genetic neuropathies. An array of examples from peripheral nerve pathology is depicted in Figure 4.49A-H.

FIGURE 4.49 Some examples of histopathology in diagnostic peripheral nerve biopsies for nonneoplastic diseases. Fibrinoid necrotizing arteritis, such as in polyarteritis nodosa (A). Amyloid neuropathy, with globular endoneurial deposits. Vascular deposition may also be seen in amyloid neuropathies (hematoxylin and eosin) (B). Amyloid neuropathy (Congo red). (C) Small fiber neuropathy in amyloid neuropathy. Selective small fiber loss may be also seen in diabetes, hereditary sensory and autonomic neuropathies, rarely in alcoholic neuropathy, and other unusual conditions (toluidine blue). (D) End-stage diabetic neuropathy showing characteristic significant thickening of endoneurial vascular basement membrane (toluidine blue). (E) Autophagic digestion chambers of “Cajal” signifying active Wallerian axonal degeneration with secondary demyelination. Note the marked loss of myelinated fibers (Luxol fast blue). (F) Axonal regeneration is easily recognizable as tightly clusters of thinly myelinated fibers and is typically seen in axonal neuropathies with Wallerian degeneration due to proximal injury such as ischemia or compression that nonetheless allows for regeneration (Toluidine blue). (G) Tomaculous (sausage-like) swellings seen in microdissected (“teased”) glutaraldehyde-fixed, osmium-treated nerve in a case of hereditary neuropathy with pressure palsies (H).

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5

Soft Tissues Jason L. Hornick

Although soft tissue sarcomas are rare, accounting for less than 1% of malignant neoplasms (~13,000 new cases in the United States in 2020) (1), soft tissue tumors overall are not uncommon; benign mesenchymal neoplasms outnumber sarcomas by more than 100 to 1 (2). These numbers do not truly indicate how often the pathologist is faced with a diagnostic problem related to this field. The first issue is definitional—only entities encountered within the soft tissues are quantified, but pathologists see identical tumors within other body sites (e.g., skin, visceral organs), and the real scope of the matter is better thought of in terms of all mesenchymal lesions. Counting organ-based mesenchymal tumors is virtually impossible. Second, pathologists consider a “soft tissue” tumor whenever they are faced with a potential mimic, and many melanomas, carcinomas, and lymphomas can arouse such thoughts. Third, even the malignancies are underestimated because tumors from the skin and uncoded locations are not counted as “connective tissue” sarcomas. For example, gastrointestinal stromal tumor (GIST) is the most common sarcoma, but it is not even included in the abovementioned estimate! Clearly, pathologists deal with questions pertaining to this field very often if these other elements are entered into the equation. This chapter provides a problem-oriented approach, which emphasizes diagnosis based on histologic appearances, integrated with clinical findings (i.e., age and anatomic site). Details for selected tumor types are summarized in tabular form, highlighting the clinical presentation and providing a guide to the natural history of these tumors. The text concentrates on histologic features, and diagnostic criteria are discussed in a practical way. The supplemental information includes an account of the differential diagnostic possibilities and ancillary diagnostic techniques, especially immunohistochemistry (IHC). References highlight only classic descriptions and key molecular and IHC findings. In this era of electronic online databases, readers are encouraged to search for much more detailed information on the numerous entities discussed in this chapter.

PHILOSOPHIC APPROACH TO THE DIAGNOSIS OF SOFT TISSUE TUMORS—AN OVERVIEW Soft tissue tumors are often overwhelming to the novice; before diagnostic competence can be achieved, a general understanding of the field is necessary. To this end, one should first become familiar with benign lesions that mimic sarcomas to prevent the possibility of overdiagnosis. Malignant lesions can then be reviewed and can be placed in context. A systematic process is a useful way to approach each case; this might be called the “skeptical approach” to soft tissue tumor diagnosis. A series of crucial questions should be answered in a defined order. First, regardless of the histology of the lesion, one must ask, “Is the lesion really a neoplasm?” Reactive myofibroblastic proliferations (e.g., postsurgical or posttraumatic) may closely mimic mesenchymal neoplasms, and pseudosarcomas (such as nodular fasciitis [NOF]) may have a high mitotic rate. Therefore, the mitotic rate alone cannot be

relied on as a criterion for neoplasia or malignancy; instead, the line of differentiation in the lesion, and sometimes the actual diagnosis, must first be decided in many instances before one considers mitotic activity. In contrast, some lesions require only a brief review before malignancy is diagnosed. Such tumors often exhibit high cellularity, necrosis, marked nuclear atypia (e.g., coarse chromatin), and a high mitotic rate. In contrast, reactive lesions are often histologically organized with no nuclear atypia. The second question is, “Is the lesion malignant?” The only presumptive sign of malignancy is necrosis; without necrosis, nuclear pleomorphism and cellularity must be evaluated in context of the diagnostic category. Answering this question can sometimes be a significant challenge. The third question is, “Is the lesion actually a sarcoma?” Could it be a carcinoma or melanoma mimicking a sarcoma? Visceral “sarcomas,” such as those in the breast, esophagus, kidney, and respiratory tract, should be considered sarcomatoid carcinomas until proven otherwise. Ancillary techniques, such as IHC, are often required to make this distinction. Melanoma is one of the great histologic mimickers; it may simulate undifferentiated pleomorphic sarcoma (UPS), leiomyosarcoma (LMS), epithelioid sarcoma, and malignant peripheral nerve sheath tumor (MPNST), among other sarcoma types. For any lesion with both an epithelioid and spindle cell component, melanoma should be excluded before a sarcoma is considered. After the first three questions have been answered, for some classes of mesenchymal lesions, one should attempt to answer the question “What is the type of differentiation displayed by the putative sarcoma?” Depending on the answer to this question, one must determine whether criteria for malignancy are met: mitotic rate determines malignancy in only a limited number of tumor types (Table 5.1). For some lineages such as smooth muscle neoplasms, both the anatomic site and mitotic rate are crucial in this determination. The use of this approach aids greatly in (1) the elimination of certain bizarre, but benign, mesenchymal lesions from the malignant roster; (2) the recognition of pleomorphic, but clearly benign, tumors; and (3) the correct classification of nonsarcomas (sarcomatoid carcinomas and melanomas), which require markedly different therapeutic approaches. TABLE 5.1 Soft Tissue Tumors for Which Mitotic Rate Determines Malignancy Solitary fibrous tumors Smooth muscle tumors Nerve sheath tumors Granular cell tumors

RECOGNITION OF PSEUDOSARCOMAS Table 5.2 lists many of the lesions that mimic sarcomas. Knowledge of the histologic appearances of these lesions is critical to prevent incorrect diagnoses of sarcoma. The following discussion provides some insight into this group of lesions. TABLE 5.2 Pseudosarcomas Nodular fasciitis Proliferative fasciitis Proliferative myositis Spindle cell/pleomorphic lipoma Lipoblastoma Cellular angiolipoma Bizarre leiomyoma Fetal rhabdomyoma

Pyogenic granuloma Papillary endothelial hyperplasia Atypical (pseudosarcomatous) fibroepithelial stromal polyp Intramuscular/cellular myxoma Myositis ossificans Nonmesenchymal lesions Sarcomatoid and pleomorphic carcinomas Carcinoma with osteoclast-like giant cells Melanoma Lymphoma (especially anaplastic large cell lymphoma) Histiocytic and dendritic cell sarcomas

Some of the most rapidly growing but relatively small mesenchymal lesions are benign. Any tumor with a tissue culture–like appearance, as is found in NOF and proliferative myositis, should be considered benign until proven otherwise. Circumscribed adipocytic lesions with either spindle cells or pleomorphic “floret” cells, when arising on the shoulders, upper back, neck, or face, should also be considered benign. Bizarre nuclear features may be encountered in a range of benign soft tissue lesions, both reactive and neoplastic; for example, schwannomas and neurofibromas may contain pleomorphic cells. Any circumscribed vascular lesion or a vascular lesion with a lobular architecture, such as that seen in pyogenic granuloma, should also be considered benign. It is helpful to remember that many sarcomas are highly vascular; relatively avascular lesions are often benign, as exemplified by intramuscular/cellular myxoma.

RECOGNITION OF OTHER TUMORS MIMICKING SARCOMAS Tumors occurring primarily in visceral organs should be considered carcinomas until proven otherwise. Carcinomas of many organs may mimic a spindle cell sarcoma or UPS with osteoclast-like giant cells; these may occur in the pancreas, breast, bladder, and other sites. Tumors resembling UPS often turn out to be sarcomatoid carcinomas, as frequently occur in the larynx. Renal cell carcinoma may be sarcomatoid; therefore, a conventional (most often clear cell) component should be sought in spindle cell and pleomorphic neoplasms of the kidney. IHC for keratins is critical in these circumstances. Lymphoma may not only mimic small round cell tumors but may also simulate UPS. Careful attention to nuclear detail (i.e., the majority of lymphomas contain cells with irregular nuclear contours) may alert one to this possibility. Also, tumors of histiocytic and dendritic cell origin, such as histiocytic sarcoma, follicular dendritic cell sarcoma, and interdigitating dendritic cell sarcoma, as well as anaplastic large cell lymphoma, may also be mistaken for soft tissue sarcomas. SPINDLE CELL (SARCOMATOID) CARCINOMA Spindle cell carcinoma is deceptive, and because IHC for keratins may be limited in extent or entirely absent, it continues to be misdiagnosed as sarcoma. The transformation of a squamous cell carcinoma into a spindle cell carcinoma is a recognized phenomenon in a variety of mucosal sites, such as the larynx, the oral and nasal cavities, and skin. This change may be accompanied by a gain of smooth muscle actin (SMA) along with the complete or nearly complete loss of keratins. The histologic appearance of a brightly eosinophilic tumor at low power is typical. Furthermore, many cases are not highly pleomorphic, but, instead, relatively uniform. The spindle cells are elongated, with open oval nuclei and prominent nucleoli, and they have “plump” cytoplasm, meaning they are wide with some cytoplasm on the sides of the nuclei (Fig. 5.1). These features are not seen in smooth muscle and many other mesenchymal tumors. If multiple broad-spectrum keratin antibodies are applied, the majority of sarcomatoid carcinomas will be

positive (3). In the skin, sarcomatoid squamous cell carcinoma should be considered before that of the mesenchymal tumors atypical fibroxanthoma (AFX) and pleomorphic dermal sarcoma (4).

FIGURE 5.1 Spindle cell squamous cell carcinoma. Note the streaming plump spindle cells with eosinophilic cytoplasm and enlarged nuclei with prominent nucleoli (A). These tumors are often positive for high-molecularweight keratins (clone 34βE12) (B), although some cases may show only focal staining, and occasional tumors are keratin negative.

SPINDLE CELL MELANOMA When a melanoma mimics a sarcoma, its recognition may be aided by the presence of highly variable cytology (including both epithelioid and spindle cells), heterogeneous architecture, and a focally nested growth pattern. Only a high index of suspicion helps one eliminate melanomas from the differential diagnosis. In particular, spindle cell or desmoplastic melanoma, whether in a skin primary tumor or a metastasis, may simulate a nerve sheath tumor (Fig. 5.2). A key helpful feature is the common diffuse S-100 protein and SOX10 expression, in contrast to MPNST, which at most shows focal or patchy staining for these markers in only 30% to 50% of cases. Even in the skin, an intraepidermal or junctional melanocytic component may be absent. Furthermore, human melanoma black-45 (HMB-45) and melan A, which are expressed frequently in ordinary (epithelioid) melanoma, are typically negative in desmoplastic melanoma (17). Thus, as in spindle cell carcinoma, caution must be used in the interpretation of negative

results. The neuroectodermal transcription factor SOX10 is particularly helpful to support a diagnosis of melanoma (5).

FIGURE 5.2 Spindle cell melanoma. Many examples have a neural-like appearance with wavy, tapering nuclei (A). This tumor arose in the dermis, an exceptionally rare site for malignant peripheral nerve sheath tumors. Note the focal lymphocytic infiltrate (lower right). Both S-100 protein (not shown) and SOX10 (B) are strongly positive; extensive staining for either marker favors melanoma over malignant peripheral nerve sheath tumor.

RECOGNITION OF FOUR MAJOR CATEGORIES The dominant histologic pattern is a helpful feature to approach the diagnosis of soft tissue tumors. These include (1) spindle cell, (2) round cell, (3) pleomorphic, and (4) epithelioid categories. These morphologic patterns are well known to surgical pathologists; identifying the dominant histology is useful for developing a differential diagnosis. IMPORTANCE OF LINE OF DIFFERENTIATION

Referring to the line of differentiation a tumor exhibits, rather than its histogenesis, is best because of lineage infidelity in tumor progression and the fact that most sarcomas likely arise from partially committed mesenchymal precursor cells, not differentiated cell types. A pathologist who correctly identifies the tumor phenotype (the line of differentiation rather than the cell of origin) of a sarcoma and who renders a specific diagnosis performs a valuable service to the clinician. Classifying rhabdomyosarcomas (RMSs) correctly, for example, is particularly important because these are treated very differently from other sarcoma types. A correct diagnosis of an unusual sarcoma (e.g., epithelioid sarcoma) likewise alerts the clinician to the likely natural history and potential prognostic factors. Thus, tumor phenotype or line of differentiation remains important, and it will become more so as additional effective targeted therapies are identified for each tumor type. However, as will be detailed later, IHC using lineage markers is insufficient to establish a specific diagnosis for many tumor types.

ORIGIN AND ETIOLOGY OF SARCOMAS SCOPE OF DIAGNOSTIC ISSUES To give some perspective on sarcoma diagnosis, there are eight groups of soft tissue tumors based on phenotype (smooth and skeletal muscle, adipocytic, vascular, etc.), besides a large group of tumors of uncertain differentiation, including around 50 distinct soft tissue sarcomas (2). OVERVIEW Although many sarcomas show a clear and relatively uniform line of differentiation demonstrable by IHC, which is maintained throughout their natural history (e.g., at metastatic sites), occasional sarcomas “dedifferentiate,” or transform to an undifferentiated state, a phenomenon that is discussed later. Sarcomas likely arise through a genetic alteration occurring in a cell of an already partially committed mesenchymal phenotype, even though such a cell may seem primitive and without visible differentiation. Sarcomas rarely arise from benign soft tissue tumors; most sarcomas arise de novo. A benign nerve sheath tumor with a long clinical duration (both neurofibromas and, much more rarely, schwannomas) may give rise to MPNST. Other notable examples of this phenomenon include “fibrosarcomatous” transformation of dermatofibrosarcoma protuberans (DFSP) and dedifferentiated liposarcoma (DD-LPS) arising from atypical lipomatous tumor (well-differentiated liposarcoma). However, the vast majority of soft tissue sarcomas have no recognizable benign precursor. UNCLASSIFIED SARCOMAS Even with technologic advances such as IHC and molecular genetics, occasional sarcomas (roughly 5%) may not be definitively characterized. Therefore, using the term unclassified sarcoma for these lesions is appropriate. However, some qualifier should be used as a clinical guide (e.g., unclassified spindle cell sarcoma or undifferentiated round cell sarcoma). Before relegating a sarcoma to such a category, particularly for the round cell sarcomas and cytologically uniform spindle cell sarcomas, next-generation sequencing (NGS) should be considered to exclude the possibility of recently described gene rearrangements (some of which have therapeutic implications). The etiology of most sarcomas is unknown, other than postradiation sarcomas (angiosarcoma, LMS, osteosarcoma, and undifferentiated sarcomas) and chronic lymphedema-associated angiosarcoma. Some sarcomas occur in the setting of familial genetic predisposition syndromes,

such as Li-Fraumeni syndrome (TP53: osteosarcoma, RMS, and others), neurofibromatosis type 1 (NF1: MPNST), retinoblastoma (RB1: osteosarcoma), and Carney complex type 1 (PRKAR1A: malignant melanotic nerve sheath tumor, formerly known as melanotic schwannoma) (2). A viral etiology of Kaposi sarcoma is well known, with the agent being the human herpesvirus-8 (HHV8) (see section “Kaposi Sarcoma”), and some smooth muscle neoplasms in patients with acquired immunodeficiency syndrome (AIDS) have been associated with the Epstein-Barr virus (EBV) (6).

TISSUE SECTIONS As a general rule, a minimum of one block per centimeter of greatest tumor dimension should be processed; more material may be submitted, depending on the variable nature of the sectioned surface. This general rule must be tempered by the size of the tumor. In small tumors, some sections could include both the marginal and peripheral areas, whereas the remaining sections are obtained from the center of the lesion; thus, in a small lesion of approximately 3 cm, four to five sections might be obtained. In larger lesions (5-10 cm), 8 to 10 sections may be required. In very large lesions (10-20 cm), 10 to 15 sections may be obtained if the lesion is relatively uniform. When sections are obtained, documenting (1) necrosis (by including it in a section with viable tissue), (2) any unusual appearance seen at gross examination, (3) the margins, and (4) the relationship to surrounding structures is important. For specimens resected after chemotherapy, it is commonplace to evaluate a complete “slab” of the tumor—a slice taken along its largest length and width—using numbered blocks, to document the percent necrosis and fibrosis. Although the relationship between extent of treatment response and outcome is well known for osteosarcoma of bone, the prognostic significance of this assessment is less established for soft tissue sarcomas, although the extent of fibrosis may have prognostic significance (7).

COMMENT ON MARGINS A thin fascial margin (even when it is 1-2 mm) is a complete (oncologically adequate) surgical margin, and such areas do not generally give rise to recurrences. These may be described as “close,” but the tumor is really contained in that compartment; it is important to document the intact fascial plane. In contrast, the cut surgical margins, if they are close, are a major concern, and in such situations, the margins should be documented in detail. Multiple ink colors are now commonly used, and reports should include distances to various margins in a properly oriented specimen, when less than 2 cm.

EVALUATION OF PROGNOSTIC FACTORS The gross and microscopic evaluation of a neoplasm is of critical importance for determining prognosis. The evaluation must begin with a gross assessment of the lesion. The entire lesion should be inked, the exterior should be dabbed with fixative to ensure that the ink adheres, and the margins should be adequately sampled. The presence and degree of necrosis should be recorded because this affects tumor grade for some sarcoma types. Communication with the surgeons is vital for orientation of complex specimens and their impression of close margins. Furthermore, a diagnosis is incomplete without an assigned grade (when appropriate) and a comment on the status of margins. The grade of a sarcoma (see section “Grade” under “Evaluation of Prognostic Factors”) is absolutely key to the subsequent clinical management of

some tumor types; for example, adjuvant radiation therapy may be reserved for intermediate- to high-grade sarcomas.

RECOMMENDATIONS FOR THE FINAL PATHOLOGY REPORT The final pathology report should contain all relevant gross and microscopic descriptive information, and it should be able to stand alone if all of the blocks and slides from a case are lost. In this day of frequent second opinions and detailed therapeutic protocols, standardizing reports of sarcomas is even more important. To that end, the ideal pathology report contains a detailed gross description, particularly of the following: (1) size of the tumor, (2) the extent of visible necrosis, and (3) how closely the tumor approaches all of the margins (with precise measurements). The diagnosis should be made in accordance with the World Health Organization (WHO) classification (2), grade should be applied (when relevant), and tumor size should be provided. When preoperative chemotherapy has been administered, the percentage of necrosis/fibrosis should be recorded, supplying the clinician with data on the effectiveness of neoadjuvant therapy. A comment on the margins should be incorporated into the report, and a tumor template is recommended.

CONSULTATION Because of the overall rarity of soft tissue tumors and the tremendous variation in histologic patterns, even within a given sarcoma phenotype, consultation may be sought in difficult cases, particularly when the original pathologist receives only a handful of cases per year. Sending a case for consultation is also important when IHC or molecular genetic studies required for diagnostic confirmation are not available locally. To save time, recut sections should be accompanied by at least one representative block or 10 unstained slides.

LIMITATIONS OF PROCEDURES FROZEN SECTION In many large cancer centers, the diagnosis of soft tissue tumors is established on core biopsy (see later), not on frozen section. One should not try to make a specific diagnosis (unless it is obvious) from intraoperative frozen sections, but, instead, one can attempt to decide whether the lesion is malignant, although this might not be possible. If this determination cannot be made with confidence, one can resort to terms such as cellular spindle cell neoplasm or atypical spindle cell neoplasm; even this noncommittal diagnosis guides the surgeon, who may then elect for a marginal complete excision or await the final report. ASPIRATION BIOPSY CYTOLOGY The use of aspiration biopsy cytology in the diagnosis of sarcomas has been successful in the hands of expert cytopathologists, particularly when ancillary techniques, such as IHC and molecular genetic analysis, are applied. Indeed, both the overall sensitivity and specificity have been estimated to be 95% (8). However, this high diagnostic yield is undoubtedly related to experience, and those who see only an occasional cytologic aspirate of a sarcoma are unlikely to match this rate. If insufficient material is obtained to generate a cell block for IHC, the diagnostic

yield is often limited. Therefore, a certain amount of interpretive caution is mandated, particularly when a distinction must be made between benign and malignant lesions. NEEDLE AND INCISIONAL BIOPSY Because of the presence of histologic variability within many soft tissue tumors, particularly sarcomas, the application of a needle biopsy technique can give rise to difficulties. Once again, those who have a great deal of experience with the technique report excellent results in predicting the ultimate diagnosis. However, sampling error with needle biopsy, or even incisional biopsy, often leads to an underestimation of tumor grade; this is often the case for LMS. Therefore, samples obtained with this approach may not be representative of a neoplasm, and this possible shortcoming should be recognized and communicated to the clinicians planning further therapy. EXCISIONAL BIOPSY Material may also be received from an “excisional biopsy”; this is often the case for superficial (dermal and subcutaneous) tumors. In such procedures, a rim of normal or marginal tissue may or may not be present, and the surgeon may mention that the lesion was “shelled out.” Even so, the excisional biopsy should be treated as if it is the definitive surgical procedure, and all margins should be inked and sampled. Experience with the excisional biopsy has shown that it is an inadequate approach to sarcomas because of the presence of tumor at or near marginal areas; the rate of local recurrence is very high following such surgeries. CURRENT SARCOMA THERAPY Wide local excision (a minimum of 1- to 2-cm margins of normal tissue, unless there is an intact fascial plane) is the most common current approach to sarcomas; a compartmental resection is often performed whenever this is possible. In the hands of an experienced sarcoma surgeon, a biopsy is performed in such a manner that it can be incorporated by this complete resection procedure. Increasingly, resections are being performed with smaller margins (2 mm-1 cm) in an attempt to spare function. The addition of postoperative radiation therapy has been effective in reducing local recurrence, allowing wide local excision to replace amputation in many instances. Chemotherapy for advanced or metastatic disease continues to have a low response rate of about 15% to 20%; complete responses are rare. For particular tumor types, preoperative chemotherapy may be administered after a diagnostic biopsy. Adjuvant chemotherapy is sometimes administered for some rare but aggressive tumors, such as angiosarcoma and synovial sarcoma, through the use of histology-specific protocols.

METHODS OF DETERMINING LINE OF DIFFERENTIATION Major advances have been made in the diagnosis of soft tissue tumors as a result of the application of ancillary techniques (e.g., IHC, cytogenetics, molecular genetics) within recent years. A brief discussion of each of these methods is presented with the intent of increasing awareness of their usefulness and limitations. CYTOGENETICS IN SARCOMAS The analysis of chromosomal patterns in sarcomas over three decades has demonstrated that many soft tissue tumors harbor recognizable nonrandom chromosomal aberrations (Table 5.3) (9). The chromosomal translocations are specific for some tumor types and often have

diagnostic value. Fluorescence in situ hybridization (FISH) to assess for gene rearrangement has largely replaced conventional cytogenetics in most practices. TABLE 5.3 Select Chromosomal Abnormalities and Gene Fusions in Soft Tissue Tumors Tumor Type

Cytogenetic Alterations

Gene Fusion

Alveolar rhabdomyosarcoma

t(2;13)(q35;q14) or t(1;13)(p36;q14)

PAX3-FOXO1A or PAX7-FOXO1A

Alveolar soft part sarcoma

der17 t(X;17)(p11;q25)

TFE3-ASPSCR1

Angiomatoid fibrous histiocytoma

t(2;22)(q33;q12) or t(12;22)(q13;q12)

EWSR1-CREB1 or EWSR1-ATF1

CIC-rearranged sarcoma

t(4;19)(q35;q13) or t(10;19)(q26;q13)

CIC-DUX4

Clear cell sarcoma

t(12;22)(q13;q12)

EWSR1-ATF1

Dermatofibrosarcoma protuberans

der(17)(17;22)(q21;q13)

COL1A1-PDGFB

Desmoplastic small round cell tumor

t(11;22)(p13;q12)

EWSR1-WT1

Epithelioid hemangioendothelioma

t(1;3)(p36;q25)

WWTR1-CAMTA1

Ewing sarcoma

t(11;22)(q24;q12), t(21;22)(q12;q12), and other rare translocations

EWSR1-FLI1, EWSR1-ERG, and other rare fusions

Extraskeletal myxoid chondrosarcoma

t(9;22)(q31;q12) and others involving 9q31

EWSR1-NR4A3 and others involving NR4A3

Infantile fibrosarcoma

t(12;15)(p13;q26)

ETV6-NTRK3

Inflammatory myofibroblastic tumor

Translocations involving 2p23

Gene fusions involving ALK

Low-grade fibromyxoid sarcoma

t(7;16)(q33;p11) or t(11;16)(p11;p11)

FUS-CREB3L2 or FUS-CREB3L1

Myxoid liposarcoma

t(12;16)(q13;p11) or t(12;22)(q13;q12)

FUS-DDIT3 or EWSR1-DDIT3

Myxoinflammatory fibroblastic sarcoma

t(1;10)(p22;q24)

TGFBR3-MGEA5

Sclerosing epithelioid fibrosarcoma

t(11;22)(p11;q12)

EWSR1-CREB3L1

Solitary fibrous tumor

inv(12)(q13q13)

NAB2-STAT6

Synovial sarcoma

t(X;18)(p11;q11)

SS18-SSX1 or SS18-SSX2

Tenosynovial giant cell tumor

t(1;2)(p13;q35)

COL6A3-CSF1

MOLECULAR DIAGNOSIS OF SOFT TISSUE TUMORS

The molecular diagnosis of soft tissue tumors is a rapidly advancing field; selected information is supplied in Table 5.3. These findings are useful diagnostically in many situations. Some tumors may harbor more than one genetic alteration, and some genes are involved in multiple tumor types (most notably, EWSR1). Recently, multiplex molecular testing of any given tumor for a panel of genetic changes has become available (using massively parallel sequencing or NGS), making analysis more straightforward and the diagnosis easier; RNA-based gene fusion panels are increasingly being applied to sarcomas. OVERVIEW OF IMMUNOHISTOCHEMISTRY The application of IHC has had a major impact on the approach to soft tissue tumors; this technique is central to diagnosis. Most laboratories use polymer-based detection techniques because of their increased sensitivity and decreased background staining (although avidin-biotin systems are still used by some practices); automated equipment is used along with a panel of markers. The utility of IHC for the diagnosis of soft tissue tumors can be attributed to the following: (1) some tumor lineages are associated with identifiable marker proteins; (2) antigens are often preserved in formalin-fixed paraffin-embedded (FFPE) tissue sections; (3) the sensitivity and specificity of many markers are high; and (4) new markers that serve as surrogates for molecular genetic alterations continue to be developed. With the increased sensitivity resulting from antigen retrieval through microwave, pressure cooker, or enzymatic pretreatment, the range of available diagnostic markers that are effective in FFPE sections is striking. However, widespread use of antigen retrieval has led to the identification of nonspecific reactions; antigen retrieval should only be used when needed, and careful validation of new markers is critical. Furthermore, a panel approach using multiple markers is generally employed. Emphasis should always be placed on interpretation within the context of the standard histologic features. Conventional (mostly lineage-associated) markers are provided in Table 5.4, and newer markers that correlate with molecular genetic alterations (or were identified through gene expression profiling) are provided in Table 5.5. In addition, typical marker profiles for selected soft tissue tumors are listed in Table 5.6. Specific markers are discussed in the context of particular tumor types and differential diagnosis throughout the chapter. TABLE 5.4 Immunohistochemical Markers for Lineage (Differentiation) in Soft Tissue Tumors Marker

Lineage

Select Tumor Types

Caldesmon

Smooth muscle

Leiomyoma/leiomyosarcoma

CD31

Endothelium

Vascular tumors

CD34

Endothelium

Vascular tumors

Fibroblast

Dermatofibrosarcoma _protuberansSolitary fibrous tumor

Claudin-1

Perineurium

Soft tissue perineurioma

Clusterin

Synovium

Tenosynovial giant cell tumor

Desmin

Smooth muscle

Leiomyoma/leiomyosarcoma

Skeletal muscle

Rhabdomyosarcomas

Myofibroblast

Low-grade myofibroblastic sarcoma

Epithelium

Epithelioid sarcoma Synovial sarcoma

Perineurium

Soft tissue perineurioma

ERG

Endothelium

Vascular tumors

HMB-45 and Melan A

Melanocyte

Clear cell sarcoma, PEComa, Malignant melanotic nerve sheath tumor

Myogenin and MyoD1

Skeletal muscle

Rhabdomyosarcomas

S-100 protein

Schwann cell

Schwannoma, neurofibroma, epithelioid malignant peripheral nerve sheath tumor

Melanocyte

Clear cell sarcoma

SATB2

Osteoblast

Osteosarcoma

SMA

Smooth muscle

Leiomyoma/leiomyosarcoma

Myofibroblast

Desmoid fibromatosis, Nodular fasciitis, Inflammatory myofibroblastic tumor

Schwann cell

Schwannoma, neurofibroma, Epithelioid malignant peripheral nerve sheath tumor

Melanocyte

Clear cell sarcoma

EMA

SOX10

EMA, epithelial membrane antigen; HMB, human melanoma black; PEComa, perivascular epithelioid cell tumor; SMA, smooth muscle actin.

TABLE 5.5 Immunohistochemical Markers That Correlate with Molecular Genetics in Soft Tissue Tumors Marker

Molecular Alteration

Select Tumor Types

ALK

ALK rearrangements

Inflammatory myofibroblastic tumor

β-Catenin

CTNNB1 mutation

Desmoid fibromatosis Glomangiopericytoma

BCOR

BCOR rearrangements or internal tandem duplication

Sarcomas with BCOR genetic alterations

CAMTA1

WWTR1-CAMTA1 fusion

Epithelioid hemangioendothelioma

CCNB3 (cyclin B3)

BCOR-CCNB3 fusion

Sarcomas with BCOR genetic alterations

CDK4

CDK4 amplification

Atypical lipomatous tumor/well-differentiated liposarcoma Dedifferentiated liposarcoma

DDIT3

FUS-DDIT3 or EWSR1-DDIT3 fusion

Myxoid liposarcoma

ETV4

CIC-DUX4 fusion

CIC-rearranged sarcoma

FOSB

FOSB fusion

Pseudomyogenic hemangioendothelioma Epithelioid hemangioma (50%)

H3K27me3

SUZ12 or EED mutations

Malignant peripheral nerve sheath tumor

INI1 (SMARCB1)

SMARCB1 deletion

Malignant rhabdoid tumor

Epithelioid sarcoma MDM2

Epithelioid malignant peripheral nerve sheath tumor MDM2 amplification

Atypical lipomatous tumor/well-differentiated liposarcoma Dedifferentiated liposarcoma

MYC

MYC amplification

Postradiation cutaneous angiosarcoma

Pan-TRK

NTRK1, NTRK2, or NTRK3 rearrangements

Infantile fibrosarcoma NTRK-rearranged spindle cell neoplasms

PLAG1

PLAG1 rearrangement

Mixed tumor, lipoblastoma

PRKAR1A

PRKAR1A deletion

Malignant melanotic nerve sheath tumor

RB1

RB1 deletion

Spindle cell/pleomorphic lipoma Cellular angiofibroma Myofibroblastoma Atypical spindle cell lipomatous tumor

ROS1

ROS1 rearrangement

Inflammatory myofibroblastic tumor (5%)

SS18-SSX

SS18-SSX1 or SS18-SSX2 fusions

Synovial sarcoma

STAT6

NAB2-STAT6 fusion

Solitary fibrous tumor

TFE3

ASPSCR1-TFE3 fusion

Alveolar soft part sarcoma

YAP1-TFE3 fusion

Epithelioid hemangioendothelioma variant

Other TFE3 fusions

PEComa (15%)

TABLE 5.6 Immunohistochemical Marker Profiles for Select Soft Tissue Tumorsa DES

SMA

CD34

S-100

KRT

EMA

CD31

ERG

MUC4

Rhabdomyosarcoma

++

−b

−

+/−

−b

−

−

−

−

Ewing sarcoma

−

−

−

−

−

−

−

−b

−

Malignant rhabdoid tumor

−

−

−

−

+

+

−

−

−

Desmoplastic SRCT

++

−

−

+/−

++

++

−

−

−

Myxoid liposarcoma

−

−

+/−

+

−

−

−

−

−

Dedifferentiated liposarcoma

+/−

+/−

+/−

−

−

−

−

−

−

Leiomyosarcoma

+

++

+

+/−

+/−

+/−

−

−

−

MPNST

+/−

−

−b

+

−b

+/−

−

−

−

Synovial sarcoma

−

−

−

+/−

+

+

−

−

+/−

Desmoid fibromatosis

+/−

++

−

−

−

−

−

−

−

Solitary fibrous tumor

−

−

++

−

−

−

−

−

−

Low-grade fibromyxoid sarcoma

−

−

−

−

−

+/−

−

−

++

Nodular fasciitis

−

++

−

−

−

−

−

−

−

BFH

−

+/−

−b

−

−

−

−

−

−

DFSP

−

−

++

−

−

−

−

−

−

UPS

−b

+/−

+/−

−

−

+/−

−

−

−

Epithelioid sarcoma

−

−

++

−

++

++

−

+/−

−

Angiosarcoma

−

−b

++

−

+/−

−

++

++

−

Epithelioid hemangioendothelioma

−

−

++

−

+/−

−

++

++

−

Kaposi sarcoma

−

+/−

++

−

−

−

+

++

−

Chondrosarcoma

−

−

−

++

−

−

−

−

−

Chordoma

−

−

−

++

++

++

−

−

−

Clear cell sarcoma

−

−

−

++

−

+/−

−

−

−

Alveolar soft part sarcoma

−

−

−

−

−

−

−

−

−

aInterpretive

caution and correlation with histology are vital; no stain is diagnostic in and of itself. negative, but positive in a small subset of cases. ++, strongly reactive in most cases; +, positive reaction; −, pertinent negatives in a panel approach; +/−, occasional, usually focal, reactions; BFH, benign fibrous histiocytoma; DES, desmin; DFSP, dermatofibrosarcoma protuberans; EMA, epithelial membrane antigen; KRT, keratins; MPNST, malignant peripheral nerve sheath tumor; SMA, smooth muscle actin; SRCT, small round cell tumor; UPS, undifferentiated pleomorphic sarcoma. bTypically

Problems with Immunohistochemistry Serious interpretative difficulties have arisen within the past several years because numerous IHC markers have been applied to the vast array of mesenchymal lesions. The work of many authors has highlighted problems relating to the lack of specificity on the one hand and aberrant immunoreactivity on the other. Marker Negativity. One crucial fact seems to have been forgotten whenever stains are assessed for diagnosis or a contribution to the literature—any given phenotypic marker does not react with 100% of its proper tumor type. In fact, many studies show that only 50% to 90% of given tumor types show reactivity for the specific marker tested. Even with antigen retrieval, not

all cases are immunoreactive. Therefore, the significant lesson that must be relearned is that one may make a correct diagnosis in the face of marker negativity, although care must be taken. For example, not all MPNSTs are S-100 protein positive, and not all monophasic synovial sarcomas express keratins. In such instances, other support is required (e.g., novel IHC markers based on molecular genetics). Therefore, perfection in an IHC marker should not be expected. Lack of Specificity. The initial rush of enthusiasm accompanying the appearance of a new “specific” marker is often replaced with harsh realism once the marker is subjected to a series of scientific studies. The results frequently show that the marker is useful, but less than completely specific. For example, as a group, the muscle markers were originally thought to be specific for the smooth and skeletal muscle cell types, but actins react with myofibroblastic and so-called fibrohistiocytic neoplasms, with SMA expected in myofibroblasts. Hence, marker reactivity alone does not lead to a correct diagnosis; cytology, architecture, tumor location, and patient information must be integrated with the results of IHC. Aberrant Immunoreactivity. The phenomenon of marker staining when it is theoretically unexpected is often referred to as aberrant immunoreactivity. For example, the normal epithelial marker keratin may be detected in nonepithelial neoplasms, such as LMS (up to 38%); keratins have been identified in a wide range of mesenchymal tumor types. Pathologists must take this phenomenon into account in the interpretation of IHC. Luckily, most instances of aberrant reactivity show only patchy rather than diffuse staining. General Markers. Vimentin has been hailed as the intermediate filament for mesenchymal tissues because it is found within essentially all normal mesenchymal tissue elements and most sarcomas. However, vimentin immunoreactivity is not informative from a diagnostic viewpoint because many tumors, including carcinomas, melanomas, and lymphomas, contain vimentin. Given its lack of specificity, vimentin has no utility in the diagnosis of soft tissue tumors; it has been entirely abandoned in diagnostic practice by many experts (including the author of this chapter). Unfortunately, there are no general IHC markers that define a neoplasm as (broadly) mesenchymal; lineage-restricted markers and specific molecular genetic surrogates should be applied (see Tables 5.4 and 5.5). ELECTRON MICROSCOPY Electron microscopy has a long history in soft tissue tumor pathology; in the past, this technique held value for identifying particular cell and tumor types. However, electron microscopy has essentially been replaced by IHC and molecular genetics for the diagnosis of soft tissue tumors; the WHO classification no longer includes ultrastructural features for particular tumor types.

EVALUATION OF PROGNOSTIC FACTORS GRADE The French Federation of Cancer Centers Sarcoma Group (FNCLCC) grading system is mostly widely used, having been adopted by the American Joint Committee on Cancer (AJCC), the WHO, and the College of American Pathologists (CAP) cancer protocols; this system is based on a combination of “differentiation,” mitotic rate, and necrosis (10). Some sarcoma types are ungradable (the diagnosis alone providing critical prognostic information; many sarcomas of uncertain lineage belong to this group), whereas others are high grade by definition (e.g., angiosarcoma, round cell sarcomas, and RMS). Some tumor types believed to be “sarcomas”

are not in fact malignant (i.e., they do not metastasize), including DFSP (of conventional type) and well-differentiated liposarcoma; these tumors belong to the category of mesenchymal neoplasms of “intermediate biologic potential, locally aggressive.” Still other tumor types are considered to be “intermediate, rarely metastasizing” (e.g., inflammatory myofibroblastic tumor and angiomatoid fibrous histiocytoma), because the metastatic potential is less than 2% and cannot be predicted on the basis of histologic features. STAGING SYSTEM The current (eighth edition) AJCC staging system for sarcomas of the extremities and trunk wall is based on histologic grade and tumor size; for staging purposes, grades 2 and 3 are grouped together (11). There are separate staging systems for retroperitoneal sarcomas, sarcomas of the head and neck, and visceral sarcomas, although the prognostic significance of the latter two systems has yet to be confirmed. In many sarcoma centers, AJCC stage per se is less important than is histologic diagnosis and grade for treatment planning. FUTURE PROGNOSTICATION Recently, prognostic nomograms incorporating histologic type, grade, tumor size, patient age, depth, and anatomic site have been developed to help stratify patients with sarcoma for risk of metastasis (12). However, most such systems were developed on the basis of the relatively common sarcoma types; it is therefore not clear whether these nomograms are reliable for rare tumor types. Tumor type–specific nomograms have also recently been established (13-15).

SOFT TISSUE LESIONS: LESIONS MIMICKING SARCOMAS (PSEUDOSARCOMAS) NODULAR FASCIITIS NOF, which is commonly mistaken for a sarcoma, is a self-limiting myofibroblastic neoplasm (16,17) characterized by extremely rapid growth (more so than the usual sarcoma); it achieves its small size of 2 to 3 cm in a matter of weeks. Lesions are only rarely larger or of longer duration (3 months-1 year). Most cases occur in persons between the ages of 20 and 50 years, with men and women being equally affected. NOF is commonly found on the forearm, upper arm, face, and shoulder, although it may occur in any somatic soft tissue location. NOF, which is usually well circumscribed, is tan to gray-white with a myxoid appearance. In NOF, several histologic findings are key, the most important of which is apparent at low power; nearly all cases have a characteristic architecture in the form of a zonation effect (Fig. 5.3). The center is hypocellular, whereas at the periphery, the appearance is more hypercellular, with small vessels in a lobular array abutting a collagenous zone. In between, spindle cells are arranged in a loose myxoid stroma. The lesion typically is located just above the muscle layer, abutting the superficial fascia. The lesional cells are elongated, plump, spindled and stellate cells with long cytoplasmic processes. By IHC, many cells exhibit strong and diffuse staining for SMA (Fig. 5.3D), which can be used to diagnostic advantage; desmin is usually negative. Although the cells may appear worrisome with their enlarged vesicular nuclei and prominent nucleoli, they are uniform with no pleomorphism. The hallmark is the loose arrangement of the cells in a “tissue culture”–like manner. Some “cellular” variants contain storiform areas, interconnecting fascicles, or cystic areas, even in the same lesion; in these tumors, small microcysts with mucin should be sought. An inflammatory component of lymphocytes and macrophages is practically always present to some degree; scattered

osteoclastic giant cells are often seen. Scattered extravasated red blood cells are also often seen. Lastly, mitotic activity may be prominent. Nonetheless, NOF is the classic example of an important dictum—benign lesions may be quite mitotically active.

FIGURE 5.3 Nodular fasciitis. Note the deep subcutaneous tumor above the fascial plane (A). The tumor contains abundant myxoid stroma with variable cellularity (B). Foamy histiocytes and lymphocytes accompany a loose array of plump spindle cells with a tissue culture–like quality (C). These cells are diffusely positive for smooth muscle actin (D).

The long-standing belief that NOF is reactive in nature has been proven incorrect; nearly all cases harbor rearrangements of USP6 (17p13), most often resulting in an MYH9-USP6 gene fusion (18). The most common diagnostic difficulty arises with other fibroblastic/myofibroblastic tumors, including desmoid fibromatosis, myofibroma, cellular dermatofibroma (benign fibrous histiocytoma [BFH]), and a spindle cell sarcoma or UPS. However, unlike NOF, each of these other tumor types has a relatively uniform pattern, none with the microcysts typical of NOF. Long, sweeping fascicles and extensive stromal collagen distinguish desmoid fibromatosis from NOF. NOF only very rarely recurs locally. PROLIFERATIVE FASCIITIS Proliferative fasciitis (19) resembles NOF in the following respects: the location within deep tissues, the small size, the presence of a zonation pattern, and the loose quality of cell growth. Strikingly different are the cells themselves. More polygonal in shape, they have abundant amphophilic cytoplasm surrounding very large, but oval to round, vesicular nuclei with prominent nucleoli that resemble ganglion cells (Fig. 5.4). They are dispersed singly within a slightly myxoid or collagenized stroma. Unlike NOF, proliferative fasciitis occurs in an older age group (>50 years), but otherwise, it has a similar presentation and also does not recur.

FIGURE 5.4 Proliferative fasciitis. Tumor cells have abundant cytoplasm and oval nuclei, mimicking ganglion cells.

PROLIFERATIVE MYOSITIS Proliferative myositis (20) is a related lesion in which ganglion-like cells proliferate between muscle fibers and separate each of them so that, at low power, a distinctive “checkerboard”

appearance is visible. Although the checkerboard pattern is occasionally mimicked by an infiltrating lymphoma or desmoid fibromatosis, in neither of these lesions are the cells dispersed and ganglion-like. PSEUDOSARCOMATOUS MYOFIBROBLASTIC PROLIFERATIONS This lesion has also been referred to as “postoperative spindle cell nodule,” although spontaneous development is also common. This tumor type typically arises in the urinary bladder, less often at other mucosal locations, and is usually an edematous ulcerating lesion with infiltrative border, composed of plump spindle cells with abundant amphophilic cytoplasm (Fig. 5.5). The mitotic rate is highly variable. Although pseudosarcomatous myofibroblastic proliferations may simulate sarcomas, the nuclei are not hyperchromatic and the myxoid quality of the stroma with cell separation, when combined with the anatomic site, are characteristic features. By IHC, the lesional cells have the staining pattern of myofibroblasts (positive for desmin and actins) and may also express keratins, which can lead to diagnostic confusion with consideration for spindle cell carcinoma. Up to 70% of cases are immunoreactive for anaplastic lymphoma kinase (ALK) (21), and a recent study detected consistent ALK gene rearrangements, most often FN1-ALK (22), suggesting that this tumor type is in fact a neoplasm.

FIGURE 5.5 Pseudosarcomatous myofibroblastic proliferation. In this bladder tumor, plump myofibroblasts with prominent nucleoli may be concerning. However, they occur singly, and they are accompanied by an inflammatory cell infiltrate.

SPINDLE CELL LIPOMA Spindle cell lipoma consists of adipocytes and bland, short spindle cells with ovoid to wavy nuclei, in markedly variable proportions (Fig. 5.6); some tumors are nearly indistinguishable from conventional lipomas, whereas others are entirely devoid of adipocytes (23). Myxoid stroma is common; however, the capillary vascular network (crow’s-feet vessels) of myxoid liposarcoma is absent. The differential diagnosis may include neurofibroma and deep fibrous histiocytoma in those cases in which spindle cells predominate. Spindle cell lipoma, however, does not exhibit a storiform pattern. It is frequently more cellular than is the usual neurofibroma, and it is negative for S-100 protein and CD34 positive. Spindle cell lipoma shows consistent loss of expression of the retinoblastoma protein (RB1) by IHC, as do other benign soft tissue tumors characterized by 13q14 chromosomal losses (myofibroblastoma and cellular angiofibroma) (24); this marker may be helpful in differential diagnosis. Its clinical presentation is characteristic, with a vast majority of lesions occurring on the back of the neck, upper back, or shoulder in elderly men; however,

occasional tumors arise in the extremities. Some examples of spindle cell lipoma contain thin bands of collagen mimicking solitary fibrous tumor (SFT); however, spindle cell lipoma is negative for STAT6.

FIGURE 5.6 Spindle cell lipoma. This tumor is typically composed of an admixture of adipocytes, short spindle cells, and ropey collagen bundles. In some case, spindle cells may predominate.

PLEOMORPHIC LIPOMA Pleomorphic lipoma is a histologic variant of spindle cell lipoma, characterized by large, pleomorphic and floret-type giant cells (Fig. 5.7) within a background of adipocytes and short spindle cells, within a variable myxoid stroma (25). Nearly all cases are superficial in subcutaneous tissue. The clinical setting of this CD34-positive tumor is similar to that of spindle cell lipoma. Cases in women or unusual locations are infrequent. Despite the worrisome histologic appearances, pleomorphic lipomas are benign and only rarely recur locally. The cytogenetic findings (with loss of nuclear RB1 by IHC) are the same as spindle cell lipoma and different from atypical lipomatous tumor (26).

FIGURE 5.7 Pleomorphic lipoma. The characteristic floret cells are large and pleomorphic with nuclei around the cell periphery. Adjacent fat cells are variably sized, and smaller spindle cells are present.

ATYPICAL SPINDLE CELL LIPOMATOUS TUMOR

Previously known as “spindle cell liposarcoma,” atypical spindle cell lipomatous tumor (ASCLT) is now known to be a benign adipocytic neoplasm with a low risk of local recurrence (10%-15%), distinct from conventional atypical lipomatous tumor and spindle cell/pleomorphic lipoma, although it often mimics both of these other tumor types (27-29). This tumor type has no potential to dedifferentiate. ASCLT usually presents on the limbs (especially hands and feet), more often superficial than deep; unlike well-differentiated liposarcoma, retroperitoneal involvement is rare. Histologically, ASCLT shows a range of appearances, with varying proportions of spindle cells, adipocytes, and lipoblasts, including hyperchromatic and sometimes pleomorphic cells, in a variably collagenous or myxoid stroma (Fig. 5.8). By IHC, ASCLT shows inconsistent staining for CD34, S-100 protein, and desmin; loss of nuclear RB1 is seen in around 60% of cases (29). Importantly, murine double-minute type 2 (MDM2) and cyclin-dependent kinase 4 (CDK4) are negative (although focal MDM2 staining is rarely seen). MDM2 amplification is absent. The differential diagnosis includes spindle cell/pleomorphic lipoma and atypical lipomatous tumor (ALT). Spindle cell/pleomorphic lipomas almost always arise in the subcutaneous tissue of the upper back, shoulders, or neck, and contain bright eosinophilic, “ropey” collagen bundles. A prominent spindle cell component is uncommon in ALT; IHC or FISH for MDM2 can be used to make this distinction.

FIGURE 5.8 Atypical spindle cell lipomatous tumor. The tumor is composed of adipocytes showing variation in cell size, spindle cells, and scattered pleomorphic cells. Note the collagenous stroma.

SUPERFICIAL CD34-POSITIVE FIBROBLASTIC TUMOR This recently described fibroblastic neoplasm arises in the skin and subcutaneous tissue, with a predilection for the extremities of middle-aged adults. This tumor type is composed of fascicles and sheets of spindled to epithelioid cells with abundant eosinophilic, often glassy cytoplasm and sometimes marked pleomorphism (Fig. 5.9); superficial CD34-positive fibroblastic tumor may be mistaken for a pleomorphic sarcoma, especially UPS (30,31). By IHC, the tumor cells show strong CD34 expression, and keratins are positive in 70% of cases (30). Some tumors contain PRDM10 gene rearrangements (32). Although data are limited, thus far, recurrence and metastasis are rare.

FIGURE 5.9 Superficial CD34-positive fibroblastic tumor. This tumor type often shows striking pleomorphism and may, therefore, mimic undifferentiated pleomorphic sarcoma. The superficial presentation and abundant glassy eosinophilic cytoplasm are clues to the diagnosis.

LIPOBLASTOMA Lipoblastoma characteristically arises on the extremities of young children, particularly boys, before the age of 3 years (33), but rare cases can occur in adulthood. In this lesion, prominent fibrous septa divide the adipocytic neoplasm into lobules, and a myxoid quality is often visible at low power. A range of differentiation, from short spindle cells within the myxoid matrix to vacuolated cells to mature adipocytes (Fig. 5.10) can be appreciated at high power. Despite the presence of these scattered developing lipoblasts, the lesion usually lacks the capillary network of myxoid liposarcoma, although in some cases, these tumor types may be essentially indistinguishable. However, myxoid liposarcoma harbors DDIT3 rearrangements (with either FUS or EWSR1), which can be detected by FISH or IHC for DDIT3, lipoblastoma is characterized by PLAG1 rearrangements (34), leading to nuclear expression of the PLAG1 protein, which can be detected by IHC (35). As patients age, lipoblastomas gradually mature, with the disappearance of the spindle cell and myxoid components. Such lesions are virtually indistinguishable from conventional lipomas, other than the presence of a lobulated architecture with thin fibrous septa.

FIGURE 5.10 Lipoblastoma. This lobulated tumor contains areas of myxoid stroma with short, hyperchromatic spindle cells, delicate capillary vessels, and a mature adipocytic component. The resemblance to myxoid liposarcoma can be striking.

CELLULAR ANGIOLIPOMA Most angiolipomas are easily recognized; these subcutaneous, small lesions are often painful, and are histologically dominated by mature adipocytes with scattered small blood vessels with occasional fibrin microthrombi. However, cellular examples include a prominent vascular component mimicking Kaposi sarcoma (36) or other vascular neoplasms. Spindle cells (pericytes) are often prominent, admixed with endothelial cells (Fig. 5.11). In contrast to Kaposi sarcoma, cellular angiolipomas are negative for HHV8. When angiolipomas arise in the subcutaneous tissue of the breast, they may be mistaken for mammary angiosarcoma; however, the latter tumor type arises in the breast parenchyma and entraps breast ducts and lobules.

FIGURE 5.11 Cellular angiolipoma. This well-circumscribed subcutaneous tumor is composed nearly entirely of a vascular proliferation; on closer inspection, scattered fat cells are noted. Some of the small vessels contain thrombi.

BIZARRE (SYMPLASTIC) LEIOMYOMA

That pleomorphism is not a sine qua non for sarcoma is exemplified by this highly pleomorphic but benign smooth muscle tumor, which is almost always found in the uterus; bizarre leiomyomas are exceptionally rarely found at other anatomic sites. These tumors are characterized by large, atypical and pleomorphic nuclei in an otherwise typical leiomyoma (Fig. 5.12). They tend to be small, and mitotic activity is typically low. Whenever nuclear atypia is encountered in a putative soft tissue or retroperitoneal leiomyoma, mitoses should be sought very carefully, and a malignant diagnosis should be seriously considered.

FIGURE 5.12 Bizarre leiomyoma. Enlarged pleomorphic nuclei were found in this uterine tumor; however, a careful search proved the well-sampled lesion to be completely devoid of mitotic activity.

FETAL AND GENITAL RHABDOMYOMA Fetal rhabdomyoma, which is occasionally confused with the rare well-differentiated embryonal RMS, occurs mainly in the head and neck of toddlers (37). A similar lesion is found in the adult female genital tract (38). Small parallel bundles of easily recognized spindled skeletal muscle cells are separated by thin strands of collagen and vascular tissue or by less differentiated cells. The groups of spindle cells intersect in a random manner, although the entire lesion retains an architectural structure of separated bundles of streaming tumor cells (Fig. 5.13). The nuclei are bland in appearance, and only occasional nucleoli can be seen. Notably, the tumor lacks mitotic activity and the infiltrative growth pattern of RMS. When the fetal type is highly cellular and compact, it becomes more difficult to distinguish from RMS. PTCH1 mutations and deletions are detected in fetal rhabdomyoma (39). Genital rhabdomyomas arising in the vagina are typically polypoid, but lack the cambium layer of botryoid embryonal RMS.

FIGURE 5.13 Rhabdomyoma, genital type. In the “separated bundle” pattern, fibrous tissue is seen on either side of groups of elongated spindle cells; note the long cytoplasmic extensions with parallel sides, peculiar to skeletal muscle tumors.

PAPILLARY ENDOTHELIAL HYPERPLASIA Papillary endothelial hyperplasia (Masson change) may commonly be mistaken for angiosarcoma; however, diagnosis is relatively straightforward if careful attention is paid to histology (40). First, most of these reactive lesions occur within a vessel or vascular malformation; therefore, unlike angiosarcoma, they are circumscribed. Second, the nuclei are bland and uniform. Third, rather than ramifying vascular channels, the papillary structures tend to occupy a contiguous large space that can be traced around much of the proliferation (Fig. 5.14). In foci, a fibrin-like matrix representing a stage of organizing thrombus is observed. Although the lesion may occur almost anywhere, it is frequently found on the fingers as a painful nodule. Pathologists should be aware that it can be found in a hematoma or blood-filled space within another tumor, causing further confusion, or that it can result from procedures such as fineneedle aspiration of organs, where it is less well circumscribed, simulating angiosarcoma even further.

FIGURE 5.14 Papillary endothelial hyperplasia. Papillary structures project into a large continuous space, unlike the less ramifying channels of angiosarcoma; cores of either cellular collagen or fibrin are lined by bland endothelial cells without the multilayering typical of angiosarcoma.

ATYPICAL (PSEUDOSARCOMATOUS) FIBROEPITHELIAL STROMAL POLYPS Fibroepithelial stromal polyps are benign lesions that occur most often in the vulvovaginal or anal locations, although they also form elsewhere. Such polyps often contain scattered small multinucleated stromal cells; on occasion, the degree of atypia in such reactive cells can be striking, in which cases the lesions superficially resemble a sarcoma (41). However, unlike sarcomas, atypical fibroepithelial stromal polyps exhibit multiple oval nuclear lobes that are usually uniform in size (Fig. 5.15). Another characteristic is that the atypical cells are scattered singly throughout the lesions. Therefore, another helpful principle of soft tissue tumor pathology is that neoplasms tend to exhibit crowding and closely apposed atypical cells, in contrast to the single-cell growth of reactive fibroblastic proliferations. The stromal cells in such polyps extend to the epidermis without a border, another clue to the diagnosis.

FIGURE 5.15 Atypical (pseudosarcomatous) fibroepithelial stromal polyp. This polypoid lesion is composed of collagen punctuated by individually scattered fibroblasts, some of which have pleomorphic nuclei including multinucleated forms.

GIANT CELL FIBROBLASTOMA

Giant cell fibroblastoma is discussed in the section “Dermatofibrosarcoma Protuberans.” INTRAMUSCULAR/CELLULAR MYXOMA Intramuscular myxoma is a gelatinous lesion that commonly appears as a relatively circumscribed mass deep within a muscle, usually in an extremity (Table 5.7, Fig. 5.16) (42). Intramuscular myxomas may reach considerable size (10-13 cm). The bulk of the lesion consists of a slightly basophilic matrix, and at high power, a few scattered cytologically uniform, stellate or bipolar cells with oval nuclei are seen. Lesions showing higher cellularity are referred to as cellular myxoma; this is simply a histologic variant of intramuscular myxoma. Unlike myxoid liposarcoma, the lesion is hypocellular, and it lacks significant vascularity. An inapparent architecture is present; toward the periphery, an increased number of spindle cells may be seen. Peripheral entrapment of skeletal muscle showing atrophy is common, characteristic of slowgrowing lesions. A GNAS mutation can be found in most examples of intramuscular/cellular myxoma (43,44). Intramuscular myxoma very rarely recurs.

FIGURE 5.16 Intramuscular myxoma. This strikingly hypocellular lesion is nearly avascular. There is no atypia.

TABLE 5.7 Differential Diagnosis of Myxoid Lesions: Histology and Immunohistochemistry Tumor

Nodules

Vesselsa

Cellularity

Pleomorphism

Mitoses

IHC

Intramuscular myxoma

S

+/−

Low

−

−

−

Superficial angiomyxoma

M

+

Moderate

−

+/−

−

Deep (“aggressive”) angiomyxoma

S

+

Moderate

−

+/−

DES+

Angiomyofibroblastoma

S

+

Moderate

−

+/−

DES+

Myxofibrosarcoma

S or M

++

High

+/++

++

SMA+/−

Myxoid liposarcoma

S or M

++

High

−

+

DDIT3+

Low-grade fibromyxoid sarcoma

S

+

Low

−

+/−

MUC4+

Extraskeletal myxoid chondrosarcoma

M

+

Moderate

−

+/−

S-100+/−

Myoepithelioma

M

+

Moderate

−

+

KRT, EMA, S-100, GFAP, p63+

aSee

text and schematic (Fig. 5.53) for vessel descriptions. +, present; ++, prominent; −, absent; +/−, rare or focal; DES, desmin; IHC, immunohistochemistry; KRT, keratins; M, multiple; S, single; SMA, smooth muscle actin.

MYOSITIS OSSIFICANS Myositis ossificans typically arises rapidly, similar to NOF (45), within the extremity musculature, usually in young male patients (Fig. 5.17). The characteristic extensive calcification of the lesion is apparent on radiographic study, and histologically, it has the configuration of lamellar bone. It is a solitary and well-circumscribed lesion with organization into the following three zones: a periphery of well-formed lamellar bone gradually maturing from poorly formed trabeculae of osteoid in the middle zone and, in the center, a fibroblastic proliferation with remarkable similarity to that in NOF. Before the lesion is well developed, it may resemble osteosarcoma, but, in extraskeletal osteosarcomas, the bone matrix is usually located more centrally, which is the reverse of myositis ossificans. Recent studies have documented recurrent COL1A1-USP6 rearrangement in this tumor type, genetically linking myositis ossificans to NOF (46).

FIGURE 5.17 Myositis ossificans. Parallel arrays of immature osteoid arise from a fasciitis-like background to form bony trabeculae; the nuclei are disturbing because of prominent nucleoli. This tumor type shows a characteristic pattern of zonation, with peripheral osteoid maturing into lamellar bone.

FIBROUS LESIONS NOF, proliferative myositis, and atypical fibroepithelial stromal polyps have already been described in the section “Soft Tissue Lesions: Lesions Mimicking Sarcomas (Pseudosarcomas).” TUMEFACTIVE FIBROINFLAMMATORY LESIONS Mass-forming nonneoplastic lesions characteristic by fibrosis and chronic inflammation are described as tumefactive fibroinflammatory lesions. Such lesions include idiopathic retroperitoneal fibrosis, sclerosing mesenteritis, and immunoglobulin G4 (IgG4)-related disease. The etiology of these lesions is unknown.

Focal myositis is another histologically bland entity with a mass-forming presentation. RETROPERITONEAL FIBROSIS Retroperitoneal fibrosis is a fibrotic process that typically shows bilateral impingement on the ureters, resulting in hydronephrosis. Fibrosis with clusters of lymphocytes and plasma cells is found. Occasionally, large presacral masses can be seen. KELOID The keloid is a hypertrophic scar typified by the presence of thick, bright eosinophilic collagen bands. The reason for mentioning this entity is that the presence of such “keloidal collagen” in another lesion, such as desmoid fibromatosis, may provide a clue to its fibroblastic nature. FIBROMAS Several distinct tumor types are referred to as fibromas. Hypocellularity accompanied by dense collagen is the main feature in the sclerotic fibroma (47), but pleomorphic fibromas of the skin have also been described (48). If a pleomorphic fibroma is a consideration, excluding desmoplastic melanoma with an S-100 stain is wise. Better known is fibroma of tendon sheath (49). In the usual clinical setting, a male patient between 30 and 50 years of age presents with a nodule on the fingers, hands, or wrist. These well-circumscribed, generally small tumors may be lobulated. These collagenous lesions contain small slitlike vessels, most prominent at the periphery; most lesions are moderately cellular, composed of thin, hyperchromatic spindled fibroblasts (Fig. 5.18). Focally, the lesions may be cellular, particularly at the periphery, but mitotic figures are scarce.

FIGURE 5.18 Fibroma of tendon sheath. This tumor is well circumscribed and composed of small fibroblasts arranged in short bundles. Note the characteristic slitlike blood vessels at the periphery.

The nuchal fibroma often occurs on the back of the neck, and presents as fibrofatty tissue with very dense collagen and occasional cartilaginous metaplasia (50). Associated with familial adenomatous polyposis (FAP) syndrome, the Gardner fibroma is a highly collagenized and hypocellular tumor (Fig. 5.19), similar to nuchal fibroma, occurring predominantly in the paraspinal back, chest wall, and flank of young children (51). Similar to

desmoid fibromatosis, Gardner fibroma usually shows aberrant nuclear immunoreactivity for βcatenin (51).

FIGURE 5.19 Gardner fibroma. This markedly hypocellular lesion contains a sparse, haphazard distribution of spindle cells within a dense collagenous stroma.

DESMOPLASTIC FIBROBLASTOMA Desmoplastic fibroblastoma, which is also called collagenous fibroma (52,53), typically arises in the subcutis. It is hypocellular, with dense collagenous stroma and scattered stellate spindle cells (Fig. 5.20). Although some lesions show irregular margins, most are circumscribed. No recurrences have been reported.

FIGURE 5.20 Desmoplastic fibroblastoma (collagenous fibroma). Stellate fibroblasts are typical of this hypocellular, collagenous tumor.

NASOPHARYNGEAL ANGIOFIBROMA, CELLULAR ANGIOFIBROMA, AND SOFT TISSUE ANGIOFIBROMA

Nasopharyngeal angiofibroma is a tumor that occurs in young male patients, particularly in the nasopharynx (see Chapter 21). It consists of bland fibroblastic cells proliferating among irregular, thin-walled blood vessels. Cellular angiofibroma has a predilection for the vulva and inguinal region. This usually cellular tumor is composed of short fascicles of bland spindle cells with prominent thick-walled, hyalinized blood vessels, and a variable adipocytic component (Fig. 5.21) (54). These lesions show considerable histologic overlap with and have similar cytogenetic features as spindle cell lipoma (55). Angiofibroma of soft tissue is composed of small hyperchromatic spindle cells in a collagenous or myxoid stroma with numerous, branching blood vessels superficially resembling those in myxoid liposarcoma but with thicker walls (Fig. 5.22) (56). Soft tissue angiofibroma has a t(5;8)(p15;q13) translocation, resulting in an AHRR-NCOA2 fusion (57).

FIGURE 5.21 Cellular angiofibroma. This vulvar tumor is composed of loosely arranged, short spindle cells in a variably edematous or collagenous stroma with large hyalinized blood vessels.

FIGURE 5.22 Angiofibroma of soft tissue. In this tumor type, branching capillary-sized blood vessels mimic those seen in myxoid liposarcoma, although the vessel walls are thicker in soft tissue angiofibroma. Note the edematous stroma and disorganized short spindle cells.

ELASTOFIBROMA Nearly every example of elastofibroma has occurred in the mid-back and scapular region (58). On low power, it appears to be a fibroblastic or fibrofatty proliferation that is densely collagenized, but at higher power, circular and snakelike eosinophilic bands of elastic tissue are noted (Fig. 5.23). Usually, these elastic fibers have a beaded appearance and constitute most of the stroma. The underlying cause of this pseudotumor produced by abnormal elastogenesis is unknown, but the surgically excised lesion has no tendency to recur.

FIGURE 5.23 Elastofibroma. Thick, wormlike fibers are embedded in this lesion, a fibrofatty mass (A). On elastic stain (B), the abnormal quantity and shapes of elastic fibers can be appreciated.

FIBROUS PROLIFERATIONS OF CHILDHOOD Numerous fibroblastic/myofibroblastic neoplasms occur in children (59,60). When such lesions are encountered, some of which may simulate fibrosarcoma or other sarcomas, consulting soft tissue or pediatric pathology texts or review articles is best. Only selected features of certain lesions are discussed here. The fibrous hamartoma of infancy is a solitary, poorly circumscribed proliferation in which organoid nodules of spindle cells with a myxoid matrix are interspersed within fatty and fibrous tissue (Fig. 5.24A) (61). Inclusion body fibromatosis (also known as

infantile digital fibroma/fibromatosis) is characterized by fascicles of fibroblasts with prominent stromal collagen (62). The pathognomonic feature is the presence of small, round intracytoplasmic inclusions approximately the size of a lymphocyte nucleus; they appear red on a trichrome stain and consist of actin filaments. In infantile myofibromatosis, small bundles of spindle-shaped myofibroblasts alternate with more cellular areas containing ovoid cells and thinwalled, branching blood vessels (63). Cutaneous nodules, gingival hypertrophy, and flexure contractures characterize the mesenchymal dysplasia known as juvenile hyaline fibromatosis (64), which is inherited as an autosomal recessive trait caused by mutations in the ANTXR2 gene (65). The amorphous hyaline substance found within it has areas resembling keloidal collagen. Fibromatosis colli presents as a rapidly growing mass in the second to fourth week of life, and microscopically, it appears as a diffuse scar within skeletal muscle. In calcifying aponeurotic fibroma (66), ill-defined and painless masses appear on the hands and feet of children between the ages of 10 and 15 years; in the classic lesion, primitive mesenchymal cells resembling fibromatosis occur in nodules surrounding central calcification (Fig. 5.24B).

FIGURE 5.24 Pediatric fibrous tumors. Fibrous hamartoma of infancy shows a triphasic appearance, including bundles of eosinophilic spindle cells, a mature adipocytic component, and small lobules of more primitive-appearing

spindle cells in a basophilic, myxoid stroma (A). Calcifying aponeurotic fibroma is dominated by spindled fibroblasts and contains calcified nodules surrounded by more rounded tumor cells (B).

ANGIOMYXOID LESIONS ANGIOMYOFIBROBLASTOMA Angiomyofibroblastoma arises in the vulvovaginal region, shows variable cellularity, and is composed of small round to spindle cells accompanied by small but widely arced vessels (67). Pleomorphism is absent, and the tumor is circumscribed (Fig. 5.25; see Table 5.7 for the differential diagnosis). Unlike deep (“aggressive”) angiomyxoma, with which angiomyofibroblastoma may be confused, it is more vascular and does not have vessels that are open and round. The immunoprofile is similar to that of deep angiomyxoma—it is positive for desmin but negative for muscle-specific actin (MSA) and SMA; thus, the distinction between the two entities must be based on the hematoxylin and eosin (H&E) appearance (68).

FIGURE 5.25 Angiomyofibroblastoma. Small rounded to spindle cells, some stellate in appearance, proliferate in a loose myxoid matrix accompanied by short capillary vessels. The cells are positive for desmin (not shown).

SUPERFICIAL ANGIOMYXOMA Superficial angiomyxoma is a cutaneous and subcutaneous multinodular hypocellular myxomalike tumor that may contain epithelial elements in the form of squamous inclusions. Thin-walled, curved blood vessels and stromal neutrophils are typical features (Fig. 5.26). The recurrence rate is 30% (69,70). Some cases are related to Carney complex (see Table 5.7 and Fig. 5.53 for the differential diagnosis).

FIGURE 5.26 Superficial angiomyxoma. This tumor involves the dermis and subcutaneous tissue and is composed of small spindle cells in a myxoid stroma with thin-walled, curved blood vessels. Note the stromal neutrophils, a characteristic feature observed in half of cases.

DEEP (“AGGRESSIVE”) ANGIOMYXOMA Deep (“aggressive”) angiomyxoma (see Chapter 51) has a predilection for the perineum and pelvis of women, but rarely also occurs in male patients (71); it can be distinguished from angiomyofibroblastoma by its large size, infiltrating border, uniformly hypocellular appearance, and thick rounded vessels (see Table 5.7 and Fig. 5.53 for the differential diagnosis). With only a 30% recurrence rate, it is not as aggressive as originally thought.

FIBROBLASTIC/MYOFIBROBLASTIC NEOPLASMS OF INTERMEDIATE BIOLOGIC POTENTIAL INFLAMMATORY MYOFIBROBLASTIC TUMOR This myofibroblastic neoplasm may be seen in a wide variety of locations in both children and adults but has a predilection for the lung, retroperitoneum, and abdominal cavity (72,73). Typically, inflammatory myofibroblastic tumor is a circumscribed lesion; it contains fascicles of spindle cells proliferating in a background of fibrosis with lymphocytes, plasma cells, histiocytes, and, occasionally, eosinophils and neutrophils (Fig. 5.27A). In some cases, the stroma is sclerotic and hypocellular, whereas in other cases, numerous SMA-positive plump spindle cells with pale eosinophilic cytoplasm are noted. Nuclear pleomorphism and atypical mitoses are absent. A rare, usually intraabdominal variant is dominated by epithelioid cells with amphophilic cytoplasm, often with myxoid stroma and stromal neutrophils (74) (Fig. 5.27B). This aggressive variant is known as epithelioid inflammatory myofibroblastic sarcoma (74). Inflammatory myofibroblastic tumor has the potential for local recurrence, especially intraabdominal lesions (up to 35%); distant metastases develop in fewer than 5% of patients (75). Inflammatory myofibroblastic tumor demonstrates fusions of the ALK gene with a wide range of partners in around 60% of cases (76); these cases are immunoreactive with ALK antibodies (77). Epithelioid inflammatory myofibroblastic sarcoma usually harbors an ALK-RANBP2 fusion and shows a nuclear membrane pattern of ALK staining (74). A small subset of tumors harbors alternate ROS1 gene rearrangements or, rarely, other tyrosine kinase receptor gene fusions (78,79). The

small subset of patients whose tumors pursue an aggressive clinical course may be treated with tyrosine kinase inhibitors such as crizotinib (80).

FIGURE 5.27 Inflammatory myofibroblastic tumor. In a tumor sprinkled with lymphocytes and plasma cells, fascicles of eosinophilic spindle cells with vesicular nuclei are seen (A). Pleomorphism and necrosis are notably absent, and mitotic activity is low. Rare tumors are dominated by polygonal cells with amphophilic cytoplasm and prominent nucleoli, embedded within a myxoid stroma often containing neutrophils (B). This aggressive variant, known as epithelioid inflammatory myofibroblastic sarcoma, usually arises within the abdomen. A nuclear membrane pattern of ALK staining is typical (not shown).

FIBROMATOSIS Several fibromatoses occur in adults, mainly in two distinct forms (81,82). First, lesions occurring on the hands and feet (palmar and plantar, respectively) appear as contractures with small nodules; cellular fibrotic lesions with variable amounts of collagen are seen among the tendinous tissue. Although they are frequently multinodular, a single dominant nodule may be present. The lesions are rarely misdiagnosed. Unlike the proliferation in scars, proliferation in fibromatosis is uniform and relatively hypovascular, and it lacks hemosiderin. Although the palmar and plantar fibromatoses may recur locally after excision, they are localized lesions without the same potential for destructive growth and infiltration that is seen in desmoid tumors. The second, or desmoid, type of fibromatosis presents clinically as a large mass, often on the abdomen or trunk. Long, sweeping fascicles and a uniformly cellular appearance are typical (Fig. 5.28). The fibroblasts in one plane are bipolar with attenuated cytoplasm and a thin, oval, pointed nucleus. In another plane, the cells appear polygonal or stellate, and the periphery of the cytoplasm appears to merge imperceptibly with the surrounding collagen. Infiltration of the surrounding tissues is typical. As a rule, the cells in the fibromatoses are evenly spaced, and the overall cellularity is low to intermediate. In other words, cellularity is never marked, and the cells rarely touch one another. Mitoses are usually rare. By IHC, SMA is typically positive. Aberrant nuclear β-catenin is a helpful finding to support the diagnosis (Fig. 5.28B), particularly in core biopsy samples, but it is neither completely sensitive nor specific. Mesenteric desmoid fibromatosis is often more myxoid, somewhat resembling NOF. In FAP (Gardner syndrome), desmoid fibromatosis is often intraabdominal and may be multifocal. Sporadic desmoid fibromatosis harbors CTNNB1 (β-catenin) gene mutations (83).

FIGURE 5.28 Desmoid fibromatosis. Fibroblasts proliferate on a continuous bed of collagen, with each cell separated from the others. In most cells, the cytoplasm is inapparent (A). Aberrant nuclear staining for β-catenin is a helpful diagnostic feature (B).

Fibromatosis often recurs, but if it is left alone, it may stop growing or even regress. The particular CTNNB1 mutation may predict recurrence (84). There is a recent trend toward a “watchful waiting” approach, reserving surgery for symptomatic patients and those whose tumors continue to grow. Congenital and infantile forms of fibromatosis are highly cellular and mitotically active, and these may mimic fibrosarcoma (85). SOLITARY FIBROUS TUMOR SFT was originally reported as localized fibrous mesothelioma (86). SFT is anatomically ubiquitous; such tumors also arise in the head and neck, retroperitoneum, abdominal cavity, and somatic soft tissues. SFT belongs to the category of fibroblastic tumors. The tumor formerly known as lipomatous hemangiopericytoma (HPC) is now known to represent SFT with adipocytic differentiation (87,88), and, indeed, nearly all tumors that were previously diagnosed as HPC are now considered (uniformly cellular) SFT. SFT is composed of nondescript bland and uniform spindle

cells dispersed among elongated, thin, parallel collagen bands in a “patternless” pattern, with scattered thin-walled, dilated, branching (“staghorn”) vessels (Fig. 5.29) (89). Around 95% of SFTs are positive for CD34 (294). SFT harbors a pathognomonic NAB2-STAT6 gene fusion (resulting from an inversion) (90-92). This specific genetic signature of SFT results in nuclear expression of the STAT6 protein (Fig. 5.29C), which can be detected by IHC (93). Although the behavior of SFT was historically believed to be unpredictable, recent studies have established a risk stratification system helpful for prognostication, based on patient age, tumor size, necrosis, and mitotic activity (94). Low-risk SFT is essentially benign, whereas high-risk tumors behave as intermediate- to high-grade sarcomas. In rare cases, SFT may show an abrupt transformation to a nondistinctive pleomorphic appearance; such tumors, designated dedifferentiated SFT, pursue a highly aggressive clinical course (95).

FIGURE 5.29 Solitary fibrous tumor. Bland, nondescript spindle cells are dispersed within sclerotic collagen in a haphazard (“patternless”) pattern (A). Note the dilated, thin-walled blood vessel. Some cases are more uniformly cellular, with a vaguely storiform architecture (B). There is perivascular collagen deposition. Nuclear reactivity for STAT6 is a highly sensitive and specific feature (C), a consequence of the NAB2-STAT6 fusion.

MYOFIBROBLASTOMAS PALISADED MYOFIBROBLASTOMA Previously known as intranodal hemorrhagic spindle tumor with amianthoid fibers, palisaded myofibroblastoma is nearly exclusively found in inguinal lymph nodes (96,97). It is composed of a spindle cell proliferation replacing the substance of a lymph node and is accompanied by

hemorrhage and irregular knots of collagen bundles (the amianthoid fibers with a crystalline appearance). The spindle cells have elongated nuclei that often show nuclear palisading similar to that of a nerve sheath tumor and eosinophilic cytoplasm (Fig. 5.30). This tumor type may mimic Kaposi sarcoma and schwannoma, but HHV8 and S-100 protein are negative. Palisaded myofibroblastoma harbors consistent CTNNB1 gene mutations; IHC for β-catenin, therefore, shows nuclear staining (98). These tumors are benign and do not recur.

FIGURE 5.30 Palisaded myofibroblastoma. This intranodal tumor resembles a schwannoma, with tapering nuclei and nuclear palisading (present elsewhere); the crystal-like amianthoid fibers are characteristic.

MAMMARY-TYPE MYOFIBROBLASTOMA Because “mammary-type” myofibroblastomas most often arise in the inguinal region (not the breast), this tumor type is now designated simply myofibroblastoma; such tumors may also arise in the trunk, extremities, and central body cavity sites. Myofibroblastoma is composed of fascicles of bland, short spindle cells with prominent stromal collagen and a variable adipocytic component (Fig. 5.31) (99-101). Expression of both desmin and CD34 is typical. This tumor shares genetic features (loss of 13q14) with spindle cell lipoma and cellular angiofibroma (55), and loss of RB1 (retinoblastoma) protein expression (24). Myofibroblastoma is benign and rarely recurs.

FIGURE 5.31 Myofibroblastoma. Also known as “mammary-type” myofibroblastoma, this tumor has a variable adipocytic component, and is composed of fascicles of bland spindle cells with a collagenous stroma. Co-expression of CD34 and desmin is typical (not shown).

OSSIFYING FIBROMYXOID TUMOR Ossifying fibromyxoid tumor (OFMT) most often arises in the subcutis, but may also arise in skeletal muscle of the extremities (102). More than 80% of cases have an incomplete shell of mature bone. Small rounded cells are arranged in cords and strands embedded in a loose fibromyxoid stroma (Fig. 5.32). Two-thirds of cases show S-100 protein positivity; approximately half express desmin. OFMT harbors PHF1 gene rearrangements in most cases (103-105). Typical OFMT has a low but unpredictable risk of local recurrence and rarely metastasizes, sometimes decades following excision of the primary tumor (102). Atypical and malignant variants have been reported (the latter with hypercellularity, high nuclear grade, and greater than two mitoses per 50 high-power fields [hpf]) (106); malignant OFMT behaves as a sarcoma with significant metastatic potential.

FIGURE 5.32 Ossifying fibromyxoid tumor. Strings, cords, and clusters of cells with modest eosinophilic cytoplasm are found in a fibromyxoid matrix. Not shown is the bone typically seen in the capsule.

FIBROSARCOMAS LOW-GRADE MYOFIBROBLASTIC SARCOMA Low-grade myofibroblastic sarcoma is composed of fascicles and sheets of spindle cells with tapering nuclei and pale eosinophilic cytoplasm, often with a markedly infiltrative growth pattern into adjacent skeletal muscle (107,108). Such tumors have a predilection for the extremities and head and neck, especially the tongue. Variable expression of SMA, and often desmin, is typical. Such tumors may closely mimic desmoid fibromatosis (although they have notable nuclear atypia) and LMS (although they lack the alternating fascicular pattern of a smooth muscle neoplasm) (Fig. 5.33). Low-grade myofibroblastic sarcoma is locally aggressive, but rarely metastasizes.

FIGURE 5.33 Low-grade myofibroblastic sarcoma. This cellular, fascicular spindle cell tumor shows mild nuclear atypia and an infiltrative margin. This example arose on the tongue (a typical site) and invades skeletal muscle.

MYXOINFLAMMATORY FIBROBLASTIC SARCOMA This tumor type occurs mainly on the distal extremities, especially the hands (109-111). The histology may mimic an inflammatory process or myxofibrosarcoma. This distinctive tumor type usually has the following constellation of morphologic findings: (1) an infiltrative subcutaneous tumor with a multinodular appearance, (2) areas of dense fibrosis with prominent chronic inflammation, (3) myxoid areas with both spindled and epithelioid cells, (4) scattered pleomorphic cells resembling Reed-Sternberg cells with prominent eosinophilic nucleoli (“virocytes”), and (5) mucin-filled cells (pseudolipoblasts) in the myxoid areas with a basophilic wispy cytoplasm similar to the extracellular matrix (Fig. 5.34). There are no helpful IHC markers. A characteristic translocation t(1;10)(p22;q24) has been identified in some cases; others harbor BRAF fusions (see Table 5.3) (112-114). Multiple local recurrences are common, seen in more than 50% of patients, but distant metastases are rare (111).

FIGURE 5.34 Myxoinflammatory fibroblastic sarcoma. An admixture of myxoid and fibroinflammatory areas is typical. Lymphocytes often predominate, but a wide range of inflammatory cells may be observed. The characteristic large mononuclear tumor cells contain inclusion-like nucleoli (“virocyte-like” or “Reed-Sternberg–like” cells).

MYXOFIBROSARCOMA The term myxofibrosarcoma was originally applied to low-grade lesions (115), but it has come to encompass cases previously termed myxoid MFH as well (see Table 5.7 and Fig. 5.34) (116). Myxofibrosarcoma is much more locally aggressive than are most other sarcomas, and shows markedly infiltrative margins along fascial planes. It often affects elderly adults, frequently arises in superficial soft tissues of the limbs and limb girdles, and typically exhibits a lobulated growth pattern with distinctive histologic features, ranging from hypocellular myxoid tumors to highly cellular, pleomorphic sarcomas (117). The lower grade myxoid component with curvilinear vessels and scattered pleomorphic cells must be identified to diagnose high-grade myxofibrosarcomas properly. The pleomorphic tumor cells in the myxoid areas often have a stellate or tripolar shape (see Fig. 5.35). The distinctive blood vessels of myxofibrosarcoma are different from those of myxoid liposarcoma, being of thicker caliber and often curved with a wide arc; the often closely adherent tumor cells add to their thick appearance at low power (see Fig. 5.50 for comparison with other myxoid tumors). Another differentiating feature is the presence of nonmyxoid UPS-like elements in many cases. There is no minimal requirement for the extent of myxoid areas, so long as the typical low-grade component is identified (117). Lowgrade myxofibrosarcoma (lacking solid, cellular areas) rarely metastasizes, whereas intermediate- or high-grade tumors have a significant metastatic risk (20%-35%). Of note, local recurrences often progress to higher histologic grade. Unlike other pleomorphic sarcomas, myxofibrosarcoma sometimes metastasizes to lymph nodes. This is one of the few sarcoma types that still sometimes necessitates amputation, after several aggressive local recurrences.

FIGURE 5.35 Myxofibrosarcoma. This myxoid neoplasm contains dispersed, markedly atypical and pleomorphic cells and curvilinear blood vessels. The vessels are longer and thicker than those of myxoid liposarcoma (see Fig. 5.53).

There is an epithelioid variant of myxofibrosarcoma containing epithelioid cells surrounded by varying amounts of myxoid matrix, growing in sheets, with pleomorphic cells; this variant recalls a differential diagnosis of melanoma, carcinoma, and other epithelioid tumors, and has a worse prognosis than do other high-grade myxofibrosarcomas, with a greater than 50% metastatic rate (118). FIBROSARCOMA The term fibrosarcoma is used for two tumor types: infantile fibrosarcoma and adult fibrosarcoma (119,120). Infantile fibrosarcoma typically arises in infants and young children as a rapidly growing, often large mass in the extremities or trunk wall; this locally aggressive tumor type is composed of intersecting, highly cellular fascicles of primitive-appearing, mitotically active spindle cells; most tumors harbor an ETV6-NTRK3 rearrangement, although recently alternate gene fusions have been identified (e.g., NTRK1, BRAF, or MET) (121-123). FISH is most often used to confirm the diagnosis; IHC using pan-TRK antibodies is an alternative strategy, although such markers are not entirely specific for tumors with NTRK rearrangements (124,125). In the past, tumors in adults labeled as fibrosarcomas constituted a high percentage of soft issue sarcomas. More recently, the total number of cases with this designation has decreased precipitously for several reasons (126). First, pleomorphic tumors in adults are now called undifferentiated pleomorphic sarcoma (UPS). Second, with the use of IHC and molecular genetic analysis, it has become apparent that several distinct tumor types may mimic fibrosarcoma in adults, particularly MPNST, monophasic synovial sarcoma, and the fibrosarcomatous variant of DFSP. Adult fibrosarcoma is a diagnosis of exclusion; true examples are exceedingly rare. After excluding mimics, this designation should be limited to lesions with the following characteristics: (1) a highly cellular, fascicular spindle cell appearance; (2) a herringbone pattern in which fascicles of cells intersect at acute angles (Fig. 5.36); and (3) an absence of pleomorphism.

FIGURE 5.36 Fibrosarcoma. Highly cellular streams of nuclei intersect at acute angles, giving rise to the nonspecific “herringbone” pattern. This is an exceptionally rare tumor type in adults, a diagnosis of exclusion. Synovial sarcoma and malignant peripheral nerve sheath tumor should be considered when this pattern is seen.

It has recently been recognized that a range of spindle cell neoplasms other than infantile fibrosarcoma also harbor NTRK fusions (provisionally designated NTRK-rearranged spindle cell neoplasms in the 2020 WHO classification) (2), as well as fusions of other genes such as RAF1 and BRAF; NTRK1 fusions are most frequent (127). These tumors are most common in children and young adults, arise in the extremities and trunk wall, and show a spectrum of morphologic patterns, ranging from moderately hypercellular tumors with monomorphic, bland nuclear morphology and an infiltrative growth pattern, to highly cellular, sheetlike tumors, often with bands of stromal collagen and perivascular hyalinization; some examples show a neural-like appearance (Fig. 5.37) (127,128). Co-expression of CD34 and S-100 protein is characteristic; pan-TRK is also positive (in the subset with NTRK fusions) with a cytoplasmic or nuclear pattern, and SOX10 is negative. The clinical behavior is variable: cytologically uniform and bland tumors have recurrent potential, whereas some examples with clearly malignant histology metastasize; however, firm criteria for malignancy in these tumors have yet to be defined.

FIGURE 5.37 NTRK-rearranged spindle cell neoplasm. Mesenchymal neoplasms with NTRK gene fusions often show a neural-like appearance. Note the perivascular hyalinization. Immunohistochemistry using pan-TRK

antibodies is strongly positive (not shown). Firm criteria for malignancy have not yet been established for this group of tumors.

LOW-GRADE FIBROMYXOID SARCOMA This deceptively bland fibroblastic sarcoma is associated with a significant risk of late local recurrence and distant metastasis to the lungs and pleura (in up to 40% of cases), often decades following primary tumor excision (129,130). Low-grade fibromyxoid sarcoma characteristically shows abrupt demarcation between a cellular component with bland and uniform, slender spindle cells with wavy nuclei arranged in a storiform-to-whorled growth pattern and a hypocellular myxoid component containing arcades of thin-walled blood vessels (Fig. 5.38).

FIGURE 5.38 Low-grade fibromyxoid sarcoma. At medium power, a giant collagen rosette is surrounded by tumor cells with small nuclei. Adjacent spindle cells lack pleomorphism and exhibit a vague storiform pattern.

Around 20% of cases contain giant collagen rosettes, collagenous nodules surrounded by epithelioid tumor cells (131). Occasional cases contain areas of sclerosing epithelioid fibrosarcoma (see later). Low-grade fibromyxoid sarcoma harbors a translocation t(7;16) (q34;p11) or t(11;16)(p11;p11), resulting in FUS-CREB3L2 (>90%) or FUS-CREB3L1 gene fusions; EWSR1 fusions are rare (132,133). Epithelial membrane antigen (EMA) is positive in up to 80% of cases, potentially leading to diagnostic confusion with soft tissue perineurioma, which is also a histologic mimic. MUC4 is a highly sensitive and specific IHC marker for this tumor type (134). Reactivity for MUC4 is helpful to distinguish low-grade fibromyxoid sarcoma from soft tissue perineurioma. SCLEROSING EPITHELIOID FIBROSARCOMA Sclerosing epithelioid fibrosarcoma typically arises in the deep soft tissues of the lower extremities of middle-aged adults, although the anatomic distribution is broad; some tumors arise in the abdominal cavity (135,136). This tumor type is composed of uniform epithelioid cells with abundant pale eosinophilic or clear cytoplasm, arranged in cords and nests within a densely hyalinized collagenous stroma (Fig. 5.39); some examples contain a minor fascicular, spindle cell component. A subset of cases of sclerosing epithelioid fibrosarcoma show areas of low-grade fibromyxoid sarcoma (132). By IHC, EMA or SMA is positive in around 40% of cases; most cases of sclerosing epithelioid fibrosarcoma (around 80%) are positive for MUC4 (134). The most common gene fusion is EWSR1-CREB3L1 (137). Sclerosing epithelioid fibrosarcomas containing a component of low-grade fibromyxoid sarcoma often harbor FUS-CREB3L2 fusions,

and may represent a form of tumor progression (132). Recent studies have shown that many MUC4-negative cases harbor fusions between YAP1 and KMT2A (138). Sclerosing epithelioid fibrosarcoma is an aggressive sarcoma type with both local recurrence and distant metastasis in around 50% of cases (139).

FIGURE 5.39 Sclerosing epithelioid fibrosarcoma. Epithelioid cells with pale cytoplasm are arranged in cords and nests within an abundant hyalinized collagenous stroma. This tumor type closely mimics metastatic carcinoma. Most cases are positive for MUC4 (not shown).

FIBROHISTIOCYTIC LESIONS It is now accepted that tumors in this category are not related to a true histiocytic lineage (monocyte/macrophage bone marrow–derived cell), but rather are probably myofibroblastic in lineage. However, given the widespread use of the fibrohistiocytic term, this group of tumors is described together in this section, as well as the undifferentiated neoplasms previously believed to be “fibrohistiocytic” in type. BENIGN FIBROUS HISTIOCYTOMA (DERMATOFIBROMA) Benign fibrous histiocytoma (BFH) is the term used most often by soft tissue pathologists, whereas dermatofibroma is preferred by dermatopathologists. BFH usually presents as a small dermal nodule at almost any age. Key diagnostic features are notable at low magnification: overlying epidermal hyperplasia and lateral entrapment of hyaline dermal collagen. The architecture may be storiform or more haphazard; in conventional BFH, the tumor cells are admixed with prominent inflammatory cells, chiefly lymphocytes and histiocytes, although hypocellular, sclerotic lesions may be devoid of inflammatory cells. The spindle cells have bland elongated nuclei with fine chromatin and scant pale eosinophilic cytoplasm. Conventional BFH is rarely a diagnostic challenge. However, the cellular variant may be mistaken for DFSP or even a spindle cell sarcoma. Cellular BFH is a more uniformly cellular lesion with a variably storiform-tofascicular architecture with less inflammation; in such cases, attention to the lateral collagen entrapment and epidermal hyperplasia is key to recognition. Large examples often extend into underlying subcutaneous adipose tissue, but they lack the diffusely infiltrative growth and “honeycomb” pattern of DFSP. Conventional BFH rarely recurs, whereas cellular BFH has a 15% to 20% rate of nondestructive local recurrence if incompletely excised (140). Patchy staining for

SMA is common; CD34 is positive in around 5% of cases, rarely extensively. Deep fibrous histiocytoma is a rare variant that arises in subcutaneous tissue or deep soft tissue (141); such tumors are relatively circumscribed, but otherwise show histologic appearances similar to that of cutaneous BFH; CD34 is more often expressed by deep fibrous histiocytoma. Related lesions include atypical fibrous histiocytoma and aneurysmal fibrous histiocytoma (Fig. 5.40) (142,143). A subset of BFH and variants harbor gene fusions involving protein kinase C (PRKCB and PRKCD) (144). Epithelioid fibrous histiocytoma is unrelated to these other variants; this tumor type is usually polypoid, well-circumscribed, harbors ALK gene fusions, and is positive for ALK by IHC (145).

FIGURE 5.40 Aneurysmal variant of benign fibrous histiocytoma (dermatofibroma). Below a hyperplastic epidermis, a dermal spindle cell lesion contains lakes of blood.

ATYPICAL FIBROUS HISTIOCYTOMA Atypical fibrous histiocytoma is distinguished by the presence of scattered atypical and pleomorphic cells (142). Again, recognition of overlying epidermal hyperplasia and lateral collagen entrapment is critical. In contrast to AFX (see later), atypical fibrous histiocytoma commonly presents on the extremities of young to middle-aged adults, rather than on sunexposed areas, including the head and neck, of older patients. The recurrence rate of atypical fibrous histiocytoma is similar to that of cellular BFH. PLEXIFORM FIBROHISTIOCYTIC TUMOR In this deep dermal and subcutaneous tumor of predominantly female children and young adults, scattered nodules of histiocytoid cells and osteoclastic giant cells are variably surrounded by fascicles of (SMA-positive) myofibroblastic spindle cells (Fig. 5.41) (146,147). Some cases are dominated by histiocytoid nodules, whereas other cases are almost exclusively composed of bands of spindle cells. This neoplasm belongs to the group of intermediate, rarely metastasizing tumors. The local recurrence rate is 20% to 35%, whereas lymph node metastases are rare (146,147).

FIGURE 5.41 Plexiform fibrohistiocytic tumor. Nodules of histiocytoid cells and occasional osteoclastic giant cells infiltrate the dermis and subcutaneous tissue. Fibromatosis-like fascicles of myofibroblastic spindle cells may be dispersed between the nodules (not shown).

DERMATOFIBROSARCOMA PROTUBERANS DFSP is discussed elsewhere in this book (see Chapter 2). It is the storiform tumor par excellence, with every field showing this characteristic pattern (Fig. 5.42). Other tumors may also show this pattern. Although the tumor is highly cellular, the nuclei are remarkably uniform with slender, hyperchromatic nuclei; cytoplasm is scant. In the conventional form, mitoses are difficult to find. A diffusely infiltrative growth pattern within the dermis and subcutis (imparting the classic “honeycomb” pattern in fat) is characteristic, distinguishing DFSP from cellular BFH. DFSP has a high risk of local recurrence, but does not metastasize in its conventional form (352). The myxoid variant is a challenge to recognize (148). DFSP may also be pigmented (“Bednar tumor”), and it may progress to a fibrosarcoma-like or UPS-like sarcoma (149-151).

FIGURE 5.42 Dermatofibrosarcoma protuberans. The cartwheel or storiform pattern without pleomorphism is typical of dermatofibrosarcoma. Note the thin nuclei, lack of perceptible cell borders, and overall tight quality of the pattern (A). This tumor type often shows plaque-like growth in the dermis and prominent layered invasion of the subcutaneous fat (B).

Transformation to the fibrosarcomatous variant is accompanied by acquisition of metastatic potential; 10% to 15% of fibrosarcomatous DFSP metastasize to the lungs (149). This tumor type is a challenging diagnosis without the presence of a component of conventional DFSP; the diagnosis should always be favored for a fibrosarcoma-like tumor of the superficial soft tissues. The fibrosarcomatous component is highly cellular and fascicular with vesicular nuclei and a higher mitotic rate (Fig. 5.43); CD34 expression may be partially or completely lost.

FIGURE 5.43 Fibrosarcomatous dermatofibrosarcoma protuberans. The fibrosarcomatous variant does not resemble conventional dermatofibrosarcoma, instead showing a fascicular architecture with vesicular nuclei and increased mitotic activity.

FISH for PDGFB can be used to confirm the diagnosis; DFSP (including the fibrosarcomatous variant) harbors COL1A1-PDGFB rearrangements (see Table 5.3) (152). Because of the pathogenetic gene fusion, patients with uncontrolled local recurrences and metastases of DFSP may be treated with imatinib mesylate (153). GIANT CELL FIBROBLASTOMA Giant cell fibroblastoma is a rare cutaneous fibroblastic tumor that occurs mainly in boys younger than 10 years of age (154). The lesions may contain collagenous or myxoid stroma with a haphazard arrangement of small spindle cells and widely scattered atypical and multinucleated cells; a characteristic feature is the presence of pseudovascular (“angiectoid”) spaces, lined by tumor cells (Fig. 5.44). Approximately half of patients experience local recurrence, but this tumor type does not metastasize. This lesion has acquired the designation childhood DFSP because occasional cases show zones consistent with the storiform adult tumor, adult DFSP may contain giant cell fibroblastoma–like areas, and the same gene fusion is detected (155).

FIGURE 5.44 Giant cell fibroblastoma. Tumor cells including multinucleated forms are scattered within a collagenous stroma. Dilated pseudovascular spaces are lined by hyperchromatic tumor cells.

UNDIFFERENTIATED PLEOMORPHIC SARCOMA UPS is one of the most common sarcoma types; UPS, myxofibrosarcoma, and DD-LPS are the most common pleomorphic sarcomas. The thigh is the most common location; this tumor type is rarely encountered outside of the extremities and trunk wall. Retroperitoneal and abdominal cavity sarcomas with similar morphology are nearly all DD-LPSs. The histologic appearances of UPS are not distinctive. The tumor often shows a variably storiform and sheetlike architecture and is composed of a combination of spindled, epithelioid, and bizarre pleomorphic and multinucleated tumor cells with marked nuclear atypia (coarse chromatin and often large nucleoli), moderate amounts of pale eosinophilic cytoplasm, and a high mitotic rate (Fig. 5.45). Some tumors contain prominent inflammation or osteoclastic giant cells. In the past, pleomorphic sarcomas with prominent neutrophils and foam cells were designated “inflammatory MFH” (Fig. 5.45D); it is now recognized that most examples of inflammatory MFH are in fact DD-LPSs (156). IHC for MDM2 and CDK4 or FISH for MDM2 amplification can be used to confirm the diagnosis of DD-LPS. UPS is a diagnosis of exclusion: before reaching such a diagnosis, the possibility of high-grade myxofibrosarcoma, pleomorphic LMS, pleomorphic RMS, and pleomorphic liposarcoma should be excluded. High-grade myxofibrosarcoma can be diagnosed on finding the lower grade component with abundant myxoid stroma and curvilinear blood vessels. Pleomorphic LMS often contains areas of conventional LMS with the characteristic fascicles of spindle cells with broad, blunt-ended nuclei and bright eosinophilic cytoplasm; IHC for SMA, desmin, and caldesmon is helpful to support the diagnosis. The cells of pleomorphic RMS are often variably epithelioid to pleomorphic and contain abundant bright eosinophilic cytoplasm; desmin, myogenin, and MyoD1 staining can confirm the diagnosis. Pleomorphic liposarcoma is indistinguishable from UPS other than the presence of lipoblasts, that is, vacuolated cells in which the clear vacuoles indent or scallop the hyperchromatic nucleus.

FIGURE 5.45 Undifferentiated pleomorphic sarcoma (UPS). At medium power, pleomorphic nuclei are scattered among smaller cells growing in a vaguely storiform pattern (A). At high power, bizarre nuclei in huge cells are often found, and they may be accompanied by an inflammatory infiltrate (B). In many cases, the smaller spindle cells exhibit irregular nuclear shapes; note also the lack of well-defined cell borders (C). UPS with prominent inflammation (inflammatory “malignant fibrous histiocytoma”) (D). In contrast to a cluster of benign foamy histiocytes, the malignant nuclei of this tumor are larger and vesicular, but not bizarre. They are immersed in a sea of neutrophils and other inflammatory cells. Most tumors with this pattern are dedifferentiated liposarcomas.

AFX is covered in detail in Chapter 2. This tumor type is restricted to sun-damaged skin of the elderly, most often presenting on the face or scalp. By definition, AFX is restricted to the dermis; this tumor type often grows rapidly, ulcerates the skin, and sometimes shows an epidermal collarette. The lower aspect of the tumor is well circumscribed; lymphovascular invasion and perineural invasion are absent. Pleomorphic cells are apparent at low power, but there is great variability in cytomorphology, often including spindle cells and epithelioid cells as well (Fig. 5.46). The mitotic rate may be very high, and atypical forms are often seen. There are no specific IHC markers for AFX. It is important to exclude sarcomatoid squamous cell carcinoma and melanoma before making such a diagnosis. Spindle cell squamous carcinoma tends to have cells that are plumper and more eosinophilic, with focal nesting (Fig. 5.1). Keratin expression may be limited in extent; multiple broad-spectrum (and high-molecular-weight) keratin antibodies may need to be applied. With melanoma, attention to the epidermal surface (searching for a junctional or intraepidermal component) and the presence of separated bundles of cells are helpful features. AFX is negative for S-100 protein and SOX10, although sometimes prominent admixed S-100– positive dendritic cells can be identified. AFX is a pseudosarcoma that does not metastasize. Tumor invading into the subcutaneous tissue or deeper should be diagnosed as pleomorphic dermal sarcoma (157). The term superficial UPS is confusing to clinicians and should be avoided. Pleomorphic dermal sarcomas have relatively low metastatic potential, only 10% to 15%.

FIGURE 5.46 Atypical fibroxanthoma (AFX). In contrast to spindle cell carcinoma (Fig. 5.1), the cell shape and size vary markedly in this lesion. The smaller cells of AFX lack prominent cell borders and have oval or angulated and bent nuclei, unlike those of most carcinomas.

Pleomorphic hyalinizing angiectatic tumor is a rare pleomorphic neoplasm that may be confused with pleomorphic sarcomas and myxofibrosarcoma (158). This tumor type also contains bizarre cells; however, it is well circumscribed, often with sclerotic stroma, and contains characteristic dilated blood vessels with mural fibrinoid change. Mitotic activity is generally absent. This diagnosis should be made with great care; pleomorphic sarcomas may also contain similar vessels. GIANT CELL TUMORS INCLUDING UNDIFFERENTIATED PLEOMORPHIC SARCOMA WITH GIANT CELLS Giant cell–rich tumors of soft tissue constitute a group of neoplasms ranging from benign to malignant (159,160). Osteoclastic giant cells are a dominant feature. Giant cell tumor of soft tissue usually arises in the superficial tissues of the extremities or trunk wall of adults. These circumscribed lesions show a multinodular architecture with intervening fibrotic septa containing hemosiderin; 50% of cases show metaplastic ossification. The tumor is composed of osteoclastic giant cells and bland, mononuclear histiocytoid or short spindle cells (Fig. 5.47). The giant cells have many (5-20 or more) nuclei; all the nuclei or nuclear lobes are small, bland, and round to oval without nuclear atypia or pleomorphism. Mitotic activity is often brisk, and vascular invasion is common. Despite these worrisome features, the local recurrence rate is only 15%, and metastases are rare. As mentioned previously, UPS may occasionally contain prominent osteoclastic giant cells; in such cases, the tumor cells show marked nuclear atypia and pleomorphism, in contrast to giant cell tumor of soft tissue. LMSs may also contain notable osteoclastic giant cells (161). Metastatic undifferentiated carcinoma (e.g., from the pancreas) should also be excluded.

FIGURE 5.47 Giant cell tumor of soft tissue. The tumor shows a multinodular architecture; the nodules are composed of mononuclear cells and osteoclastic giant cells. The appearances are similar to those of giant cell tumor of bone.

ANGIOMATOID FIBROUS HISTIOCYTOMA Angiomatoid fibrous histiocytoma is a mesenchymal neoplasm of intermediate biologic potential (rarely metastasizing), not a sarcoma (162,163). This often cystic and hemorrhagic tumor most often occurs in the subcutaneous tissue of the extremities (especially antecubital and popliteal fossae), with a peak incidence in children and young adults. This tumor type may mimic a lymph node, owing to the often-striking lymphoid cuff (Fig. 5.48). Histologically, sheets and nodules of histiocytoid or somewhat myoid-appearing spindle cells with ovoid nuclei, small nucleoli, and eosinophilic cytoplasm with a syncytial appearance are seen within the walls of the cysts, surrounded by lymphoid follicles. Pleomorphism is rare. By IHC, desmin, EMA, CD99, and CD68 are variably positive, each in around 50% of cases. Angiomatoid fibrous histiocytoma most often harbors an EWSR1-CREB1 fusion; occasional examples harbor EWSR1-ATF1 or FUS-ATF1 fusion genes (164,165). Cutaneous aneurysmal BFH is unrelated (166). Angiomatoid fibrous histiocytoma recurs locally in 15% to 20% of cases, and rarely metastasizes (2%-3%), most often to regional lymph nodes.

FIGURE 5.48 Angiomatoid fibrous histiocytoma. At low power, a key feature is the dark cuff of lymphoid cells, often with germinal centers; paler tumor nodules proliferate in the center and at the top left.

LESIONS OF ADIPOSE TISSUE Lipoblastoma, spindle cell lipoma, and pleomorphic lipoma were discussed earlier in this chapter (see Figs. 5.6, 5.7, 5.8). LIPOMA VARIANTS Conventional lipomas are usually easily recognized as circumscribed proliferations of mature adipocytes. Unlike the adipocytes in atypical lipomatous tumor (well-differentiated liposarcoma), the adipocytes in lipoma have lipid vacuoles that are uniform in size. Some lipomas exhibit prominent vascularity, usually at the periphery. These are called angiolipomas, and they are one of the few painful soft tissue tumors (see Table 5.8). Angiolipomas usually affect young adults and may be multifocal, often presenting on the arm as multiple, small, firm subcutaneous nodules. The peripheral vessels often contain fibrin microthrombi. In occasional cases, the vascular component may predominate (see Fig. 5.11), mimicking either a capillary hemangioma or even Kaposi sarcoma. However, the circumscription of the lesion, the scattered adipocytes, and the fibrin microthrombi are helpful distinguishing features. Occasionally, deep vascular tumors are incorrectly called angiolipomas; these are in fact intramuscular angiomas, and they often consist of venous, arterial, and capillary vascular components with associated fat (167). Angiomyolipomas are also unrelated; these tumors contain perivascular epithelioid cells with granular eosinophilic cytoplasm within the walls of large vessels, in addition to the adipose tissue and vascular elements. These tumor cells are positive for HMB-45 and for smooth muscle markers as well. Although it characteristically occurs in the kidney (sometimes as part of the tuberous sclerosis complex), occasional cases have been reported elsewhere. Deep intramuscular forms of lipoma also occur, particularly in the paraspinal region and limb girdles (168). Intramuscular lipomas characteristically show entrapment of skeletal muscle fibers; unlike subcutaneous lipomas, intramuscular tumors frequently recur. TABLE 5.8 Frequently Painful Soft Tissue Tumors Glomus tumor Angiolipoma Traumatic neuroma Angioleiomyoma (vascular leiomyoma) Atypical intradermal smooth muscle neoplasm (formerly “cutaneous leiomyosarcoma”) Clear cell sarcoma Calcified synovial sarcoma

Some cases of hibernoma, a tumor of brown fat with variably multivacuolated and granular cytoplasm (Fig. 5.49), may contain large areas of white fat elements, making the diagnosis challenging and potentially raising concern for liposarcoma (169).

FIGURE 5.49 Hibernoma. This benign adipocytic tumor is composed of an admixture of white fat cells, multivacuolated lipoblast-like cells, and granular eosinophilic cells in varying proportions.

CHONDROID LIPOMA As the name chondroid lipoma suggests, this unusual tumor has two components—mature adipose tissue and cords and nests of eosinophilic vacuolated cells resembling both lipoblasts and chondroblasts (170,171). The tumor is well circumscribed and often has a myxoid matrix (Fig. 5.50). By IHC, the lesion is positive for S-100 protein to variable extent; keratins are rarely positive. A consistent translocation t(11;16)(q13;p13) has been identified (172). The differential diagnosis may include liposarcoma and extraskeletal myxoid chondrosarcoma. Chondroid lipomas are benign and rarely recur.

FIGURE 5.50 Chondroid lipoma. The combination of mature adipocytes with clusters of less differentiated cells in a loose chondroid matrix is typical.

LIPOSARCOMAS

The term liposarcoma applies to four (or five) distinct tumor types: (1) atypical lipomatous tumor (ALT)/well-differentiated liposarcoma (WD-LPS) and the related tumor dedifferentiated liposarcoma (DD-LPS) (173-175); (2) myxoid liposarcoma (including tumor formerly known as round cell liposarcoma); (3) pleomorphic liposarcoma; and (4) the recently described myxoid pleomorphic liposarcoma. ATYPICAL LIPOMATOUS TUMOR Atypical lipomatous tumor (ALT), a synonym for well-differentiated liposarcoma (WD-LPS), is the term applied to tumors of the extremities and trunk wall, for which curative surgery is relatively straightforward. However, central body cavity (such as retroperitoneal and abdominopelvic) tumors are designated well-differentiated liposarcoma. This common tumor type cannot metastasize (Table 5.9). Even for peripheral tumors, clinicians should be alerted to the significant potential for local recurrence, and complete excision should be encouraged; there is a small risk of dedifferentiation for extremity tumors. The distinction between ALT, pleomorphic lipoma, and ASCLT is discussed earlier in the chapter. TABLE 5.9 Liposarcomas: Subtypes and Natural History ALT/WD-LPS

DD-LPS

Myxoid

High-Grade Myxoid (“Round Cell”)

Pleomorphic

Relative Percentage

45%

15%

25%

10%

5%

Average Age (yr)

55

65

45

45

60

Sites

Retroper; extremities; inguinal region

Retroper

Extremities (thigh)

Extremities (thigh)

Extremities

Other

Wide local excision needed

Multiple local recurrences

Multiple local recurrences

Aggressive clinical course

Aggressive clinical course

Local Recurrence

15%-60% (depends on site)

80%

40%-50%

80%

50%

Metastases

0

15%-20%

20%-30%

High rate

50%

5 yr

70%-85% (retroper)

70%

60%-80%

20%-40%

30%-50%

10 yr

50% (retroper)

20%

50%-60%

10%-20%

20%

Survival

ALT/WD-LPS, atypical lipomatous tumor/well-differentiated liposarcoma; DD-LPS, dedifferentiated liposarcoma; retroper, retroperitoneum.

Histologically, ALT/WD-LPS is usually easily recognized as adipocytic in nature, being dominated by adipocytes; variation in adipocyte size and thickened fibrous septa are characteristic features. The septa may be cellular, and they contain small spindle cells and occasional enlarged, pleomorphic cells, a feature that may be noted at low power. Lipoblasts can be identified by the lipid vacuoles indenting the nucleus (the definition of a lipoblast) (Fig. 5.51). Lipoblasts are neither necessary nor sufficient to make the diagnosis. Two other histologic

variants of WD-LPS deserve mention. The sclerosing variant (observed in the retroperitoneum and spermatic cord) is characterized by scattered pleomorphic cells within a hypocellular, fibrillary collagenous stroma (Fig. 5.51C); adipocytic differentiation may be limited (or even absent). The inflammatory variant (also typically seen in the retroperitoneum) can be quite difficult to recognize and is characterized by a dense chronic inflammatory infiltrate, often obscuring the adipocytic component; the presence of occasional pleomorphic cells is a helpful diagnostic clue. It is common for an individual tumor to contain an admixture of these variants and the more common adipocytic (lipoma-like) variant. ALT/WD-LPS usually harbors ring and giant marker chromosomes; these structures contain amplified material from chromosome 12q13~15, including the genes MDM2, CDK4, and others (176,177). IHC for MDM2 and CDK4 can, therefore, be used to support the diagnosis (178), although available antibodies are difficult to optimize; many practices prefer FISH for MDM2 amplification. A potential diagnostic pitfall is the common labeling of histiocytes for MDM2 in foci of microscopic fat necrosis in conventional lipomas.

FIGURE 5.51 Well-differentiated liposarcoma (atypical lipomatous tumor). Fibrous septa with more cells than in ordinary fibrous tissue (cellular septa) are often found in this tumor (A). At high power, pleomorphic nuclei are widely scattered among fat cells of variable size (B). The sclerosing variant contains fibrillary collagen, within which scattered hyperchromatic cells are dispersed (C). In this variant, an adipocytic component may be very limited (or entirely absent). A well-differentiated liposarcoma on the exterior of the body is referred to as an atypical lipomatous tumor, for which complete excision should be attempted.

Dedifferentiation in ALT/WD-LPS is usually characterized by an abrupt transition to solid tumor foci without adipocytes. DD-LPS is characterized by marked intratumoral heterogeneity, often including nondistinctive, highly pleomorphic, and spindle cell components; myxoid stroma may also be prominent, mimicking myxofibrosarcoma or myxoid liposarcoma. Unlike ALT/WD-LPS,

DD-LPS has metastatic potential; FNCLCC grading of DD-LPS is prognostic (179,180). Occasionally, the surgeon resects only the firm dedifferentiated area, creating the possibility of misdiagnosis as UPS; thus, any pleomorphic or spindle cell sarcoma in the retroperitoneum or abdominal cavity should be considered a possible DD-LPS and the fatty tissue at the edge of the tumor should be examined carefully for the presence of atypical cells or fibrous septa. When such a tumor is encountered, IHC or FISH for MDM2 can be helpful to support a diagnosis of DD-LPS, even in the absence of a well-differentiated component. MYXOID LIPOSARCOMA Of all liposarcomas, the myxoid type is the second most common (Table 5.9). Myxoid liposarcoma usually presents as a large, painless tumor in the deep soft tissue of the extremities (especially thigh) of young to middle-aged adults. This sarcoma shows variable cellularity, often with prominent myxoid stroma and scattered uniform short spindle cells and a diagnostically useful rich plexiform, thin-walled, branching capillary network (“chicken-wire” or “crow’s-feet” vessels) (Fig. 5.52A). Univacuolated and bivacuolated lipoblasts are often found, most frequently at the periphery of tumor lobules. Diagnosis of low-grade myxoid liposarcomas is usually straightforward. The vascular pattern is distinctive in comparison with that of other myxoid tumors (Fig. 5.53). The tumor cell nuclei are small and ovoid, with finely dispersed chromatin and indistinct nucleoli. Mitoses are uncommon, except in the more cellular examples. The uniformity of this tumor directly contrasts with the pleomorphism of myxofibrosarcoma. A guide to the differential diagnosis of myxoid lesions is provided in Table 5.7. A very helpful diagnostic feature is the presence of pulmonary edema–like or lymphangioma-like mucin pools.

FIGURE 5.52 Liposarcoma, myxoid and pleomorphic types. A delicate, branching capillary network courses through the uniform and bland small cells of myxoid liposarcoma (A); lipoblasts, which are typically bivacuolated or

univacuolated, can be difficult to locate, and the lymphangioma-like cystic degeneration (bottom) is characteristic. High-grade myxoid liposarcoma may be dominated by large, uniform round cells (B). This histologic variant was formerly known as round cell liposarcoma. Pleomorphic liposarcoma (C) contains large cells with multiple grapelike vacuoles, in variable numbers within an otherwise nondistinctive pleomorphic sarcoma.

FIGURE 5.53 Schematic of vessels in myxoid tumors. Circular vessels with thick rims set deep (“aggressive”) angiomyxoma apart from other angiomyxoid lesions. Myxoma is nearly always hypovascular. The very frequent small capillaries seen in myxoid liposarcoma form a network, and they have a small arc—in other words, they would form small circles if the arc were complete. In contrast, the vessels of myxofibrosarcoma are less frequent and thicker because pericytes or tumor cells are attached, and they have wider arcs.

Myxoid liposarcoma may contain hypercellular areas, sometimes acquiring round cell morphology. Greater than 5% hypercellularity (with or without round cell features) is associated with an aggressive clinical course; such tumors are high grade with significant metastatic potential. Pure “round cell” liposarcomas, composed of sheets of larger rounded cells with scant eosinophilic cytoplasm, sometimes arranged in cords within a somewhat sclerotic stroma, are difficult to recognize and may be mistaken for other round cell sarcomas (Fig. 5.52B). The characteristic vascularity in the form of a capillary network is a helpful diagnostic feature, although this is sometimes obscured (181,182). The natural history of “round cell” liposarcoma is extremely aggressive, as Table 5.9 indicates. Most (>90%) cases of myxoid liposarcoma harbor the translocation t(12;16)(q13;p11), resulting in FUS-DDIT3 (see Table 5.3) (183); in around 5% of cases, EWSR1 substitutes for FUS. Recent studies have demonstrated that IHC for DDIT3 is highly sensitive and specific for myxoid liposarcoma, including high-grade (round cell) examples (184,185). PLEOMORPHIC LIPOSARCOMA Pleomorphic liposarcoma shows a range of histologic appearances. In the majority of cases, tumors resembling UPS contain scattered or, occasionally, sheets of pleomorphic lipoblasts (186,187). In some cases, multivacuolated giant cells (Fig. 5.52C) are present in a lesion that somewhat resembles round cell liposarcoma with rounded or epithelioid tumor cells. The vacuoles are characteristically small and numerous within the tumor giant cells. Occasionally, the smaller cells have plump cytoplasm diffusely throughout a tumor, giving rise to the epithelioid subtype, which may closely resemble adrenal cortical carcinoma or renal cell carcinoma (188). Tumors that have features of WD-LPS and a second component of pleomorphic sarcoma are sometimes mistaken for pleomorphic liposarcoma; these are DD-LPS, with the distinctive cytogenetic aberrations and clinical behavior of the latter tumor type. DD-LPS may rarely show

“homologous” lipoblastic differentiation in the otherwise nonlipogenic dedifferentiated component, closely mimicking pleomorphic liposarcoma (189); when a pleomorphic liposarcoma-like tumor is encountered in the retroperitoneum, DD-LPS should be considered. Pleomorphic liposarcoma is a high-grade sarcoma with behavior similar to that of UPS; around 50% of cases metastasize. MYXOID PLEOMORPHIC LIPOSARCOMA Myxoid pleomorphic liposarcoma is a recently described, rare sarcoma type that most often arises in the mediastinum of children and young adults, sometimes in patients with Li-Fraumeni syndrome (i.e., germline TP53 mutation) (190,191). This unusual tumor contains relatively hypocellular, myxoid areas that resemble conventional myxoid liposarcoma and cellular areas resembling pleomorphic liposarcoma with marked nuclear atypia and pleomorphic lipoblasts (Fig. 5.54). Unlike myxoid liposarcoma, these tumors lack DDIT3 gene rearrangement. Myxoid pleomorphic liposarcomas are highly aggressive, with a high rate of local recurrence and distant metastasis and poor outcomes.

FIGURE 5.54 Myxoid pleomorphic liposarcoma. This sarcoma has a predilection for the mediastinum of young patients. The myxoid stroma and vascular pattern mimic myxoid liposarcoma; however, the presence of markedly atypical and pleomorphic tumor cells distinguishes this tumor type.

SMOOTH MUSCLE LESIONS Bizarre leiomyoma is discussed earlier in this chapter. LEIOMYOMA Most benign smooth muscle tumors are easily recognizable, with intersecting fascicles of elongated spindle cells with bright eosinophilic cytoplasm and variably tapering and broad, bluntended nuclei. In leiomyomas, the nuclei tend to be finely stippled, whereas in LMS, the cells often have vesicular nuclei with prominent nucleoli and significant mitotic activity. The pattern of growth is also characteristic; the cells are grouped together as bundles and fascicles that tend to intersect at right angles (Fig. 5.55).

FIGURE 5.55 Leiomyoma. In the center, the blunt-ended elongated nuclei typical of a smooth muscle neoplasm are seen along with cells coursing perpendicular to the plane of section above and below.

Subcutaneous tumors with fascicles of well-differentiated smooth muscle cells, focally arranged around thick-walled blood vessels are angioleiomyomas (within the family of perivascular tumors). Epithelioid leiomyomas composed of tumor cells with rounded morphology are nearly exclusive to the uterus. The differential diagnosis of the latter tumor is provided in Table 5.10. In the female genital tract, distinctive benign smooth muscle neoplasms occur either throughout the myometrium and vasculature (intravenous leiomyomatosis) or throughout the peritoneal cavity (leiomyomatosis peritonealis disseminata). TABLE 5.10 Sarcomas and Selected Benign Soft Tissue Tumors with Epithelioid Morphologya Alveolar soft part sarcoma Epithelioid angiosarcoma Epithelioid hemangioma Epithelioid hemangioendothelioma Epithelioid malignant peripheral nerve sheath tumor Epithelioid sarcoma Glomus tumor Leiomyosarcoma, epithelioid variant (uterus) Malignant rhabdoid tumor Myoepithelioma/myoepithelial carcinoma Myxofibrosarcoma, epithelioid variant PEComa Pleomorphic liposarcoma, epithelioid variant Sclerosing epithelioid fibrosarcoma aCarcinoma

and melanoma should always be considered. PEComa, perivascular epithelioid cell tumor.

Although leiomyomas of deep soft tissue and retroperitoneum exist (192,193), they are very uncommon, and extreme caution should be exercised when such a diagnosis is made. When nuclear pleomorphism or necrosis is encountered (along with any mitotic activity), LMS should be diagnosed (Fig. 5.56). Minimal criteria for malignancy in well-differentiated smooth muscle neoplasms differ according to anatomic site and gender (194,195). Essentially, any mitotic activity in a well-differentiated smooth muscle neoplasm of somatic soft tissue (extremities and trunk wall) should lead to a diagnosis of low-grade LMS. In the pelvis and retroperitoneum in

women, “uterine-type” leiomyomas may show mitotic activity (up to five per 50 hpf); benignappearing smooth muscle tumors at these sites with higher mitotic rates should probably be regarded as of “uncertain malignant potential” (195). Well-differentiated retroperitoneal smooth muscle tumors in men are nearly all LMSs.

FIGURE 5.56 Leiomyosarcoma. The alternating fascicular pattern (parallel and perpendicular to the plane of section) and blunt-ended nuclei are recognizable in this subcutaneous tumor, which had a high mitotic rate; only mild pleomorphism is present.

LEIOMYOSARCOMA LMS is the most common sarcoma type. Many LMSs occur in the uterus; these are discussed in detail in Chapter 54. The cellular characteristics of the benign smooth muscle tumors that were previously described are retained in most LMSs. Significantly, the definition of a malignant smooth muscle tumor varies according to the site, with the criteria between uterine and nonuterine lesions differing drastically (see preceding section “Leiomyoma”). In the soft tissues, LMS may arise from medium or large veins (196), such as the inferior vena cava (197). Retroperitoneal cases show a female predominance (198), and are highly aggressive with metastases to the lungs, liver, and bones. Tumors often referred to as “cutaneous leiomyosarcomas,” when confined to the dermis, have no metastatic potential; many soft tissue pathologists, therefore, prefer to designate such tumors as atypical intradermal smooth muscle neoplasms (199). These dermal tumors show a male predominance, a predilection for the extremities, and are usually composed of bundles of atypical and pleomorphic smooth muscle cells ramifying through dermal collagen (199). In contrast, subcutaneous LMSs metastasize in 30% to 40% of cases. LMS may also arise in deep subfascial and intramuscular locations. Rarely, LMS may contain myxoid stroma (200); such examples are exceptional outside of the uterus. LMS is one of the few conventional sarcoma types that not infrequently contain prominent osteoclastic giant cells (161). Children may rarely harbor LMS (201). Inflammatory LMS is a rare low-grade sarcoma that was originally buried within the “inflammatory MFH” wastebasket, and previously included as a variant of LMS; inflammatory LMS has now been pulled out as a separate entity in the 2020 WHO classification (2). This tumor type arises in the extremities and trunk, with a peak incidence in young adults (202). The relative uniform fascicles of eosinophilic spindle cells with mild atypia are admixed with a prominent inflammatory infiltrate of lymphocytes and foamy histiocytes, often obscuring the neoplastic cells (Fig. 5.57). These tumors have a characteristic near-haploid genotype (203) and lack the

complex karyotypes and tumor suppressor gene inactivation of conventional LMS. The metastatic risk is low. Inflammatory LMS may be related to a recently described tumor type, socalled histiocyte-rich rhabdomyoblastic tumor; there has been a proposal to rename both tumor types as “inflammatory rhabdomyoblastic tumor” (204).

FIGURE 5.57 Inflammatory leiomyosarcoma. Numerous lymphocytes infiltrate this fascicular spindle cell neoplasm with nuclear atypia. Desmin is typically strongly positive (not shown). This tumor type has a much better prognosis than does conventional leiomyosarcoma.

LMS can also be pleomorphic (205). However, it must be stressed that pleomorphism is neither a requirement nor a criterion for a malignant diagnosis, as was noted earlier in this chapter. A distinction must also be made between (1) pleomorphic LMS, in which the pleomorphism occurs within the bounds of recognizable smooth muscle features and (2) dedifferentiated LMS, in which two patterns are present, with the second pattern being UPS-like with loss of smooth muscle marker expression (206). EPSTEIN-BARR VIRUS–ASSOCIATED SMOOTH MUSCLE TUMORS This distinctive group of smooth muscle tumors usually arises in immunosuppressed patients (HIV-associated or posttransplant); the anatomic distribution of EBV-associated smooth muscle tumors is unusual, including the central nervous system and visceral sites, such as lung, liver, and adrenal gland (207). In addition to fascicles of eosinophilic spindle cells, some cases contain a more primitive-appearing, round cell component; prominent intratumoral lymphocytes are common. Multicentricity is common; most of these tumors do not metastasize.

LESIONS OF STRIATED MUSCLE Fetal rhabdomyoma was described earlier in this chapter (see Fig. 5.11). ADULT RHABDOMYOMA In addition to fetal rhabdomyoma, which has already been discussed, an adult form that consists of highly differentiated cells growing in sheets occurs. The cytoplasm is abundant, and it is distinctly granular and fibrillar (Fig. 5.58). It is usually easily recognized despite the presence of cytoplasmic vacuolization and the formation of so-called spider cells. Typically, adult

rhabdomyoma develops in the head and neck region, particularly in the pharynx and larynx. The cardiac rhabdomyoma is a small lesion encountered in association with tuberous sclerosis, and it is probably a result of a hamartomatous process. A rare paratesticular rhabdomyoma variant has also been described; in this variant, well-differentiated skeletal muscle cells are embedded in a hyalinized collagenous stroma (208).

FIGURE 5.58 Rhabdomyoma, adult type. The skeletal muscle nature of this tumor is easily appreciated. The tumor cells are large and polygonal, with granular cytoplasm. Occasional cells show cytoplasmic vacuolization.

RHABDOMYOSARCOMA The vast majority of RMSs do not occur in the extremities, where the bulk of skeletal muscle resides; this is a good illustration of the fallacy of the concept of “histogenesis” for soft tissue tumors: most mesenchymal tumors do not arise from differentiated cell types. Rather, a host of classic locations have been described, each with its own typical histology and natural history. Most cases of embryonal RMS occur in the first decade of life; alveolar RMS has a peak incidence in adolescents (Table 5.11). Rarely, RMS cases occur congenitally or in adults older than 40 years; such tumors belong to distinct histologic categories. RMSs are highly aggressive sarcomas with a high mortality rate, although some pediatric cases are cured by modern chemotherapy. Prognosis depends on factors such as site, histology, and group (a type of staging; applicable only to pediatric patients). In general, patients in groups I or II, who have either completely resected disease or microscopic disease remaining, respectively, are frequently cured. In contrast, patients in groups III or IV, who have locally unresectable or metastatic disease, respectively, frequently die despite chemotherapeutic measures. TABLE 5.11 Sarcomas by Peak Age Group Younger than 10 years Rhabdomyosarcoma Neuroblastoma Infantile fibrosarcoma Malignant rhabdoid tumor Between 11 and 40 years Synovial sarcoma Epithelioid sarcoma Clear cell sarcoma

Malignant peripheral nerve sheath tumor Epithelioid hemangioendothelioma Mesenchymal chondrosarcoma Extraskeletal Ewing sarcoma Alveolar soft part sarcoma Older than 40 years—all other types

Rhabdomyosarcoma Classification The Intergroup Rhabdomyosarcoma Study designates pediatric tumors as either favorable (botryoid, well differentiated, or spindle cell, and most embryonal RMS) or unfavorable (209). The unfavorable tumors are those that have anaplastic features or alveolar histology or those that are poorly differentiated with monomorphous round cell morphology. With this designation, the unfavorable category comprises approximately 20% of cases; the 2-year disease-free survival rate for this group is around 70%, versus 90% for favorable cases. Embryonal Rhabdomyosarcoma Most pediatric RMSs are classified as embryonal; these tumors consist of sheets of round to short spindle cells. In contrast to the nucleus in Ewing sarcoma, the nucleus in embryonal RMS is eccentric (Fig. 5.59A), and the cytoplasm in occasional cells appears more granular and eosinophilic. As the cells become larger, the cytoplasm may appear fibrillar, and it may encircle the nucleus. Cross-striations are uncommon, and need not be sought to make the diagnosis. Again, in contrast to Ewing sarcoma, RMS exhibits occasional spindling, and in such fields, the characteristic “strap” cells may be identified. The cytoplasmic borders run in parallel without tapering. When such cells are present throughout embryonal RMS, the tumor mimics the myotubular stage of normal muscle development, and the lesion is considered well differentiated. The embryonal pattern is the most common type of RMS in practically all sites. Embryonal RMS cases that occur near a lumen or a space (e.g., genitourinary tract, female reproductive tract, and conjunctiva) are most often of the botryoid type. This much more myxoid tumor has a very typical low-power appearance in which the density of cells is greatest just below the adjacent epithelium (Fig. 5.59B,C). This area is known as the cambium layer. Usually, the characteristic small strap cells are located at the junction of this layer with the more myxoid underlying regions. In all, this type accounts for approximately 25% of all RMS cases; roughly 75% of all botryoid tumors occur in the genitourinary tract.

FIGURE 5.59 Rhabdomyosarcoma. In embryonal rhabdomyosarcoma (A), round cells with small oval nuclei and scanty eosinophilic cytoplasm are seen. The eccentric placement of the nucleus in the cytoplasm is a helpful diagnostic characteristic. Such cells grow in sheets without other distinctive features. The botryoid type bulges into a space, which, in this case, is the orbital conjunctiva (B) (courtesy of Dr. Ralph Eagle, Temple University, Philadelphia, PA); the dense aggregation of cells below the epithelium (C) is its hallmark (known as the cambium layer). Underneath, the loose texture is pronounced, and differentiating strap cells can be found. In some examples of alveolar rhabdomyosarcoma, the alveolar morphology is obvious and striking (D). The tumor cells either grip fibrous septa around the alveoli or appear to lie in the space; large multinucleated cells with abundant cytoplasm are infrequent but diagnostic, especially in the solid variant or when alveoli are less conspicuous.

Alveolar Rhabdomyosarcoma

Alveolar RMS affects patients at an older mean age than does embryonal RMS, it is frequently observed in adolescents with lesions on the extremities and trunk. This histologic type has the worst prognosis of pediatric and adolescent RMS when all other variables are held constant. The tumor is sometimes divided by small fibrous septa between which some of the tumor cells appear to detach into oval or elongated spaces (Fig. 5.59D). Cells at the periphery of the alveoli hug these septa. Most of the tumors are dominated by rounded cells with minimal cytoplasm, and only scattered eosinophilic cells can be seen. Multinucleated wreath-like giant cells are a frequent finding in alveolar RMS, but not in the other types. By IHC, desmin and the skeletal muscle transcription factors myogenin and MyoD1 are positive. Myogenin typically shows strong and diffuse nuclear reactivity, which can be a helpful feature to distinguish alveolar from embryonal RMS in a limited biopsy (210). The solid variant fails to form alveolar spaces. This form can be recognized by the presence of the aforementioned tumor giant cells. Alveolar RMS may present with bone marrow metastases or rarely a leukemic presentation. The diagnosis can be confirmed by FISH for FOXO1, reverse transcription polymerase chain reaction (RT-PCR), or NGS; around 85% of cases have PAX3-FOXO1 (80% of fusion-positive tumors) or PAX7-FOXO1 fusions (20%). Molecular Prognosis Molecular analysis, which is helpful in classifying round cell sarcomas, also has prognostic value because alveolar RMSs with PAX7-FOXO1A gene fusions have a more favorable clinical course than do tumors with PAX3-FOXO1A fusions (211). Spindle Cell/Sclerosing Rhabdomyosarcoma Spindle cell RMS includes tumors dominated by intersecting fascicles of elongated spindle cells that may mimic LMS (Fig. 5.60), although rare rhabdomyoblasts are often detectable (212,213). Childhood cases of spindle cell RMS have a predilection for paratesticular locations and an excellent prognosis, whereas tumors in adults most often arise in the head and neck or extremities and are highly aggressive (213). Congenital and infantile spindle cell RMS often harbor NCOA2, VGLL2, or CITED2 gene rearrangements (214), whereas spindle cell (and sclerosing) RMS in older children and adults often contain MYOD1 mutations (215-217). Sclerosing RMS contains abundant hyalinized collagenous stroma within which tumor cells are arranged in nests and small alveolar structures (Fig. 5.60B) (218,219). Spindle cell and sclerosing patterns not infrequently coexist in a single tumor. In addition to desmin, by IHC spindle cell/sclerosing RMS typically shows diffuse nuclear staining for MyoD1, whereas myogenin often shows limited staining.

FIGURE 5.60 Spindle cell/sclerosing rhabdomyosarcoma. Spindle cell rhabdomyosarcoma shows a fascicular growth pattern and closely mimics leiomyosarcoma (A). Sclerosing rhabdomyosarcoma contains abundant hyalinized collagenous stroma, and shows a more nested architecture (B). Many examples show both histologic patterns. In addition to desmin, MyoD1 is typically strongly positive (not shown).

Pleomorphic Rhabdomyosarcoma Pleomorphic RMS is rare; most of the examples designated as such in the past were likely UPS. RMS tumors in children with some pleomorphism are, by current convention, subsumed into the embryonal group. Pleomorphic RMS is an aggressive sarcoma of middle-aged to elderly adults, most often arising in the deep soft tissue of the extremities (220,221). The tumor cells contain abundant bright eosinophilic cytoplasm and range from polygonal to rounded or spindled, often including large, bizarre forms. Diffuse desmin expression is typical of this tumor type, but myogenin and MyoD1 staining is often limited. Pleomorphic RMS has the highest metastatic rate of all adult pleomorphic sarcomas.

VASCULAR LESIONS Papillary endothelial hyperplasia was described earlier in this chapter (see Fig. 5.12). LOBULAR CAPILLARY HEMANGIOMA (PYOGENIC GRANULOMA) This relatively common, reddish-blue, small cutaneous or mucosal lesion may appear in almost any site, but it tends to occur on the lips and face of pregnant women (222). The frequent mitoses and enlarged endothelial nuclei may initially be disturbing at high power, but a low-power view discloses an organized architecture for this lesion (Fig. 5.61). Particularly at the lateral edges, a lobular arrangement wherein groups of capillaries proliferate but abruptly stop is noted. A thin collagen layer surrounds each lobule. This arrangement is disrupted at the base, where irregularly shaped larger vascular channels reside. At higher power, another discriminating feature of benign vascular lesions is apparent; the small capillary endothelium-lined spaces are also surrounded by a pericytic cell layer. This double layer of cells, which is also seen in angiomatosis, is not seen in angiosarcoma. Frequently, pyogenic granuloma, which is also called lobular capillary hemangioma, contains a superficial region of ulceration in which neutrophils abound in the more superficial lobules.

FIGURE 5.61 Lobular capillary hemangioma (pyogenic granuloma). At medium power (A), the lobules are sharply demarcated by fibrous septa that are hypocellular; at high power (B), dilated capillary vessels have an eosinophilic outline resulting from a pericyte layer.

HEMANGIOMAS A tremendous variety is seen in hemangiomas, which range from (1) the cavernous type, in which very large spaces are separated by fibrous tissue, to (2) the capillary type, which lacks fibrous septa and may be quite cellular. The venous hemangioma is another form containing smooth muscle encircling the vascular spaces. Occasional hemangiomas develop within muscle (intramuscular type). These are noncircumscribed tumors that particularly form in the muscles of the thigh in young adults; pain is a frequent symptom. The intramuscular type is often accompanied by adipose tissue, and it may recur. Special subtypes of hemangioma exist, including the multicentric cutaneous glomeruloid hemangioma; this has a characteristic appearance within dilated vascular channels, and it is associated with Castleman disease and POEMS (polyneuropathy, organomegaly, endocrinopathy, M-protein, skin changes) syndrome (223). An encyclopedic coverage of the vast array of benign vascular lesions and malformations is beyond the scope of this chapter; selected lesions of skin and soft tissue are discussed later. Infantile Hemangioma Infantile hemangioma is cellular capillary hemangioma that is exclusive to young children younger than 1 year of age. It may be found in a variety of sites, but it is common in the cheek or periparotid region. The histologic appearance is characteristic, with a lobular proliferation of tightly apposed compressed capillaries lined by plump endothelial cells surrounded by pericytes; the lobules are demarcated by thin fibrous septa. Numerous mitotic figures may be noted. Infantile hemangioma almost invariably slowly regresses; during involution, the capillaries become progressively dilated. In contrast to other forms of capillary hemangioma, the endothelial cells are consistently positive for glucose transporter 1 (GLUT1) by IHC. Epithelioid Hemangioma

Epithelioid hemangioma is a distinctive benign vascular neoplasm within which the well-formed blood vessels are lined by plump, epithelioid endothelial cells (Table 5.10). This tumor type has a marked predilection for the head and neck (especially the preauricular region and forehead); multifocality is seen in 10% to 20% of cases. Epithelioid hemangioma is the accepted term for the lesion formerly known as angiolymphoid hyperplasia with eosinophilia (224), nomenclature that reflects the inflammatory infiltrate observed in a subset of cases. Plump endothelial cells with often glassy eosinophilic cytoplasm containing occasional vacuoles (Fig. 5.62) line rounded blood vessels and occasionally form sheets; some tumors are accompanied by lymphoid aggregates and prominent eosinophils. Gene rearrangements of FOS or FOSB are detected in around half of cases (225,226); by IHC, FOSB shows strong nuclear staining in just over 50% of tumors (227). Given the eosinophils, epithelioid hemangioma may be mistaken for Kimura disease; however, Kimura disease is not a hemangioma, lacks epithelioid morphology, and is essentially geographically restricted to Japan (228). Epithelioid hemangioma may recur in around one-third of cases. Epithelioid endothelial cells may also be identified in other hemangiomas, epithelioid hemangioendothelioma (EHE; see section “Epithelioid Hemangioendothelioma”), and some angiosarcomas.

FIGURE 5.62 Epithelioid hemangioma. Plump epithelioid endothelial cells with glassy eosinophilic cytoplasm and occasional vacuoles line small blood vessels. Note the prominent inflammatory infiltrate of lymphocytes and eosinophils.

Spindle Cell Hemangioma Spindle cell hemangioma may resemble nodular Kaposi sarcoma; distinguishing between the two may sometimes be difficult (229). Spindle cell hemangioma typically presents as multiple bluish nodules on the distal extremities of young adults. These tumors involve the dermis and subcutis and histologically are composed of dilated, irregular, thin-walled cavernous vessels, bundles of bland spindle cells, and clusters of epithelioid endothelial cells with intracytoplasmic vacuoles (Fig. 5.63). Intravascular thrombi and phleboliths may be seen. The spindle cell component is positive for SMA. Unlike Kaposi sarcoma, spindle cell hemangioma is negative for HHV8. Spindle cell hemangiomas harbor mutations in IDH1 or IDH2 (230). Multiple spindle cell hemangiomas are often seen in patients with Maffucci syndrome (along with multiple enchondromas); somatic mosaic IDH1 mutations underlie this disorder (231).

FIGURE 5.63 Spindle cell hemangioma. Although the spindle cells can mimic Kaposi sarcoma, the vacuolated endothelial cells are unique to spindle cell hemangioma. A cavernous hemangioma-like component is characteristic.

Tufted Angioma Tufted angioma is an acquired benign cutaneous vascular neoplasm that is most often seen in infants or young children; this tumor type is essentially the superficial variant of kaposiform hemangioendothelioma (see later) (2,232). The lesions present as indolent, but occasionally progressive, tender, ill-defined erythematous macules and papules on the neck or upper trunk. Despite their enlargement over time, with some reaching more than 10 cm in diameter, the clinical course is benign. A characteristic “cannonball” distribution of the vascular nodules is seen, with a pattern somewhat reminiscent of lobular capillary hemangioma. A crescent-shaped or dilated vascular channel is often seen at the edge of each nodule. Microvenular Hemangioma Microvenular hemangioma is an acquired cutaneous hemangioma that presents as an asymptomatic, small, well-circumscribed red papule on the limbs and trunk of young adults, and it may clinically mimic Kaposi sarcoma (233). Irregular, narrow branching vessels are lined by flat or slightly plump endothelial cells without atypia. They dissect a sclerotic stroma in an infiltrative manner. Sinusoidal Hemangioma Sinusoidal hemangioma is a variant of cavernous hemangioma that occurs in middle-aged adults, predominantly women, often on the breast (234). It has a characteristic appearance and grows as a nodule in the dermis or subcutaneous tissue. This circumscribed hemorrhagic lesion has a lobular growth pattern and intercommunicating thin-walled blood vessels containing pseudopapillary structures. Thrombosis with dystrophic calcification can occur. Hobnail Hemangioma Occurring on the trunk or extremities of young to middle-aged adults, hobnail hemangioma (also known as targetoid hemosiderotic hemangioma) presents as a small, solitary purple papule sometimes surrounded by a pale halo and occasionally by a peripheral ring (235). In the dermis, an irregular, dilated, thin-walled proliferation of vascular channels is lined by prominent, often hobnail endothelial cells with occasional papillary projections. Extravasated red blood cells and inflammatory cells may be seen in the surrounding stroma. In the deeper subcutaneous component, the vascular channels become narrowed, they dissect the collagen, and they are lined by flat endothelial cells. The lesion appears to be self-limited, and excision is curative.

ANGIOMATOSIS The involvement of a large portion of the body (skin to deep muscle or multiple muscle compartments) with a benign but diffuse vascular proliferation is termed angiomatosis (236). Small, meandering capillaries or small venules may be seen, or the lesion may be composed of a mixture of larger venous channels and capillaries. Some cutaneous forms of angiomatosis appear reactive (“reactive angioendotheliomatosis”), associated with systemic disorders such as renal failure and cardiac valvular disease (237). BACILLARY ANGIOMATOSIS Bacillary angiomatosis is an inflammatory lesion that may arise in immunosuppressed patients, most often in patients with AIDS, but also in the posttransplant setting (238); this lesion is currently rarely encountered. Clinically, bacillary angiomatosis most commonly presents as a cutaneous disorder characterized by multiple friable angiomatous papules or erythematous papules with and without crusts. Systemic symptoms, such as fever, chills, night sweats, and weight loss, are common. Other organs and systems may be involved, including the liver, spleen, lymph nodes, and mucosal surfaces of the conjunctivae and respiratory tract. Histologically, bacillary angiomatosis superficially resembles lobular capillary hemangioma; the lesions are often polypoid with epidermal collarettes. Granulation tissue–like clusters of small rounded vessels are present, separated by bands of fibrosis; plump endothelial cells and neutrophils are prominent. Pericytes surround the small vessels. Granular eosinophilic or, less often, basophilic material representing the bacilli may be observed, but this can be mistaken for fibrin; the Warthin-Starry stain can be used to detect and to confirm the bacterial nature. The lesion is caused by several agents, including the Bartonella species. VASCULAR TRANSFORMATION OF SINUSES Vascular transformation of sinuses is a vascular proliferation that is typically limited to the sinuses of lymph nodes (Fig. 5.64). At low power, most cases are recognizable because of the prominence of red blood cell–filled spaces limited to the nodal sinuses. At high power, in most cases, the spaces are dilated and circular with very few, if any, spindled cells among them. No evidence of intraparenchymal or intracapsular involvement has been found. However, the morphologic spectrum also includes cases with spindle cells, which may display an almost identical appearance to Kaposi sarcoma with parenchymal involvement (239). However, fibrosis between spindle cells is usually present in vascular transformation of sinuses, but not in nodal Kaposi sarcoma; in addition, the well-formed spindle cell fascicles of Kaposi sarcoma are rarely seen in vascular transformation of sinuses. Vascular transformation of sinuses may infiltrate the nodal capsule, another difference from Kaposi sarcoma. Unlike Kaposi sarcoma, the spindle cells in vascular transformation of sinuses are negative for HHV8.

FIGURE 5.64 Vascular transformation of sinuses. In this lymph node, open vascular channels occupy the sinuses beneath the uninvolved capsule. In addition, spindle cells proliferate along the sinuses and in the parenchyma. Note the small foci of fibrosis.

INTERMEDIATE ENDOTHELIAL TUMORS KAPOSIFORM HEMANGIOENDOTHELIOMA Kaposiform hemangioendothelioma, a distinctive, locally aggressive vascular tumor often occurring in the deep soft tissues of the extremities, head and neck, and retroperitoneum of young children, commonly present with profound thrombocytopenia and consumptive coagulopathy (Kasabach-Merritt syndrome) (240). Large, invasive tumors contain coalescing solid lobules of spindle cells with slitlike spaces similar to those of Kaposi sarcoma; however, the lobules are surrounded by fibrous bands, unlike those seen in Kaposi sarcoma. The skin is another common site for this tumor, where it presents as ill-defined plaques; superficial cutaneous tumors are known as tufted angioma. The tumor may present as intraabdominal masses, or it may surround the internal organs, such as the kidneys, pancreas, adrenal glands, and the gut. The large size of these lesions, their position in the body, and the lack of HHV8 distinguish them from Kaposi sarcoma. Similar tumors rarely arise in adults (241). RETIFORM HEMANGIOENDOTHELIOMA AND COMPOSITE HEMANGIOENDOTHELIOMA Both retiform hemangioendothelioma and composite hemangioendothelioma typically arise in the skin and subcutaneous tissue of the distal extremities of young adults. Retiform hemangioendothelioma forms narrow, arborizing vascular channels lined by hyperchromatic endothelial cells with naked nuclei showing a hobnail appearance, sometimes with papillary tufting into the lumina (Fig. 5.65) (242). Composite hemangioendothelioma almost invariably contains a component of retiform hemangioendothelioma, along with EHE-like and low-grade angiosarcoma-like components (243). A neuroendocrine variant of composite hemangioendothelioma has recently been reported; this variant often includes a component with a nested appearance and is positive for synaptophysin (244). A subset of both retiform and composite hemangioendotheliomas harbor YAP1 gene rearrangements, usually YAP1-MAML2 (245). Retiform hemangioendothelioma is associated with a significant risk for multiple local recurrences (>50%), but only very rarely metastasizes to lymph nodes. Composite hemangioendothelioma is also associated with local recurrences and rare metastasis; the neuroendocrine variant is considerably more aggressive with visceral and bone metastases.

FIGURE 5.65 Retiform hemangioendothelioma. The vascular structures in this lesion may be open or closed. In open vessels, the small, bland hobnail nuclei are often seen protruding into the lumen; no mitoses or atypia are seen. Closed vessels resemble ramifying cords of cells within the fibrous tissue.

PSEUDOMYOGENIC HEMANGIOENDOTHELIOMA This distinctive tumor type was originally described as the “fibroma-like” variant of epithelioid sarcoma (246) and later as “epithelioid sarcoma–like” hemangioendothelioma (247). The clinical and pathologic features were more thoroughly characterized in a larger series, in which the term pseudomyogenic hemangioendothelioma was introduced, owing to the tumor’s histologic resemblance to a myoid neoplasm (248). Pseudomyogenic hemangioendothelioma typically presents as multiple nodules in the limb of young adults, with a striking male predominance, although the age and anatomic distribution is wide. Many patients present with discrete lesions in multiple tissue planes (most often skin, followed in frequency by skeletal muscle and bone) (248). Despite the ominous clinical presentation, the rate of distant metastasis is low, although the development of additional nodules in the same anatomic region is common. The tumor is composed of loose fascicles of plump spindle cells, with or without epithelioid cells with abundant, often brightly eosinophilic cytoplasm, sometimes closely mimicking rhabdomyoblasts (Fig. 5.66). Despite these appearances, muscle markers are negative. The tumor cells are positive for keratins (AE1/AE3), ERG, and often for CD31. Interestingly, other broad-spectrum keratin antibodies (e.g., MNF116) are usually negative. In contrast to epithelioid sarcoma, pseudomyogenic hemangioendothelioma is negative for EMA and CD34 and is positive for SMARCB1 (INI1) (i.e., nuclear expression is retained). FOSB gene rearrangements are a consistent finding, within either SERPINE1-FOSB or ACTB-FOSB fusions (249,250). IHC for FOSB shows nuclear staining in nearly all cases (227).

FIGURE 5.66 Pseudomyogenic hemangioendothelioma. This tumor is composed of loose fascicles of spindle cells with abundant eosinophilic cytoplasm. Nuclear atypia is usually only mild. The tumor cells are positive for keratins AE1/AE3 and ERG (not shown).

MALIGNANT ENDOTHELIAL TUMORS EPITHELIOID HEMANGIOENDOTHELIOMA EHE is a malignant vascular neoplasm composed of epithelioid endothelial cells; in its conventional form, EHE cannot form vascular channels (251,252). Soft tissue cases commonly occur in deep tissues of the extremities and often display striking angiocentricity, with growth within and around large veins or arteries. However, EHE may also occur within organs, such as the liver and lungs, and bone; visceral and bone tumors are often multifocal at presentation. Regardless of the anatomic site, the histology is similar. The tumor is composed of epithelioid cells with glassy eosinophilic cytoplasm, arranged in cords, strands, and nests within a sclerotic or myxohyaline stroma (Fig. 5.67). Intracytoplasmic vacuoles are a characteristic finding and a helpful diagnostic clue (Fig. 5.67). Notably, true vascular channels are not a feature of this tumor type. Although the nuclei may vary from small and pyknotic to large and oval with nucleoli, occasional cases have prominent nuclear pleomorphism. IHC for endothelial markers such as CD31 and ERG is helpful to confirm the diagnosis. Expression of keratins is common and represents a potential diagnostic pitfall; EHE can closely mimic metastatic poorly cohesive gastric carcinoma or lobular breast carcinoma. EHE harbors a characteristic WWTR1-CAMTA1 gene fusion, as a consequence of the translocation t(1;3)(p36;q25), in 85% to 90% of cases (253,254); IHC for CAMTA1 is a highly sensitive and specific diagnostic marker (255). CAMTA1 can be helpful to distinguish EHE from cellular epithelioid hemangiomas and epithelioid angiosarcoma. A small subset of tumors instead contains a YAP1-TFE3 fusion, associated with nuclear TFE3 expression (256). This EHE variant shows distinct histology, including large epithelioid cells with voluminous pale cytoplasm, a nested architecture, and occasional vascular channels (Fig. 5.68).

FIGURE 5.67 Epithelioid hemangioendothelioma. This is a sclerosing tumor in which thin strands of tumor cells proliferate. Some of the cells have vacuoles, typical of epithelioid endothelial neoplasms.

FIGURE 5.68 Epithelioid hemangioendothelioma variant with YAP1-TFE3. This distinctive variant shows a nested architecture with occasional vascular channels; the tumor cells contain voluminous pale cytoplasm. An immunostain for TFE3 is positive (not shown).

Most of these tumors have a low mitotic rate, with an average of one or two mitotic figures per 10 hpf. In the soft tissues, the recurrence rate is 10%, and more than 20% of cases metastasize to the lymph nodes, lungs, or liver. Risk stratification for soft tissue EHE has been proposed on the basis of mitotic rate and tumor size; high-risk EHE pursues a more aggressive clinical course (257). ANGIOSARCOMA The majority of angiosarcomas occur in the skin, particularly sun-damaged skin of the head and neck of elderly patients, with a male predominance (258). Cutaneous angiosarcoma appears as either reddish-blue nodules or, more commonly, flat spreading bruises. They may expand to a large size, and their full extent is frequently underestimated by the clinician. Surgical excision is usually ineffective, and aggressive recurrences are common. Early metastatic disease develops

in a high percentage of patients, and the overall prognosis is poor. Less common are those cases that develop in the deep soft tissue, sometimes in association with major vessels (259); deep soft tissue angiosarcomas are often epithelioid. Two other presenting forms of angiosarcoma are less common. In patients with long-standing lymphedema, the risk of the development of angiosarcoma in the affected extremity is increased. In the days of radical breast surgery for cancer, postmastectomy angiosarcoma developed in the arm after a latent period of about 10 years. These patients were frequently in the seventh decade of life, and they had a dismal survival, usually 2 years. Angiosarcomas may also arise primarily within the breast (260); this form affects patients in the third and fourth decades of life. The histology can be deceptively bland; diffuse growth within and entrapment of normal breast ducts and lobules is characteristic. An increasing number of cutaneous angiosarcomas have been reported after radiation therapy for localized breast cancer, mostly arising in the dermis (261); in the same postradiation setting, so-called atypical vascular lesions of the mammary skin have also been reported, and form irregular spaces without significant nuclear atypia (261). MYC gene amplification is common in postradiation angiosarcomas, leading to overexpression of the MYC protein; FISH or IHC can be used in particularly challenging cases (262-264), because atypical postradiation vascular proliferations are negative for MYC. Histologically, most angiosarcomas are easily recognizable as vascular in nature, because they form dissecting vascular channels. Unlike those of benign tumors, these channels extensively anastomose and branch. They show endothelial multilayering, and the cells are often plump, with marked nuclear atypia in the form of hyperchromatic nuclei and sometimes prominent nucleoli. In addition, tufts of endothelial cells may protrude into the vascular channels. In the skin, the existing collagen bundles frequently serve as a “scaffold” for the vascular proliferation (Fig. 5.69A). Occasionally, the cutaneous forms are epithelioid in appearance, with round to polygonal cells growing in diffuse sheets with vesicular chromatin and amphophilic or eosinophilic cytoplasm (Fig. 5.69B). Epithelioid angiosarcoma should be distinguished from EHE, a tumor with less aggressive behavior. In this more poorly differentiated form, the cells commonly have enlarged nuclei with prominent nucleoli; sometimes the only hints of the vascular nature of the neoplasm are found at the edge of the lesion with vessel formation or with occasional cytoplasmic vacuoles (Fig. 5.69B). Deep soft tissue variants of epithelioid angiosarcoma also occur (265). Some tumors may contain extensive hemorrhage, mimicking a hematoma. Highly cellular cases may show fascicular, spindle cell morphology, but open vascular channels are usually visible in foci. Rare cases show marked stromal sclerosis (Fig. 5.69C,D). Poorly differentiated epithelioid and spindle cell angiosarcomas can be difficult to diagnose without the application of IHC; CD31 and ERG (266) are the most sensitive and specific markers in this context. Similar to EHE, epithelioid angiosarcoma is often positive for keratins.

FIGURE 5.69 Angiosarcoma. The existing collagen serves as a “scaffold” for the hyperchromatic and tufting endothelial cells in angiosarcoma (A). Too many cells line the anastomosing spaces, and atypia is present. In poorly differentiated epithelioid cases (B), vacuoles are often seen, and when two vacuoles abut each other, a septate vacuole is produced. This is a clue to the endothelial nature of the lesion. A sclerosing angiosarcoma (C,D) has a double row of nuclei in a branching pattern (C); open channels and septate vacuoles are found focally (D).

The differential diagnosis includes pseudoangiomatous carcinoma when the skin is involved and mesothelioma when the serous membranes are involved. Regardless of the site, angiosarcoma is a highly aggressive sarcoma type, and in most instances, the degree of differentiation or grade does not affect overall survival.

KAPOSI SARCOMA Three points should be made about Kaposi sarcoma. First, it may or may not be a fully malignant neoplasm, and it may be genetically polyclonal. Second, its clinical forms are misunderstood. In early reports, more internal visceral involvement was found in elderly patients than is currently appreciated; in cases associated with transplantation, regression is common if the immunosuppression is halted (267). Furthermore, in the AIDS population, regression and long survival may occur, and survival is related more to immunologic status than to the bulk or distribution of the lesions. Third, although many cases are histologically straightforward (a uniform and bland proliferation of eosinophilic spindle cells showing directional streaming) (Fig. 5.70A), Kaposi sarcoma may be mistaken for the following in the skin: (1) inflammatory

dermatosis; (2) pyogenic granuloma, which lacks a spindle cell element; (3) angiodermatitis or pseudo-Kaposi sarcoma, which is a lobular proliferation of capillaries separated by fibrous tissue rather than by spindle cells below hyperplastic or ulcerated epithelium and that contains prominent hemosiderin; (4) bacillary angiomatosis, an epithelioid, vascular tumor–like lesion that lacks spindle cells and that is the result of a bacterial infection; and (5) angiosarcoma, in which a spindle cell component accompanies the formation of anastomosing channels in some cases. In the lymph node, the vascular transformation of sinuses should be considered; in contrast to Kaposi sarcoma, this is a hemangioma-like lesion with blood-filled spaces and mild fibrosis.

FIGURE 5.70 Kaposi sarcoma. This cellular tumor contains fascicles and sheets of elongated spindle cells with mild nuclear atypia and pale cytoplasm (A). Slitlike spaces and hemorrhage are typical features. The diagnosis can be confirmed by staining for human herpesvirus-8 (HHV8) (B).

Kaposi sarcoma has been shown to be caused by the virus HHV8 (268). Confirmation of Kaposi sarcoma is straightforward by IHC for HHV8, which is a highly specific marker that shows a nuclear pattern of staining (Fig. 5.70B).

PERICYTIC (PERIVASCULAR) TUMORS GLOMUS TUMOR Glomus tumor, which is derived from a specialized pericyte, frequently occurs in the subungual portion of the finger and less frequently on other parts of the extremities and trunk (269). The histology is variable, with prominent vasculature noted in most cases. Sometimes, these vessels are surrounded by a sharply demarcated cuff of cells with prominent eosinophilic or clear cytoplasm (Fig. 5.71). Well-defined cell borders, which give an epithelioid appearance, are often seen, but the cell borders may not always be distinct. In some cases, the sheets of cells appear to be punctuated by vascular spaces. In still others, the dilated vascular spaces are large (glomangioma), and they may have muscular walls (glomangiomyoma). The nucleus is usually small and round, with finely dispersed chromatin. Strong and diffuse SMA reactivity can be an aid to diagnosis. NOTCH gene rearrangements (most often NOTCH2) have been identified in the majority of glomus tumors (270). Most patients are young adults, who present with pain, particularly on exposure to cold. Rare glomus tumors that are cellular with mitoses and nuclear atypia are called atypical glomus tumors or malignant glomus tumor (glomangiosarcoma) (271); metastases occur in nearly 40% of these patients. The differential diagnosis of painful soft tissue tumors is listed in Table 5.8.

FIGURE 5.71 Glomus tumor. Small rounded cells with eosinophilic cytoplasm cluster around and between gaping vascular spaces. The nuclei are round to slightly oval.

LYMPHANGIOMYOMA OR LYMPHANGIOMYOMATOSIS Lymphangiomyoma is often associated with the tuberous sclerosis complex (272) with underlying TSC2 mutations (273). The tumor, which was originally described long ago in isolated case reports, has been incorporated into a family of neoplasms with perivascular epithelioid cell differentiation that are known as perivascular epithelioid cell tumors (PEComas) (see section “Perivascular Epithelioid Cell Tumors”). Lymphangiomyomatosis (LAM) is a well-recognized clinical entity characterized by a chylous pleural effusion or ascites that appears almost exclusively in female patients. Tumors in the mediastinum and lung are common, but they occasionally occur in the retroperitoneum. In the lung, they typically are multiple, and they appear as stellate lesions containing bundles of smooth muscle–like cells around dilated spaces. The smooth muscle–like cells in LAM are predominantly spindled, but may also be epithelioid, with granular eosinophilic cytoplasm (Fig. 5.72). In addition to expression of SMA and desmin, the lesional cells show reactivity for HMB-45 (although the latter may be limited in extent). Such tumors may be found diffusely throughout the lungs, and they frequently replace the local lymph nodes. The clinical course is variable, with resectable cases doing well; those patients with diffuse pulmonary involvement usually have a progressive course and succumb to disease after long periods. Recent studies have demonstrated the efficacy of mammalian target of rapamycin (mTOR) inhibitors such as sirolimus in stabilization of disease in patients with pulmonary LAM (274). Single-lung transplants have also been performed.

FIGURE 5.72 Lymphangiomyoma. This retroperitoneal tumor is composed of clear to granular eosinophilic cells growing within dilated lymphatic channels. The nuclei are uniform, small, and bland.

MYOPERICYTOMA Tumors of true pericytic lineage have been described (275,276); these lesions express myoid markers. Myopericytomas are composed of ovoid to spindle cells with eosinophilic cytoplasm and show a characteristic concentric ring–like perivascular growth pattern, which is seen best in the less cellular areas of the tumor or at its periphery (Fig. 5.73). By IHC, SMA is often reactive, and desmin may be found in some tumors. Myopericytoma forms a morphologic spectrum with myofibroma and angioleiomyoma (vascular leiomyoma) (24). A special form of pericytoma in the nasal sinuses (glomangiopericytoma) harbors CTNNB1 mutations, and shows aberrant nuclear β-catenin (277).

FIGURE 5.73 Myopericytoma. Eosinophilic spindle cells with ovoid nuclei are arranged around and between rounded and dilated, thin-walled vessels.

LESIONS OF PUTATIVE SYNOVIAL ORIGIN

TENOSYNOVIAL GIANT CELL TUMOR This lesion occurs in the following two clinically distinct forms: a localized form, which is also called giant cell tumor of tendon sheath, and a diffuse form clinically known as “pigmented villonodular synovitis” (PVNS) (278). The localized type is usually a small, circumscribed, lobulated mass affecting mainly the fingers. Most of the tumors are cellular, with numerous multinucleated osteoclastic giant cells dispersed throughout the lesion (Fig. 5.74). Accompanying these are variable numbers of inflammatory cells, namely, lymphocytes and small histiocytes. Scattered larger epithelioid cells with eosinophilic cytoplasm and, often, eccentric nuclei are a typical feature (Fig. 5.67). Hemosiderin is typically present, and it occasionally is prominent. The lesion has cleftlike spaces, and scattered mitotic figures may be found. The neoplastic epithelioid cells are positive for clusterin, which is a helpful diagnostic marker for diffuse cases with few or no giant cells (279). Desmin-positive, dendritic-like cells can be seen in around half of cases (280). Local recurrence develops in approximately 20% of cases.

FIGURE 5.74 Tenosynovial giant cell tumor. This diffuse-type tumor arose in extra-articular soft tissue. Sheets of small histiocytoid cells are punctuated by osteoclastic giant cells and larger mononuclear cells with vesicular, eccentric nuclei and small nucleoli. Note the stromal lymphocytes and hemosiderin deposition.

In the diffuse form, which is less common, the knee, hip, or ankle joints are affected by a florid papillary intra-articular proliferation, with identical histology to the localized type. The tumor often extends into the periarticular soft tissue. These lesions are much more locally aggressive, eroding joints and recurring in nearly half of cases. Extra-articular diffuse-type tenosynovial giant cell tumors also occur; these tumors are sometimes a diagnostic challenge, especially when the osteoclastic giant cells are few or absent (281). Tenosynovial giant cell tumors of both types contain the COL6A3-CSF1 gene fusion (282). MALIGNANT TENOSYNOVIAL GIANT CELL TUMOR Very rarely, tenosynovial giant cell tumors show a more aggressive appearance at the time of recurrence, and they may occasionally metastasize (281). Such recurrences often have areas without multinucleated giant cells, more tightly compact mononuclear cells with more notable nuclear atypia arranged in sheets, necrosis, and a high mitotic rate (>10 mitotic figures/10 hpf). Even more rarely, the primary tumor may be associated with a histologically malignant

component (283). Without the history (or conventional tenosynovial giant cell tumor component), it is essentially impossible to distinguish this tumor from UPS.

PERIPHERAL NERVE SHEATH TUMORS SCHWANNOMA Encapsulation combined with a dimorphic histology (cellular or Antoni A regions with loose myxoid Antoni B areas) defines this benign nerve sheath tumor. Schwannoma can be found at almost any site, but it is more common on the head and neck and extremities, where the tumors arise from small- to medium-sized nerves. Most lesions are painless, and only those that are larger or are undergoing cystic degeneration may cause pain. Uncommonly, large tumors are found in the posterior mediastinum or the retroperitoneum. The typical tumor is solitary, occurring in a middle-aged adult. An occasional cutaneous or subcutaneous schwannoma may exhibit a plexiform growth pattern, which should not cause confusion with neurofibroma; this type can be seen in neurofibromatosis type 2 or schwannomatosis, but it is usually sporadic. Histologically, a helpful clue to the diagnosis is the dimorphic growth pattern (Fig. 5.75). The Antoni A regions are cellular and fascicular with a low mitotic rate. The nuclei are plump, elongated, and wavy, occasionally pointed. Nuclear palisading is common, as are Verocay bodies (structures in which eosinophilic cell bodies are nearly encircled by parallel rows of nuclei). The Antoni B areas are hypocellular, and the cells lack orientation. They are loosely arranged in a myxoid matrix accompanied by thin strands of collagen. Occasional mast cells may be identified in the Antoni B areas. In addition, thick-walled vessels with hyalinized walls are often prominent and represent a helpful diagnostic feature. Degeneration with cyst formation should not be mistaken for necrosis, and rare atypical and pleomorphic nuclei (so-called ancient change) should not be taken as a sign of malignancy; these nuclei often contain smudgy chromatin and nuclear pseudoinclusions. Hypercellular tumors lacking Antoni B zones have been termed cellular schwannomas (see section “Cellular Schwannoma”). Strong and diffuse S100 protein and SOX10 reactivity is constant; the perineurial capsule can be highlighted with EMA. Rare variants include neuroblastoma-like schwannoma (284); benign glandular schwannoma, in which normal skin adnexal glands are entrapped in the superficial tumor (285); pseudoglandular schwannoma (286); and microcystic/reticular schwannoma (287). So-called melanotic schwannoma has recently been renamed malignant melanotic nerve sheath tumor in the 2020 WHO classification (see section “Malignant Melanotic Nerve Sheath Tumor”) (2,288).

FIGURE 5.75 Schwannoma. A dimorphic pattern of cellular (Antoni A) and loose myxoid (Antoni B) areas is typically seen. Note the parallel rows of nuclei (nuclear palisading).

CELLULAR SCHWANNOMA Occasional highly cellular nerve sheath tumors may be mistaken for malignant neoplasms, of which cellular schwannoma is a prime example (289-291). This lesion has a predilection for the paravertebral retroperitoneum, pelvis, and posterior mediastinum and may be large. Cellular fascicles and sheets of plump eosinophilic spindle cells with mild nuclear variability and bulletshaped nuclei are characteristic; hyalinized thick vessels and foamy histiocytes are also seen. A helpful diagnostic feature is a thick capsule, often with prominent lymphoid aggregates. Unlike MPNST, cellular schwannomas exhibit strong, diffuse S-100 protein and SOX10 reactivity. GRANULAR CELL TUMOR This Schwann cell neoplasm is composed of epithelioid cells with abundant granular eosinophilic cytoplasm and bland, often pyknotic nuclei, which are arranged in sheets and trabeculae (Fig. 5.76). Cytoplasmic granularity may be seen in several other tumor types, most often smooth muscle tumors and melanoma. The tumor cells are strongly positive for S-100 protein and SOX10. Granular cell tumors very rarely recur, even following marginal or incomplete excision. A malignant counterpart has similar morphology but with prominent nucleoli and mitotic activity (see section “Malignant Granular Cell Tumor”).

FIGURE 5.76 Granular cell tumor. Sheets and nests of epithelioid cells contain abundant granular eosinophilic cytoplasm and small, pyknotic nuclei. An infiltrative growth pattern is typical. Strong and diffuse expression of S-100 protein is observed (not shown).

NEUROFIBROMA Unlike schwannoma, neurofibroma is an unencapsulated nerve sheath tumor that may occur in the following forms: (1) a solitary localized, often cutaneous nodule; (2) a diffuse thickening of the skin and subcutaneous tissues; or (3) a plexiform tumor, with a wormlike, multinodular growth within major or minor nerves. Although only the last form is nearly pathognomonic of type 1 neurofibromatosis, that autosomal dominant disease may also produce solitary or diffuse lesions. Neurofibromas may be found virtually anywhere in the skin or subcutaneous tissues.

Neurofibroma differs from schwannoma in that a uniform histology is usually notable at low power (Fig. 5.77A). The borders infiltrate the adjacent dermis and adipose tissue, even in the apparently circumscribed solitary type. In both the solitary and diffuse forms, the skin appendages are frequently surrounded by the proliferation. Although deeper tumors may be either quite myxoid or extensively collagenized, the more superficial cutaneous types are moderately cellular. The cells have scanty but elongated cytoplasm, appearing as extremely thin, pale eosinophilic fibrillar structures. The nucleus may be oval, but it often is wavy or comma shaped (Fig. 5.77B). Randomly dispersed, pleomorphic nuclei in the absence of mitotic activity should be ignored; they represent a type of degenerative phenomenon, with such lesions called ancient neurofibroma (or “neurofibroma with atypia”).

FIGURE 5.77 Neurofibroma. One of several expanded nerves is seen in plexiform neurofibroma (A). Neurofibromas are usually uniformly hypocellular and slightly myxoid, with an undulating fibrillar background (B); they may show nuclear atypia (“ancient change”), which should not be misconstrued as a sign of malignancy.

There has been a recent consensus classification of the minimal criteria for MPNST with the establishment of a new diagnostic term, atypical neurofibromatous neoplasm of uncertain biologic potential (ANNUBP), which only applies to neurofibromas in patients with type 1 neurofibromatosis. ANNUBP is characterized by at least two of the following features: cytologic atypia, hypercellularity, loss of neurofibroma architecture (on H&E and/or CD34 IHC), and a mitotic count less than three per 10 hpf (292). This diagnostic category is considered premalignant; such lesions appear to have an increased risk of progression. Scattered thin axonal processes usually can be demonstrated with neurofilament IHC. In contrast to schwannoma (which is a pure Schwann cell neoplasm), S-100 protein is often positive in only 50% to 70% of cells in neurofibromas. Reactivity for CD34 is common, and scattered EMA-positive cells may also be observed. The overall cellularity of a neurofibroma, unlike that of many other tumors, is quite uniform throughout, and the typical benign tumor is never highly cellular. Very rarely, these tumors may be pigmented, granular, epithelioid, or even cellular in appearance; however, any epithelioid morphology should be viewed with caution, and it should be accompanied by a search for mitotic figures. In the plexiform type (Fig. 5.77A), the cellular proliferation expands contiguous nerves, which, at low power, appear as small nodules with a dense eosinophilic rim representing the remnant of the original nerve (293). Circular structures of fibrillar eosinophilic material outlined by nuclei reminiscent of Wagner-Meissner corpuscles are characteristic of diffuse neurofibroma, although these structures may be scarce. Approximately 10% of patients with the diffuse form have neurofibromatosis type 1. This diffuse form may be confused with spindle cell lipoma or DFSP when it occurs predominantly within fatty tissue. IHC for S-100 protein confirms the diagnosis in this differential. Tumors with hybrid morphology (combined schwannoma and neurofibroma) can

also be identified, in which a schwannomatous nodule arises within an otherwise typical neurofibroma (294). SOFT TISSUE PERINEURIOMA Soft tissue perineurioma is an uncommon benign nerve sheath neoplasm that presents over a wide anatomic distribution with no gender or age predilection (295). The majority of tumors arise in subcutaneous tissue, although deep soft tissue and cutaneous primary tumors may also be encountered. These tumors show a storiform, whorled, and lamellar architecture (Fig. 5.78) and are composed of ovoid to elongated spindle cells with delicate bipolar cytoplasmic processes, which may not be evident on H&E. The perineurial cell of normal nerves and the corresponding tumor are positive for EMA, although in some cases, reactivity may be quite weak. CD34 is also positive in around 60% of cases, and staining for the tight junction–associated protein claudin-1 is seen in a subset of cases (296). Thus, if the pathologist encounters a neurofibroma-like, S-100 protein–negative nerve sheath tumor, it may still be of nerve sheath origin, and EMA should be applied. The sclerosing variant shows a marked predilection for the fingers and hands, and is characterized by dense fibrosis within which small, rounded cells arranged in cords and strands are embedded (297). EMA highlights the cytoplasmic processes in this variant. The reticular variant shows a delicate, netlike arrangement of slender spindle cells in a myxoid stroma (298). An unusual hybrid nerve sheath tumor composed of an intimate admixture of Schwann cells and perineurial cells has recently been described (299). This hybrid schwannoma/perineurioma shows architectural features similar to that of soft tissue perineurioma; however, the dominant cell population is Schwannian in nature, containing plump, tapering nuclei. By IHC, S-100 protein–positive Schwann cells alternate with EMA and CD34-positive perineurial cells. Perineuriomas and hybrid schwannoma/perineuriomas are benign and very rarely recur.

FIGURE 5.78 Soft tissue perineurioma. This moderately cellular spindle cell tumor shows a storiform-to-whorled growth pattern. The tumor cells contain slender nuclei and eosinophilic cytoplasm. Epithelial membrane antigen (EMA) is positive, although staining can be weak (not shown).

NEUROFIBROMATOSIS Neurofibromatosis, a single-gene disorder, is a relatively common autosomal dominant condition affecting 1 in 3000 live-born children (300). Although its penetrance is nearly 100%, the expression of the gene varies greatly. The disease takes two forms. In the better recognized

form, which is also referred to as von Recklinghausen disease (neurofibromatosis type 1), patients have multiple café au lait spots and dermal neurofibromas. In the less common neurofibromatosis type 2, patients have predominantly bilateral acoustic “neuromas” (schwannomas), which are associated with other brain and spinal cord tumors. The gene for neurofibromatosis type 1 is located on chromosome 17 (NF1), and its large protein product is designated neurofibromin 1. The gene for neurofibromatosis type 2 (NF2) maps to chromosome 22; it encodes a protein designated neurofibromin 2 (formerly known as merlin). Neurofibromatosis type 1 is diagnosed on the basis of the presence of any two of the following: 1. 2. 3. 4. 5. 6. 7.

At least five café au lait spots larger than 5 mm (6 > 15 mm if the patient is prepubertal) Two or more neurofibromas of any type or one plexiform type Multiple large freckles in the axillary or inguinal regions Sphenoid wing dysplasia Bilateral optic nerve gliomas Multiple iris nodules (Lisch spots) A first-degree relative with the preceding criteria

In addition to cutaneous tumors, patients may exhibit solitary deep tumors; diffuse gastrointestinal ganglioneuromatosis; brain and spinal cord tumors, including meningiomas and ependymomas; GISTs; and, occasionally, malignant progression within a neurofibroma (to MPNST). The lifetime risk of MPNST in patients with neurofibromatosis type 1 is around 10%. The phenomenon of divergent differentiation in MPNST is far more common in patients with neurofibromatosis type 1. A significant percentage of patients with neurofibromatosis type 1 die early, but a much higher percentage has a favorable long-term survival. MALIGNANT PERIPHERAL NERVE SHEATH TUMOR MPNST may arise sporadically (50%), in patients with neurofibromatosis type 1 (40%) or following therapeutic radiation therapy (10%). The diagnosis is straightforward for histologically malignant spindle cell neoplasms that arise from large nerves, from a preexisting neurofibroma, or in patients with neurofibromatosis (301,302). Outside of these associations, the diagnosis is challenging, and careful attention must be paid to characteristic architectural and nuclear features. With many MPNSTs, the first impression is that of a highly cellular, fibrosarcoma-like tumor, such as the one depicted in Figure 5.79. A search for more specific features should ensue. The morphologic features which, if they are present in combination, help confirm the diagnosis of MPNST include the following: (1) alternating hypocellular and hypercellular regions; (2) the appearance of the thin, wavy, comma-shaped, or bullet-shaped nuclei (Fig. 5.79A), particularly in hypocellular areas; (3) perivascular accentuation of cellularity; (4) nervelike whorls (Fig. 5.79B); (5) a prominent, thick-walled vasculature; and (6) heterologous elements. For example, isolated RMS elements in a spindle cell sarcoma (“Triton” tumor; see section “Divergent Differentiation”) practically identify the spindle cell element as being of nerve sheath origin. By IHC, only 40% to 50% of MPNST show limited staining for S-100 protein or SOX10. Recent studies have demonstrated that loss of lysine 27 trimethylation of histone H3 (H3K27me3), which can be detected by IHC, is a highly specific and moderately sensitive diagnostic marker, most helpful for high-grade tumors (303, 304).

FIGURE 5.79 Malignant peripheral nerve sheath tumor. In this mitotically active and cellular tumor, occasional nuclei have a bullet shape—blunt at one end and pointed at the other (A). In a spindle cell sarcoma, nervelike whorls (B) are another clue to nerve sheath differentiation.

With regard to distribution, MPNSTs occur almost anywhere in the body, including the head and neck and retroperitoneum, but a peripheral location on the extremities is more common in the sporadic form, whereas central lesions on the trunk, paravertebral region, or head and neck predominate in neurofibromatosis. The limb-girdle regions are commonly affected in either type. Tumors in patients with neurofibromatosis type 1 tend to occur at an earlier age, and a male predominance is noted. When MPNSTs are associated with neurofibromatosis type 1, they can develop from long-standing benign neurofibromas. Otherwise, documentation of an MPNST arising from a precursor lesion is exceedingly rare. Occasional MPNSTs are associated with pain and paresthesias, although most are without other symptoms. Several histologic types of MPNST exist. Some cases resemble fibrosarcoma, composed of a dense population of spindle cells with fascicles intersecting at acute angles (the herringbone pattern). This is perhaps the most common pattern, but hypocellular areas can generally be found with the characteristic wavy (but also bullet-shaped) nuclei. The presence of these alternating hypercellular and hypocellular areas is suggestive of nerve sheath origin. Other tumors resemble neurofibromas. In this subtype, the pathologist must ignore the bizarre nuclear changes in assessing malignancy and instead search for nuclear atypia, hypercellularity, and mitotic activity (see ANNUBP in the section “Neurofibroma”). A recent consensus statement established three or more mitotic figures per 10 hpf, along with cytologic atypia, hypercellularity, or loss of neurofibroma architecture as minimal criteria for low-grade MPNST (292). Occasional cases display extreme nuclear pleomorphism with a UPS-like appearance, a type found almost exclusively in cases of neurofibromatosis type 1. The purely epithelioid type (305), which accounts for approximately 5% of all MPNSTs, is a difficult variant to diagnose. Epithelioid MPNST is pathogenetically distinct and may easily cause confusion with carcinoma or especially metastatic melanoma (Table 5.10). The epithelioid type grows in a multinodular pattern and is composed of sheets of epithelioid cells with rounded nuclei, vesicular chromatin, prominent nucleoli, and moderate amounts of eosinophilic cytoplasm. Unlike conventional MPNST with spindle cell morphology, which shows at most patchy or focal reactivity for S-100 protein or SOX10 in only 40% to 50% of cases, these markers are strongly and diffusely positive in epithelioid MPNST, although melanocytic markers (HMB-45, melan A) are negative. Of note, the majority of epithelioid MPNSTs show loss of expression of SMARCB1 (INI1) (306), distinguishing this tumor type from melanoma, although nuclear staining for H3K27me3 is retained (i.e., normal). DIVERGENT DIFFERENTIATION

The capacity of nerve sheath tumors to exhibit heterologous elements is derived from their origin in the neural crest (307,308), where the mesenchymal tissue is pluripotent, forming the entire soft tissue of the head. Thus, in addition to RMS elements (malignant Triton tumor) (309), one may find cartilaginous, osteoblastic, and, rarely, angiosarcomatous differentiation. Very rarely, the entire tumor may be replaced by rhabdomyoblastic cells; such tumors closely mimic spindle cell RMS (310). In such cases, identifying loss of H3K27me3 is critical. A peculiar finding is the rare case with glandular (epithelial) differentiation (311). The glandular component, which usually has intestinal characteristics, can occur in isolation; it can appear benign or malignant; and it can contain evidence of neuroendocrine differentiation. Tumors with heterologous rhabdomyoblastic differentiation have the worst prognosis of all MPNSTs, and they occur more frequently in patients with neurofibromatosis type 1. MALIGNANT MELANOTIC NERVE SHEATH TUMOR New nomenclature has recently been introduced for the tumors formerly known as melanotic schwannomas, because these tumors often demonstrate aggressive clinical behavior. Now known as malignant melanotic nerve sheath tumor, these rare lesions are composed of Schwann cells with melanocytic differentiation and are frequently associated with Carney complex; the majority of both sporadic and Carney complex–associated tumors have biallelic inactivating mutations in the tumor suppressor gene PRKAR1A (312). This tumor type often arises in the paraspinal region, associated with spinal nerves. These tumors are composed of sheets of relatively uniform, variably ovoid, spindled, or epithelioid cells with eosinophilic cytoplasm, ovoid nuclei often with nuclear grooves, and coarse or fine melanin pigment that can obscure the nuclear morphology (Fig. 5.80). Psammoma bodies are common and have no clinical significance. Some tumors contain cells with prominent melanoma-like nucleoli. By IHC, S-100 protein, SOX10, and the melanocytic markers HMB45 and melan A are positive; distinguishing this tumor type from melanoma can be a significant challenge. PRKAR1A expression is frequently lost, which is helpful to confirm the diagnosis (313). Histologic features do not correlate well with clinical behavior; local recurrence is common, and more than 40% of tumors metastasize (313).

FIGURE 5.80 Malignant melanotic nerve sheath tumor. This often aggressive malignant neoplasm was formerly known as melanotic schwannoma. In many cases, abundant melanin pigment obscures the tumor cell morphology (A). In less heavily pigmented areas, the characteristic nuclear grooves can be appreciated in this case (B).

OSTEOCARTILAGINOUS LESIONS

Myositis ossificans is discussed in the section “Soft Tissue Lesions: Lesions Mimicking Sarcomas (Pseudosarcomas).” EXTRASKELETAL CHONDROMAS AND OSTEOMAS Easily recognizable benign cartilaginous and osseous tumors may occur within the soft tissues. Soft tissue chondromas are more common on the hands and in the head and neck region, and because of the lack of pleomorphism or mitotic figures, they are easily identified as benign lesions. The combination of osteoclastic giant cells, bland spindle cells in a pericytomatous pattern, grungy calcification, variable chondroid matrix, and sometimes lamellar bone is typical of the rare tumor associated with osteomalacia and hypophosphatemia, known as phosphaturic mesenchymal tumor (314). EXTRASKELETAL CHONDROSARCOMAS Two types of soft tissue tumors are designated “chondrosarcomas.” Extraskeletal myxoid chondrosarcoma is not, in fact, a chondrosarcoma; it is a translocation-associated neoplasm of uncertain differentiation; nevertheless, it is discussed here (315-317). This tumor type most often presents in the deep soft tissue of the limb girdles and proximal extremities of adults. Extraskeletal myxoid chondrosarcoma has an abundant, hypovascular myxoid stroma and a lobulated or multinodular growth pattern, in contrast to most other myxoid lesions (see Table 5.7); the lobules are demarcated by fibrous septa. This tumor type is composed of uniform spindled or small epithelioid cells that display a characteristic reticular or trabecular arrangement, with interconnecting, slender, eosinophilic cytoplasmic processes (Fig. 5.81). S-100 immunoreactivity is seen in only a minority of cases, as is synaptophysin; a recent study suggested INSM1 is a highly sensitive and relatively specific marker (318). Molecular analysis (see Table 5.3) shows the presence of NR4A3 gene rearrangements, most often with EWSR1, but sometimes with other fusion partners (319,320). Its clinical course is often indolent. Conventional histology is associated with late recurrences and predominantly lung metastases, often developing 10 to 15 years after diagnosis; however, a more rapid course is possible, particularly with the rare, high-grade cellular variants, some of which show rhabdoid morphology. Non-EWSR1 NR4A3 rearrangements appear to correlate with high-grade histology and more aggressive behavior (320). Extraskeletal myxoid chondrosarcoma may be confused with soft tissue myoepitheliomas (see section “Myoepithelioma and Mixed Tumors”). Myoepithelial tumors often show more histologic heterogeneity, including nested and solid areas, and reactivity for keratins, EMA, S-100, and sometimes glial fibrillary acidic protein (GFAP).

FIGURE 5.81 Extraskeletal myxoid chondrosarcoma. In these lobules at medium power, the myxoid substance is occupied by thin cords or strands of cells radiating toward the periphery, like spokes of a wheel.

Mesenchymal chondrosarcoma may arise in bone or soft tissue (40%); the most common soft tissue sites include head and neck and meninges (321). On excision, recognition is generally straightforward, when the nodules of well-differentiated hyaline cartilage within an otherwise primitive-appearing population of small round or short spindle cells with prominent staghorn vessels are appreciated (Fig. 5.82). The tumor is more difficult to recognize when the cartilaginous foci are extremely small and widely scattered. In these cases, the diagnosis rests on recognition of the sometimes minute areas of cartilaginous differentiation. By IHC, mesenchymal chondrosarcoma is often positive for both CD99 and NKX2.2 (322), potentially leading to confusion with Ewing sarcoma. Mesenchymal chondrosarcoma harbors a consistent HEY1-NCOA2 gene fusion (323). Patients with mesenchymal chondrosarcoma experience a protracted course, often with late metastases 5 to 15 years after primary excision.

FIGURE 5.82 Extraskeletal mesenchymal chondrosarcoma. Islands of cartilage are dispersed within sheets of uniform small round cells. If the cartilaginous component is not sampled, the diagnosis can be particularly challenging and includes Ewing sarcoma as well as other round cell neoplasms.

MYOEPITHELIOMA AND MIXED TUMORS Myxoid tumors of soft tissue with myoepithelial differentiation are termed soft tissue myoepitheliomas and mixed tumors (324,325); a tumor is mixed if it shows ductal differentiation and resembles pleomorphic adenoma of salivary glands. These often myxoid tumors simulate extraskeletal myxoid chondrosarcoma, but express keratins, EMA, S-100 protein, GFAP, p63, and SOX10 in various combinations; SMA and calponin are often positive as well, but are not diagnostically helpful. In addition to a reticular growth pattern with epithelioid cells, soft tissue myoepitheliomas may show nested or solid architecture and contain spindled, plasmacytoid (hyaline), or clear cells. Myoepitheliomas composed of large epithelioid cells with vacuolated cytoplasm were formerly known as parachordomas. Myoepithelial tumors with moderate to severe nuclear atypia (often accompanied by a high mitotic rate) behave in an aggressive manner with frequent metastases; such tumors are known as myoepithelial carcinomas (Fig. 5.83) (325). Myoepithelial tumors of soft tissue usually contain EWSR1 gene rearrangements, similar to extraskeletal myxoid chondrosarcoma, but with different fusion partners (POU5F1, PBX1, or ZNF444, among others) (326); mixed tumors usually harbor PLAG1 gene rearrangements, similar to their salivary gland and cutaneous counterparts (pleomorphic adenoma and chondroid syringoma, respectively) (327,328). Tumors with PLAG1 gene fusions show nuclear staining for the PLAG1 protein (328). A subset of myoepithelial carcinomas shows loss of SMARCB1 (INI1) expression, usually as a consequence of gene deletion (329).

FIGURE 5.83 Myoepithelial carcinoma of soft tissue. Myoepithelial tumors often show variations in cellularity and cytology; this deep soft tissue tumor contains myxoid areas with spindle cells and solid, nested areas with epithelioid cells. The latter component can easily be mistaken for metastatic carcinoma. The presence of moderate or severe nuclear atypia warrants a diagnosis of malignancy.

EXTRAOSSEOUS OSTEOSARCOMA In the soft tissue, osteosarcoma may exhibit all the various histologic features found in primary bone tumors. However, in contrast to bone tumors, most soft tissue osteosarcomas occur in middle-aged and elderly patients (330,331). They may present with pain of long duration in the affected region. Extraskeletal osteosarcoma is an aggressive sarcoma type with a high risk of distant metastasis. The zonation pattern of myositis ossificans is not found; instead, the advancing edge of this tumor is highly cellular, often without osteoid formation. As with osseous and cartilaginous tumors arising in bone, the pathologist should be reluctant to diagnose

extraskeletal osteosarcoma in the digits; in such locations, benign fibro-osseous pseudotumor (332) or reactive periostitis is more common, and it may be difficult to diagnose because it lacks the typical zonation of myositis ossificans. Extraskeletal osteosarcoma can arise in the postradiation setting, and on rare occasions, it can exhibit a markedly well-differentiated morphology or small cell features. The distinction between hyalinized collagen and osteoid can be challenging; in such cases, nuclear staining for SATB2 supports the presence of osteoblastic differentiation and a diagnosis of osteosarcoma (333).

UNUSUAL LESIONS SYNOVIAL SARCOMA Synovial sarcoma is a misnomer because this translocation-associated mesenchymal neoplasm shows no evidence of synovial differentiation; instead, this tumor type shows variable epithelial differentiation (334). In the distant past, only the biphasic variant was recognized. The monophasic variant outnumbers the biphasic type by a ratio of at least 3 to 1. Synovial sarcomas most often affect adolescents and young adults, and typically arise in the deep soft tissue of the extremities, followed by the trunk, and head and neck, although this sarcoma type also arises in older adults and visceral locations. Synovial sarcoma often pursues an aggressive course, similar to other common adult sarcomas. Some patients die of disease early, but in a fair proportion of cases, the clinical course is prolonged, with late recurrences, metastases, and death even beyond 10 years. Unlike most other adult sarcomas, synovial sarcoma shows relative chemosensitivity, with even metastatic disease showing regression in some cases. The prognosis in synovial sarcoma is related to tumor size and margin status (335). The molecular analysis of synovial sarcoma is diagnostic: the SS18-SSX1 or SS18-SSX2 fusion gene resulting from t(X;18) translocations is specific (336,337). Biphasic synovial sarcoma is histologically distinctive and straightforward to diagnose. The glandular component (Fig. 5.84A) may be prominent or obscure (in some cases, only evident by IHC for keratins or EMA); the tumor cells in the glands contain moderate amounts of pale eosinophilic cytoplasm and bland, oval nuclei. The usually dominant spindle cell component is usually highly cellular and fascicular with remarkably uniform, often overlapping nuclei with fine chromatin and scant cytoplasm. Thin-walled, dilated, branching (staghorn) vessels are often prominent. As in MPNST, alternating hypocellular and hypercellular regions can sometimes be seen.

FIGURE 5.84 Synovial sarcoma. The biphasic type frequently has an obvious glandular component, which here is noted by its lighter cytoplasm and intraluminal material. Note the small, oval but overlapping nuclei in the spindle cell component (A). Many monophasic cases contain thin-walled, dilated (“staghorn”) vessels (B); a careful search in this case revealed focal cell clusters (C), indicating epithelial differentiation. Keratins are strongly positive in the glands with limited staining in the spindle cell component (D).

Monophasic synovial sarcoma may easily simulate MPNST, cellular SFT, mesenchymal chondrosarcoma, and fibrosarcoma. A herringbone pattern or a hemangiopericytoma-like pattern (Fig. 5.84B) may predominate. However, clues to the nature of the tumor include the presence of uniform, small, oval, overlapping nuclei (Fig. 5.84A), the identification of rare clusters of more plump and eosinophilic (epithelial) cells (Fig. 5.84C), the lack of the wavy nuclei, and the lack of cartilaginous differentiation. Essentially, any highly cellular tumor with the aforementioned differential diagnosis occurring on the extremity of a young adult should be considered monophasic synovial sarcoma until proven otherwise. By IHC, nearly 90% of monophasic synovial sarcomas show at least focal staining for keratins (Fig. 5.84D) and EMA. Synovial sarcomas are also sometimes positive for S-100 protein (30%); they are negative for CD34. TLE1 shows strong nuclear staining in around 90% of cases, but this marker shows only modest specificity (338). Recently, an SS18-SSX fusion-specific antibody has been developed for IHC; this marker is 95% sensitive and entirely specific for synovial sarcoma (339). Poorly differentiated synovial sarcoma is a particularly aggressive variant that is often dominated by uniform round cells with a high mitotic rate (Fig. 5.85) (340,341); this variant may be mistaken for Ewing sarcoma. A helpful clue to the diagnosis is the presence of staghorn vessels. A rare example is myxoid synovial sarcoma (342), which is a challenge to recognize.

FIGURE 5.85 Poorly differentiated synovial sarcoma. This highly aggressive variant, which often shows round cell morphology, may be mistaken for Ewing sarcoma or other round cell sarcomas.

ALVEOLAR SOFT PART SARCOMA Despite its rarity, alveolar soft part sarcoma is generally easy for pathologists to recognize, given its unique histologic appearances (343,344). This sarcoma type has a marked predilection for the deep soft tissue of the extremities and a female predominance. In adults, the thigh is the most common site, whereas in children, there is a predilection for the head and neck (especially tongue). Most lesions contain large areas with a nested and alveolar pattern; the tumor cells are uniform and epithelioid, with large, round nuclei with a single prominent nucleus, and granular eosinophilic cytoplasm (Fig. 5.86). The nests are surrounded by a delicate capillary vasculature. A common feature is vascular invasion. Mitoses are generally scarce. Lingual tumors often show a predominantly solid growth pattern (345). The tumor may closely resemble areas in renal cell carcinoma, which should be included in the differential diagnosis, along with melanoma, metastatic adrenal cortical carcinoma, and clear cell sarcoma in some cases. The natural history can be prolonged; lung, bone, and brain are the most common metastatic sites, often developing more than a decade following diagnosis.

FIGURE 5.86 Alveolar soft part sarcoma. A thin microvasculature surrounds the nests of large polygonal cells, giving the impression of solid alveoli or an organoid appearance; the cells have uniform round nuclei with prominent nucleoli and abundant granular eosinophilic cytoplasm.

Alveolar soft part sarcoma characteristically harbors an unbalanced translocation der(17)t(X;17), with a corresponding ASPSCR1-TFE3 fusion gene, specific for this tumor type (346). TFE3 immunoreactivity is useful in the diagnosis of alveolar soft part sarcoma (347). MALIGNANT GRANULAR CELL TUMOR Although well-documented examples of malignant granular cell tumor exist, many reported tumors likely represent misdiagnosed metastatic melanomas. The tumor closely resembles conventional granular cell tumor and grows in sheets, nests, and cords (Fig. 5.87), without the organoid pattern of alveolar soft part sarcoma. Granular cell tumors with three or more of the following features should be diagnosed as malignant: (1) necrosis; (2) vesicular nuclei with large nucleoli, which is in contrast to the small and pyknotic nuclei of benign tumors; (3) pleomorphism; (4) high nuclear/cytoplasmic ratio; (5) spindle cell morphology; and (6) any appreciable mitotic rate (348). Tumor size is not a helpful distinguishing feature. A tumor with one or two of the preceding features can be labeled as atypical or a tumor of uncertain malignant potential to ensure close follow-up. Malignant granular cell tumors pursue an aggressive course with a high metastatic rate.

FIGURE 5.87 Malignant granular cell tumor. Unlike its benign counterpart, this tumor has vesicular nuclei with prominent nucleoli; mitoses were found elsewhere. These features alone are worrisome; spindling areas and necrosis confirm the diagnosis of malignancy.

MALIGNANT RHABDOID TUMORS First described in the kidneys, malignant rhabdoid tumor also arises in deep soft tissues of the axial region, retroperitoneum, abdominal cavity, pelvis, and liver. This tumor type is characterized by sheets of epithelioid cells with vesicular nuclei, prominent nucleoli, and intracytoplasmic hyaline inclusions (Fig. 5.88), which contain whorls of intermediate filaments (349,350). However, similar cytology has been documented in a wide variety of tumors in adults, most often metastatic melanoma, but also in various sarcomas, carcinomas, and mesothelioma. The distinction between malignant rhabdoid tumor and epithelioid sarcoma (particularly the proximal type) may at times be difficult, if not impossible; patient age is a helpful distinguishing feature. Malignant rhabdoid tumors affect infants and young children nearly exclusively; when a morphologically similar tumor is encountered in adults, the possibility of melanoma or the other

tumor types listed earlier should be considered. Malignant rhabdoid tumors are highly aggressive and usually lethal.

FIGURE 5.88 Malignant rhabdoid tumor. Vesicular nuclei with prominent nucleoli and hyaline eosinophilic cytoplasmic inclusions are characteristic features. Loss of nuclear staining for SMARCB1 (INI1) confirms the diagnosis (not shown). This histologic pattern may also be seen in a range of tumors in adults.

By IHC, malignant rhabdoid tumors are often positive for vimentin, keratins, and EMA; desmin and other muscle markers are not expressed. These neoplasms nearly always exhibit biallelic inactivation of the SMARCB1 gene at 22q11.2 (351). The diagnosis can be confirmed by demonstrating loss of SMARCB1 (INI1) expression by IHC (352). It should be noted, however, that several other tumor types also show loss of SMARCB1, including epithelioid sarcoma, epithelioid MPNST, and a small subset of myoepithelial carcinomas of soft tissue. EPITHELIOID SARCOMA Epithelioid sarcoma often presents with a distinctive clinical appearance, and most often affects adolescents and young adults with a male predominance (353). Many cases have a subcutaneous or deep dermal location, and these cases often appear as prominent nodules raised above the skin surface with central ulceration. Fingers and hands are more commonly affected than are lower extremities. When the diagnosis is delayed, multiple nodules of this type may develop in a line from the wrist progressing toward the elbow. Still other cases have a more deep-seated location, and their presentation is less obvious. Histologically, the tumor is a mixture of spindled and rounded or polygonal eosinophilic cells with small nuclei and small nucleoli. These spindled and epithelioid cells appear to merge imperceptibly (Fig. 5.89). The growth pattern is often nodular with central ischemic-type necrosis, and, frequently, an inflammatory cell infiltrate in the form of lymphocytes and plasma cells occupies the periphery. For this reason, the differential diagnosis may include inflammatory (necrobiotic) processes, such as a rheumatoid nodule. However, the cells of epithelioid sarcoma are more densely eosinophilic, the nuclei are more vesicular than those of palisading histiocytes, and the IHC profile is completely different. Broad areas of fibrosis and hyalinization may occur in some cases, wherein the cells appear to radiate from a central scarred area. Rhabdoid cells may be apparent, and a pseudoangiomatous pattern may rarely be noted. The so-called proximal type of epithelioid sarcoma typically arises in the inguinal region and axilla of somewhat older adults and contains sheets of epithelioid cells with prominent nucleoli and amphophilic or eosinophilic cytoplasm (Fig. 5.90), resembling poorly

differentiated (large cell) carcinoma (354), more often with a rhabdoid component than conventional epithelioid sarcoma. Epithelioid angiosarcoma, melanoma, and poorly differentiated carcinoma may be considered in the differential diagnosis (see Table 5.10). By IHC, most cases are positive for keratins and EMA; CD34 reactivity, which is found in 50% to 60% of cases (355), helps distinguish epithelioid sarcoma from carcinomas. SMARCB1 (INI1) is particularly helpful diagnostically because around 90% of epithelioid sarcomas show loss of nuclear staining (356).

FIGURE 5.89 Epithelioid sarcoma. At low power, a tumor nodule is enveloped by a fibroinflammatory cuff (A). Note the central necrosis. At high power (B), epithelioid to spindle cells with eosinophilic cytoplasm form a sheet at the periphery of the nodule. Small nucleoli can be seen.

FIGURE 5.90 Proximal-type epithelioid sarcoma. This inguinal tumor is composed of sheets of large epithelioid cells with prominent nucleoli. This tumor type is typically positive for keratins and epithelial membrane antigen (EMA; not shown) and may closely mimic metastatic poorly differentiated (large cell) carcinoma.

Local recurrence is highly likely without early detection and surgery, with the subsequent appearance of metastases to lymph nodes and lung in a high percentage of cases. The indolent yet persistent nature of this tumor should be a factor in determining therapy. Amputation, although not part of the management of most other sarcomas, is often a consideration, but attempts to avoid it are increasingly successful. CLEAR CELL SARCOMA Clear cell sarcoma characteristically develops in deep soft tissues (tendons and aponeuroses) of the lower extremities (especially feet and ankles) of young adults, commonly causing pain. The most recognizable feature of this sarcoma type is its growth pattern (357,358). In most cases, nests and fascicles of tumor cells are divided by thin fibrous septa (Fig. 5.91A). The cells may be epithelioid or spindled and typically have round to oval nuclei with vesicular chromatin and prominent central nucleoli. The nuclei are remarkably uniform without pleomorphism. Scattered wreath-like giant cells are a helpful diagnostic feature. Although the name implies a cell with clear cytoplasm, most cases have a pale eosinophilic cytoplasm; in only a small subset of tumors is the cytoplasm notably clear. A previous designation for this tumor type was melanoma of soft parts (359). As with melanomas, the tumors frequently contain melanin. Also similar to melanomas, the lesions are positive for S-100 protein (Fig. 5.91B), SOX10, HMB-45, and melan A. The typical cytogenetic abnormality is t(12;22)(q13;q12), resulting in an EWSR1-ATF1 fusion (360) (Table 5.3). FISH for EWSR1 is often applied to confirm the diagnosis and to distinguish this tumor from melanoma. This is typically a slowly growing lesion of long duration; metastases to lymph nodes and lung often develop after a long disease-free interval, often 10 years or longer. The prognosis is more favorable if the tumor is small ( 8 cm in size and confined to a single pelvic segment with no extraosseous extension T2a A tumor 8 cm or less in size and confined to a single pelvic segment with extraosseous extension or confined to two adjacent pelvic segments without extraosseous extension T2b A tumor > 8 cm in size and confined to a single pelvic segment with extraosseous extension or confined to two adjacent pelvic segments without extraosseous extension T3a A tumor 8 cm or less in size and confined to two pelvic segments with extraosseous extension T3b A tumor > 8 cm in size and confined to two pelvic segment with extraosseous extension T4a Tumor involving three adjacent pelvic segments or crossing the sacroiliac joint to the sacral neuroforamen T4b Tumor encasing the external iliac vessels or gross tumor thrombus in major pelvic vessels Note: The four pelvic segments are the sacrum lateral to the sacral foramen, iliac wing, acetabulum/periacetabulum and pelvic rami, and symphysis and ischium. N—Regional Lymph Nodes NX Regional lymph nodes cannot be assessed N0 No regional lymph node metastasis N1 Regional lymph node metastasis M—Distant Metastasis M0 No distant metastasis M1 Distant metastasis M1a Lung M1b Other distant sites STAGE GROUPING (APPENDICULAR SKELETON, TRUNK, SKULL AND FACIAL BONES) Stage

Tumor (T)

Node (N)

Metastasis (M)

Grade (G)

Stage IA

T1

N0

M0

G1, GX low grade

Stage IB

T2

N0

M0

G1, GX low grade

T3

N0

M0

G1, GX low grade

Stage IIA

T1

N0

M0

G2, 3 high grade

Stage IIB

T2

N0

M0

G2, 3 high grade

Stage III

T3

N0

M0

G2, 3 high grade

Stage IVA

Any T

N0

M1a

Any G

Stage IVB

Any T

N1

Any M

Any G

Any T

Any N

M1b

Any G

Note: The 2017 TNM staging eighth edition can be applied to all primary malignant bone tumors, except malignant lymphoma, multiple myeloma, surface osteosarcoma, and periosteal chondrosarcoma. Regional lymph node involvement is rare in bone tumors. Adapted from Kneisl JS, Rosenberg AE, Anderson PM, et al. Bone. In: Amin MB, Greene FL, Edge S, et al., eds. AJCC Cancer Staging Manual. 8th ed. Springer; 2017:471-486.

CHONDROGENIC TUMORS Osteochondroma Osteochondroma is a benign cartilage–capped bony projection arising on the external surface of bone containing a marrow cavity that is continuous with that of the underlying bone (1). The base of osteochondroma can be broad (sessile) or small (pedunculated) (Fig. 8.1). The lesion is entirely surrounded by periosteum. Osteochondromas are the most frequent cartilaginous lesions, most often found on the femur (21%), humerus (17%), and tibia (11%) (8). Osteochondromas develop and increase in size during the first decade of life and stop growing when growth plates close during puberty. The majority of osteochondromas are asymptomatic. Sporadic (solitary) osteochondromas are six times more common than those occurring within the hereditary multiple osteochondroma syndrome (previously called hereditary multiple exostoses) (Fig. 8.2). Multiple osteochondromas syndrome is an autosomal dominant condition caused by mutations in EXT1 or EXT2 (9). Radiologically, the osteochondroma is characterized by its typical localization at the transition of the metaphysis to the diaphysis, projection away from the joint, the continuity of the cortex of the underlying bone with the cortex of the osteochondroma stalk, and the presence of spongiosa within the stalk (Figs. 8.1 and 8.3). Histologically, three layers are seen: perichondrium, cartilage, and bone (Fig. 8.4). The organization of chondrocytes in columns, as is normally observed in the growth plate, can usually also be recognized in the cartilaginous layer of osteochondroma (Fig. 8.5). Endochondral ossification is seen at the cartilagebone interface. Cellularity is variable, depending on the age of the patient. Binucleated cells, calcification, necrosis, nodularity, and cystic changes can be seen (Fig. 8.6). Malignant transformation to secondary peripheral ACT (atypical cartilaginous tumor)/CS1 (chondrosarcoma grade 1) occurs in 1% to 5%. There are no accepted histologic criteria to distinguish osteochondroma from low-grade secondary peripheral chondrosarcoma (10); the imaging studies and gross macroscopic documentation of the thickness of the cartilaginous cap are extremely important, as a cartilage cap exceeding 2 cm in adults is suggestive of malignancy (1).

FIGURE 8.1 Gross specimen of an osteochondroma. The lesion is pedunculated and formed by a small stalk of medullary bone covered by a smooth, pale, blue-gray cartilage cap that has a thickness of less than 1 cm. Overview (left) and cut surface (right).

FIGURE 8.2 Multiple osteochondromas. Multiple bony lesions around the knee, projecting away from the joint.

FIGURE 8.3 Osteochondroma of the femur. Note that the medullary cavity of the lesion is continuous with the medullary cavity of the bone.

FIGURE 8.4 Osteochondroma. The tumor consists of three layers: perichondrium, cartilage, and bone.

FIGURE 8.5 Osteochondroma. The cartilage cap in osteochondroma usually displays a growth plate–like columnar arrangement of chondrocytes. Endochondral ossification is present at the cartilage-bone interface (left).

FIGURE 8.6 Osteochondroma. The cartilage cap contains small chondrocytes as well as hypertrophic cartilage cells. Trabecular bone is seen at the base of the cartilage cap.

Enchondroma Enchondroma is a benign hyaline cartilage neoplasm of medullary bone. Most tumors are solitary; however, they occasionally involve more than one bone or site in a single bone (1). Enchondromas are

relatively common and have a wide age range. Enchondromas are located in the long bones, and 50% of all cases are located in the bones of the hands and feet (1). Chondrosarcomas are only rarely seen at this location (11). The size of an enchondroma is usually less than 3 cm. Radiologically, the presence of matrix mineralization in the form of “popcorn” calcifications is most typical. The edge of the lesion is most often lobular and sharply demarcated. Histologically, enchondroma consists of lobular, relatively cell-poor hyaline cartilage, often demarcated by a zone of reactive bone formation (Fig. 8.7). The chondrocytes have nuclei with condensed chromatin and are evenly dispersed. Binucleated cells are infrequent, and mitoses are absent. Occasionally, degenerative features such as ischemic necrosis or calcifications are found (Fig. 8.8). The distinction between an enchondroma and ACT/CS1 can be difficult radiologically (12). Moreover, this distinction is also difficult on histology (13), causing high interobserver variability (14,15). Mucomyxoid matrix degeneration and infiltrative growth (entrapment of preexisting host bone) are most predictive of ACT/CS1 (14). Immunohistochemistry or molecular diagnostics cannot help in this differential diagnosis. Radiologic presentation, localization, and age should also be taken into account. For instance, in the phalanges, the matrix of enchondromas can contain more myxoid features, and the lesion may be more cellular. At this location, the main characteristics of malignancy are the presence of mitoses, cortical breakthrough, and soft-tissue involvement (11). Malignant transformation of enchondromas is thought to be extremely rare ( females

Age range (d)

1st-8th

1st-7th

1st-8th

1st-8

2nd-8th

Peak age (d)

4th-5th

within first three decades of life

4th

2nd

6th-8th

Location

Skull, vertebrae

Shoulder, hip

Long tubular bones

Long tubular bones of extremities

Long tubular bones of extremities, spine

Multifocality (%)

5%-18%

100%

18%

50%-64%

33%

Vascular spaces

Vascular spaces

Lobular growth pattern

Strands/cords or solid nests

Heterogeneous: vasoformative to solid

Periphery: arteriolar-like vessels

Epithelioid endothelial cells

Macronucleolus

Central: epithelioid cells

Intracytoplasmic vacuoles Myxoid/hyalinized stroma

0.22 mm and/or >200 cells): Number with isolated tumor cells (ITCs) (≤0.2 mm or ≤200 cells): Size of largest metastatic deposit: Extranodal extension (if present give extent)

Macrometastatic carcinoma is most significant clinically (influences axillary surgery, radiation, systemic treatment decisions) Micrometastases count toward the total pN stage only if there is at least one macrometastasis also present (otherwise it is pNmi) ITCs are not clinically relevant and do not count toward the overall pN stage (pNi+)

Treatment effect

Report if posttreatment. Can be descriptive or use standardized reporting system such as Residual Cancer Burden (RCB)

The presence of residual cancer is highly correlated with outcomes in HER2-positive and triple-negative cancers. Additional treatments may be offered if residual cancer present

Pathologic stage (AJCC eighth edition)

Use modifiers if appropriate: m = multiple foci r = recurrent y = posttreatment See staging manual for details of pTNM grouping

Staging documentation. Differences in clinical management Prognostic

Ancillary studies: ER, PR, and HER2

List as pending if to be performed as an addendum; see tables on reporting hormone receptors and HER2

Prognostic and predictive markers

aOptional.

ADH, atypical ductal hyperplasia; AJCC, American Joint Committee on Cancer; ALH, atypical lobular hyperplasia; DCIS, ductal carcinoma in situ; FEA, flat epithelial atypia; LCIS, lobular carcinoma in situ; NST, no special type.

MORPHOLOGIC AND MOLECULAR/BIOMARKER-DEFINED SUBTYPES

The most widely used classification of invasive breast cancers is that of the WHO (fifth edition) (227). This classification scheme is based on the growth pattern and cytologic features of the invasive tumor cells. However, the most common histologic type of invasive breast cancer by far is IDC-NST, which is commonly referred to as invasive ductal carcinoma (IDC) (Table 9.15). In general, special-type cancers comprise approximately 20% to 30% of invasive carcinomas, and at least 90% of a tumor should demonstrate the defining histologic characteristics of a special-type cancer before it is designated as being of that histologic type (a mixed special IDC cancer is diagnosed otherwise). TABLE 9.15 Relative Frequencies of Invasive Breast Carcinomas by Histologic Subtype Subtype

Frequency

Invasive breast carcinoma of no special type

>70%

Invasive lobular carcinoma

5%-15%

Tubular carcinoma

1.6%

Cribriform carcinoma

0.4%

Mucinous carcinoma

2%

Invasive micropapillary carcinoma

0.9%-2%

Carcinoma with apocrine differentiation

1%

Metaplastic carcinoma

0.2%-1%

Neuroendocrine tumor

12

3

hpf, high-power field. Total score: 3-5, grade 1, well differentiated; 6-7, grade 2, moderately differentiated; 8-9, grade 3, poorly differentiated.

With any grading system, appropriate sampling is important because some tumors vary markedly in appearance from area to area. The highest grade area typically drives prognosis and so should be reported. Tubule/glandular formation is assessed as a percentage of the whole cancer at low magnification. Of note, only structures exhibiting clear central lumina surrounded by polarized neoplastic cells are counted. Cutoff points of 75% and 10% are used to determine the overall differentiation score. Nuclear pleomorphism is graded in the area showing the worst pleomorphism. The preferred magnification for nuclear scoring is ×40. Nuclei that are very similar in size to the nuclei of background normal epithelial cells, with rounded to elongated regular nuclei, an even chromatin pattern, and nucleoli that are either not visible or inconspicuous are given a score of 1. Score 2 nuclei are larger (1.5-2 times normal epithelial cell nuclei), with mild-tomoderate pleomorphism and visible but small and inconspicuous nucleoli. Score 3 nuclei are pleomorphic, obviously enlarged (>two times normal epithelial cell nuclei) and contain vesicular chromatin with often prominent nucleoli. The mitotic activity score involves assessing both the total number of mitoses per 10 high-power fields (HPF), adjusted by the field diameter of the microscope used on the ×40 objective (see Table 9.17). Scoring is performed on the area exhibiting the highest frequency of mitotic figures (hotspots), typically the peripheral leading edge of the cancer. TABLE 9.17 Mitotic Activity Score Categories by Field Diameter and Mitotic Counts Field Diameter (mm)

Area (mm2)

Mitoses/10 Fields Corresponding to

SCORE 1

SCORE 2

SCORE 3

0.40

0.125

≤4

5-9

≥10

0.41

0.132

≤4

5-9

≥10

0.42

0.139

≤5

6-10

≥11

0.43

0.145

≤5

6-10

≥11

0.44

0.152

≤5

6-11

≥12

0.45

0.159

≤5

6-11

≥12

0.46

0.166

≤6

7-12

≥13

0.47

0.173

≤6

7-12

≥13

0.48

0.181

≤6

7-13

≥14

0.49

0.189

≤6

7-13

≥14

0.50

0.196

≤7

8-14

≥15

0.51

0.204

≤7

8-14

≥15

0.52

0.212

≤7

8-15

≥16

0.53

0.221

≤8

9-16

≥17

0.54

0.229

≤8

9-16

≥17

0.55

0.238

≤8

9-17

≥18

0.56

0.246

≤8

9-17

≥18

0.57

0.255

≤9

10-18

≥19

0.58

0.264

≤9

10-19

≥20

0.59

0.273

≤9

10-19

≥20

0.60

0.283

≤10

11-20

≥21

0.61

0.292

≤10

11-21

≥22

0.62

0.302

≤11

12-22

≥23

0.63

0.312

≤11

12-22

≥23

0.64

0.322

≤11

12-23

≥24

0.65

0.332

≤12

13-24

≥25

0.66

0.342

≤12

13-24

≥25

0.67

0.353

≤12

13-25

≥26

0.68

0.363

≤13

14-26

≥27

0.69

0.374

≤13

14-27

≥ 28

Grading on CNB samples is possible and often necessary, given the frequency of neoadjuvant therapy prior to surgical removal of cancers. Factors that can affect grade in these samples, such as crush artifact resulting in low mitotic counts in high-grade cancers, should be recognized. In heterogeneous cancers, initial CNB grade may be lower than in subsequent surgical specimens. Because neoadjuvant therapies can modify the appearance of a cancer, grading postneoadjuvant therapy likely does not have the same clinical relevance. The pretreatment grade may be more relevant to report in these scenarios. LOCAL RECURRENCE RISK FACTORS (ANGIOLYMPHATIC SPACE INVASION, EXTENSIVE DUCTAL CARCINOMA IN SITU, MARGINS) The presence or absence of angiolymphatic space invasion (cancer emboli in lymphatic or small vascular channels) should be reported in breast cancer cases because it is associated with risk of local recurrence (for which radiation may be offered even in the mastectomy setting) and distant recurrence, especially for node-negative patients (269). These foci are typically found at the periphery of the invasive cancer adjacent to small vascular spaces (Fig. 9.96). When in doubt about stromal retraction around a nest of cancer vs. true angiolymphatic space invasion, immunohistochemical stains such as D240 or CD31 can be used to clarify. Occasionally, large boluses of cancer filling angiolymphatic spaces can mimic DCIS because of their large rounded contours. In this setting, care should be taken not to misinterpret myoepithelial vs. vascular staining patterns.

FIGURE 9.96 Lymphatic space invasion. Multiple small lymphatic channels contain clusters of malignant cells. In the center is a venule.

As mentioned previously, extensive ductal in situ carcinoma in association with invasive breast carcinomas is regarded as a risk factor for local recurrence following conservation therapy, particularly when the resection margins are involved, with many considering this an indicator for reexcision before radiotherapy (148). There has been an evolution of thought in regard to adequate margins for invasive carcinoma. Historically, wide margins were considered necessary to reduce local recurrence rates. However, with the advent of additional local and systemic therapies, the only margin status proven to have a significant effect on local recurrence after breast-conservation surgery is a positive margin with cancer at ink (270,271). Therefore, most guidelines endorse the adequacy of a “no invasive tumor at ink” margin for invasive breast cancer and associated DCIS (and a 2-mm margin for DCIS alone) (157,272). However, the distance to close margins should be reported. Some surgeons have adopted the technique of using additional “shave margins” that are submitted from the lumpectomy cavity to reduce the frequency of positive margins (273). Cancer summary templates should include a final margin status that includes any separately excised shave margins. POSTNEOADJUVANT RESPONSE Achievement of pathologic complete response after neoadjuvant therapy is highly prognostic for HER2-positive and triple-negative breast cancers (274). Additional therapy may be considered if there is residual disease posttreatment

(with agents such as capecitabine, an oral chemotherapy) (256). The RCB index can be used to establish risk classes associated with recurrence in cases with residual disease (254). The parameters to be quantified and reported, as well as a calculator, are available online (www3.mdanderson.org/app/medcalc). Residual cancer, if present, can have a variety of appearances. In therapyresistant cancers, no morphologic alteration may be detected. More commonly, carcinomas become less cellular, sometimes present as scattered small nests across a fibrotic scarred area with associated histiocytes and chronic inflammation (Figs. 9.97 and 9.98) (275). Residual calcifications may be present in ducts that previously contained DCIS. In a case with no residual carcinoma identified, care should be taken that the tumor bed was well sampled and identified microscopically. Sometimes, immunohistochemical studies may be helpful for distinguishing cancer cells from benign histiocytes and invasive carcinoma from CIS. DCIS may be present (with or without treatment effect) in the absence of residual invasive carcinoma, and this finding does not exclude pathologic complete response because these patients have a good prognosis (276,277). Primary neoadjuvant endocrine therapy is sometimes administered, which may help identify patients who can avoid chemotherapy, but it rarely results in pathologic complete response (278-280). Cells postneoadjuvant endocrine therapy may be quite small, even smaller than normal epithelial cells in the background.

FIGURE 9.97 Postneoadjuvant chemotherapy changes. In this case, the tumor bed has no residual carcinoma. The tumor bed is composed of loose fibrous stroma with patchy chronic inflammation and histiocytes and minimal normal TDLUs. Large calcifications are present in sclerotic ducts that likely used to harbor DCIS. DCIS, ductal carcinoma in situ; TDLU, terminal duct lobular unit.

FIGURE 9.98 Postneoadjuvant chemotherapy tumor bed with residual nests of invasive carcinoma.

TUMOR-INFILTRATING LYMPHOCYTES Tumor-infiltrating lymphocytes (TILs) also appear to have prognostic power in HER2-positive and triple-negative cancers. HER2-positive cancers with higher TIL levels are reported to have better response rates to neoadjuvant treatment, which typically correlates with overall survival rates as well (281-285). Similarly, TILs have shown prognostic relevance in determining better response to neoadjuvant chemotherapy (281,282,286-288). The hypothesis is that more TILs present may create a “primed” immune environment, helping to create durable responses to eliminating a cancer. Although TILs reporting has not uniformly become standard practice, there are guidelines for standard evaluation and reporting (281,286-288) (www.tilsinbreastcancer.org). PREDICTIVE FACTORS Hormone Receptors ER is both a major prognostic factor and a predictive factor in breast cancer. Prognostically, ER-negative breast cancers have a significantly worse 5-year

overall survival than ER-positive cancers, and there are vast differences in the underlying biology between ER-positive and ER-negative breast cancers (244,289-293). ER has predictive value because cancers with greater than or equal to 1% ER expression by IHC testing receive a statistically significant disease-free and overall survival benefit when treated with hormone-targeted therapies, such as tamoxifen or selective estrogen receptor modulators (SERMs) (294,295). Therefore, the results of ER testing on a breast cancer will determine whether the patient is a candidate for hormonal therapies as well as overall treatment pathways. The most recent guidelines for HR testing, such as the ASCO/CAP HR testing breast cancer guideline, should be followed to ensure this is performed to the standards of this important predictive test. However, it is important to note that thresholds for a positive result were created for their predictive value, not necessarily for overall treatment pathways decisions. Nuclear staining only should be scored for ER and PR stains (see Figs. 9.999.101 and Table 9.18). The percentage reported should reflect the percentage of positive cells in the entire sample of the invasive cancer tested (not just the area of highest expression). Intensity of staining is also reported, but ASCO/CAP only uses percent cells staining for determining whether the assay is positive or negative. When the intensity is variable, it can be reported as a range or an average intensity, with many systems using a range of 0, 1+, 2+, and 3+. Different scoring systems can be used to combine the intensity and percentage information for an overall score (e.g., the H-score or Allred score), but it should be clearly reported what the raw percentage and intensity scores are as well. Low-power scanning is often insufficient for the detection of focal or weak levels of HR expression; therefore, in cases that appear negative on low power, a higher power scan of the slide is also appropriate, to rule out weak HR positivity.

FIGURE 9.99 Positive ER stain (>95%, 3+) in a well-differentiated invasive ductal carcinoma.

FIGURE 9.100 Negative ER stain (0%) with positive internal controls. Normal ducts have variable numbers of ER-positive cells and serve as a nice internal control to ensure that the stain worked in the setting of negative or low positive result.

FIGURE 9.101 Low positive ER stain (1%-10%, 1+) in a high-grade invasive ductal carcinoma. Weak-intensity nuclear staining may only be apparent on high power.

TABLE 9.18 Hormone Receptor and HER2 Reporting in Breast Cancer

ER

Uses

Test Type

Reporting Categories

Scoring Criteria (ASCO/CAP)

Prediction of benefit from hormonal therapies if positive Categorization for overall treatment pathways Characterization as the IHC luminal group if positive Poor prognostic marker if negative

IHC

Positive Low positivea Negative

>10% of cancer has nuclear staining (any intensity) 1%-10% of cancer has nuclear staining (any intensity) 10% of tumor cells

Predictive of benefit from HER2-targeted therapy if positive (administered with chemotherapy)

IHC

Uses

Test Type

In situ hybridization (dual probe)

Reporting Categories

Scoring Criteria (ASCO/CAP)

Negative

Incomplete membrane staining that is faint/barely perceptible and in >10% of tumor cells (1+) No staining is observed or membrane staining that is incomplete and is faint/barely perceptible and in ≤10% of tumor cells (0).

Positive

HER2/CEP17 ratio ≥2.0 and average HER2 copy number ≥4.0 signals/cell (group 1)

Uses

Test Type

Reporting Categories

Scoring Criteria (ASCO/CAP)

Positiveb

HER2/CEP17 ratio ≥2.0 and average HER2 copy number

tricuspid > pulmonic). In the second half of the 20th century, the demographics of IE changed as antibiotic therapy became routine and degenerative/senile aortic valve disease increased. Valves with regurgitant alterations are particularly at risk, as are patients with prosthetic valves and pacemaker/intracardiac cardioverterdefibrillator wires, immunosuppressed patients, and intravenous drug abusers. A variety of Gram-positive and Gram-negative bacterial, fungal, mycobacterial, rickettsial, and chlamydial organisms are responsible for IE. The gross and microscopic changes are a result of, in large part, the virulence of the organisms. The liberal use of Gram stains, fungal stains, and stains for acid-fast organisms is essential, although many patients will have received antibiotic therapy before surgical excision of the native or prosthetic valve. Microbiologic culture of excised tissue should be routinely performed. Currently, molecular techniques are available and aide in the small percentage of cases where both tissue and microbiologic analyses are unsuccessful (344). Grossly, the lesions can vary in size and shape, and particularly virulent organisms can cause perforations of the leaflets or rupture of the chordae (Fig. 29.59). Extension from the site of initiation at the cusp apposition line (atrial surface of AV valves and ventricular surface of semilunar valves) can proceed to the leaflets, chordae, and annular regions to form abscesses. In the

untreated, early phase of organization, acute fibrinous exudates with neutrophils and necrotic changes and tissue destruction in the valve are observed (Fig. 29.60). In the healing phases or in the setting of an insidious low-grade infection, neovascularization, chronic inflammation, fibrosis, and calcification replace the damaged tissue.

FIGURE 29.59 Infective endocarditis. Perforation of the mitral leaflet caused by Streptococcus viridans infection.

FIGURE 29.60 Microscopic section of the base of the aortic valve leaflet from Figure 29.47. Upper inset: Dense acute inflammatory exudates are present (*). Lower inset: The Gram stain demonstrates numerous colonies of Gram-positive organisms.

NONINFECTIVE ENDOCARDITIS Two patterns of non-IE are recognized but rarely require surgical excision. Nonbacterial thrombotic or marantic endocarditis is usually seen at autopsy and is defined as platelet-fibrin vegetations that are devoid of inflammatory cells or bacteria (345). The vegetations are arranged as continuous, linear aggregates along the lines of closure of the left-sided valves (Fig. 29.61). By definition, no inflammatory cell infiltrates or tissue destruction of the underlying valve tissue is present.

FIGURE 29.61 Marantic endocarditis. Upper panel: Linear red fibrinous exudates on the atrial surface along the lines of closure. Lower panel: Aortic valve showing fibrinous aggregates on the central nodules. The underlying valves are normal.

In SLE and antiphospholipid syndrome, flat vegetations can develop on the atrial aspect of the posterior mitral valve leaflet and the ventricular aspect of the aortic valve. These may expand to cover

both aspects of the valve or extend along the atrial or ventricular endocardium. These Libman-Sacks lesions can mimic IE on account of the presence of fibrin, cores of fibrinous necrosis of the valve, inflammatory cell infiltrates, and hematoxylin bodies. PROSTHETIC VALVES Currently, two main types of prosthetic valves are used in clinical medicine: tissue valves and mechanical valves. Tissue-engineered valve replacement is the subject of research and development for future application (310). Tissue valves are composed of either porcine aortic valve or bovine pericardium that has been treated in a dilute aldehyde solution and sewn onto a metallic frame. More than half fail within 10 years because of calcification and/or tissue degeneration, leading to tears, perforations, and leakages (Fig. 29.62). Mechanical valves are classified as ball and cage valves, caged disc valves, and tilting disc valves. Fractures of strut components, annular ring abscess formation, and dehiscence and thrombus formation are recognized complications (Fig. 29.63). A specimen photograph is helpful, and in some cases, the specimen should be retained for manufacturer evaluation or medical-legal issues. IE of prosthetic valves is also a known complication, and any vegetation should be carefully examined for the presence of infectious organisms. As in the case of IE of native valves, a host of different microbial organisms has been reported (346).

FIGURE 29.62 Bioprosthetic tissue valve with marked fibrocalcific degeneration and tearing of the leaflets.

FIGURE 29.63 Mechanical valve with thrombi on the metal leaflets and at the opening.

PERICARDIUM ACUTE PERICARDITIS There are many and varied causes of acute inflammation of the pericardium (Table 29.12). The relative proportions of these different etiologies will change with the site of practice (347). In general, acute pericarditis is more common in men than in women. Tissue injury and the resultant inflammation may be the result of direct damage by microorganisms or the result of inflammatory mediators released by inflammatory cells. Hypersensitivity and autoimmune mechanisms

are most likely causative after MI, surgery, or drug reactions and in association with systemic autoimmune disease. TABLE 29.12 Causes of Acute and Chronic Pericarditis ACUTE PERICARDITIS Infectious Bacterial (Gram-positive bacteria, Gram-negative bacteria, mycobacteria, spirochetes) Fungal Viral Parasitic Idiopathic Post–myocardial infarction Iatrogenic (postsurgical, radiation therapy, drug reaction) Metastatic neoplasm Systemic disease (autoimmune, renal failure, endocrine) Traumatic CHRONIC/CONSTRICTIVE PERICARDITIS Idiopathic Following episode of acute pericarditis Infectious (mycobacteria, fungal) Postsurgical (including transplantation) Systemic disease (autoimmune, renal failure) Radiation therapy

Neoplasms (usually metastatic tumors)

The pathology of acute pericarditis includes vascular dilation and prominence, exudates of fibrin, and inflammatory cells (Fig. 29.64). In infectious causes, microorganisms may be present, and there may be specific histopathologic features reflecting pathogenesis (granulomatous inflammation, vascular endothelial changes after radiation, lupus erythematosus [LE] cells in association with SLE, etc.).

FIGURE 29.64 Fibrinous pericarditis. Right panel: Blood and layered fibrin on the visceral pericardium. Left panel: High-power magnification showing scattered inflammatory cells, blood, and fibrin.

Acute pericarditis after viral infection, MI, or cardiac surgery is generally a self-limited process. Complications of all forms of acute pericarditis (in order of frequency) include recurrence, tamponade, pericardial constriction, and combined effusion and constriction. These complications may be predicted as more likely to occur in patients with fever less than 38°C, subacute course, large effusion or tamponade, or failure to respond to aspirin or nonsteroidal antiinflammatory therapy. CONSTRICTIVE PERICARDITIS Some versions of acute pericarditis will organize and heal with dense fibrosis and calcifications, resulting in a thickened pericardium that encases the heart and interferes with diastolic filling. The clinical distinction of constrictive pericarditis from restrictive myocardial disease caused by amyloid or hemochromatosis may be challenging; EMB is often used to exclude infiltrative myocardial disease. In the past, tuberculosis was the most often recognized cause of constrictive pericarditis. Currently, the cause is usually unknown, but multiple sections should be reviewed to exclude tuberculosis and fungal and other known etiologies for pericarditis (Fig. 29.65). Management includes medical therapy to limit volume expansion and antiinflammatory drugs. For patients who fail conservative therapy, surgical pericardiectomy is usually indicated (348). An uncommon form of constrictive pericarditis is idiopathic cholesterol pericarditis (Fig. 29.66). Originally described in 1919, it can be associated with recurrent pericardial effusions that yield a “scintillating gold paint appearance” on account of the cholesterol crystals in the fluid (349). Other known associations include hypothyroidism, rheumatoid arthritis, and tuberculosis.

FIGURE 29.65 Fungal constrictive pericarditis. Pericardiectomy specimen showing a thickening parietal pericardium measuring up to 1 cm in thickness. Inset: Granulomatous pericarditis with Coccidioides immitis.

FIGURE 29.66 Cholesterol pericarditis. Golden yellow collections of cholesterolrich deposits were found in this pericardial stripping specimen.

PRIMARY PERICARDIAL MASSES Primary pericardial tumors are much less common than metastatic malignancies. Benign masses include pericardial or mesothelial cysts (Fig. 29.67). These may be congenital or form after surgical procedures or infarct as the result of trapping of small inclusions of benign mesothelium that continues to function. Pericardial pseudocysts, lacking a mesothelial lining, have walls of collagen and granulation tissue and are the result of trauma or prior pericarditis. Mesothelial papillomas are composed of epithelioid mesothelial cells on papillary connective tissue cores. Like mesothelial/macrophage incidental cardiac excrescence, these rare lesions are likely reactive proliferations (350). Other benign lesions include teratomas in infants, bronchogenic cysts, benign fibrous tumors, lipomas, lymphangiomas, and giant lymph node hyperplasia. Foci of ectopic thyroid or thymic tissue can also be found in the pericardium.

FIGURE 29.67 Pericardial cyst. (A) Incidental 4.0-cm smooth-walled cyst on a thin pedicle found during a Type A aortic dissection repair. (B): Calretinin immunostaining highlights the mesothelial cells. (C) H&E stain showing single layer of flattened mesothelial cells admixed with scattered inflammatory cells.

Primary malignant tumors of the pericardium are rare. Most are sarcomas; angiosarcoma is the most commonly seen, but other

types including synovial sarcoma and malignant fibrous histiocytoma are reported. Malignant mesotheliomas of the pericardium occur in a similar frequency to sarcoma, although they comprise less than 1% of all malignant mesotheliomas (351). There is an association with asbestos exposure. The tumors may present with symptoms and signs of pericarditis or constriction. Tumor nodules may stud the pericardial surfaces, and at later stages, the pericardial cavity may be filled with tumor-causing constriction. The pericardial fluid is usually sanguineous. The histology and diagnostic criteria for pericardial malignant tumors are the same as for the lesions when found in other sites.

FUTURE DIRECTIONS The impact of new molecular technologies and genomic/proteomic findings in the wide spectrum of cardiac disorders has expanded the role of the diagnostic surgical pathologist. The importance of clinicopathologic correlation in the evaluation of these insights and the application of new technologies are predicated on accurate and thorough macroscopic and microscopic examinations. Further, emerging infectious diseases and reemergence of remote diseases require familiarity with uncommon lesions. The EMB remains the “gold” standard for the diagnosis, classification, and monitoring of acute allograft rejection in heart transplantation recipients. It is also an essential diagnostic tool in the distinction of primary and secondary cardiomyopathies. The applications of novel digitalization techniques and artificial intelligence provide new avenues for both clinical practice and investigative inquiry. The breadth of cardiovascular pathology continues to expand, and the role of the diagnostic surgical pathologist remains essential.

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30

Blood Vessels Renu Virmani ■ Maria Romero ■ Yu Sato

Since the advent of cardiac bypass and new techniques in vascular surgery, surgical pathologists now commonly encounter vascular specimens of numerous and varied types. This chapter focuses on a broad spectrum of vascular diseases that can be diagnosed by surgical biopsy or resection. The chapter begins with the pathologic findings of inheritable diseases of the blood vessels, followed by noninflammatory disease, including atherosclerosis of the aorta and carotid arteries. Inflammatory diseases (vasculitides) of the large, medium, and small vessels are also discussed, and a brief description of vascular tumors is provided. Aortitis can be varied, and without the clinical background and appropriate radiologic and blood laboratory testing, the true diagnosis may remain elusive. A simple classification and approach to vasculitis are presented that are likely to be helpful to the practicing pathologist in the differential diagnosis of inflammatory vascular disease. Although some of these disorders have specific pathognomonic histologic features, many require correlation with clinical, serologic, and radiologic information, especially for intracranial lesions that are best appreciated by magnetic resonance imaging or computed tomography to arrive at an appropriate diagnosis. Finally, although coronary artery disease remains the leading cause of death in the Western world, coronary specimens are typically encountered at autopsy; therefore, discussion of this important topic is beyond the scope of this chapter.

HEREDITARY DISEASES OF BLOOD VESSELS There are two major types of thoracic aortic aneurysms: sporadic (degenerative/atherosclerotic) and hereditary thoracic aortic aneurysms and dissections (TAAD) (1). The hereditary type is found in 20% to 40 % of TAAD cases when at least two subjects within a single family are diagnosed with

TAAD (2,3). TAAD are a part of a well-characterized genetic syndromes such as Marfan syndrome (MFS), Loeys-Dietz syndrome (LDS), vascular type of Ehlers-Danlos syndrome (EDS), Shprintzen-Goldberg syndrome—all named syndromic TAAD. However, most TAAD patients do not meet the criteria of those syndromes and are defined as nonsyndromic TAAD. Both are associated with autosomal dominant transmission in nearly 80% of cases (Table 30.1) (4). TABLE 30.1 Classification of Heritable Thoracic Aortic Conditions Gene symbol

Protein

Phenotypes

Associated Histologic Findings

GENES SPECIFYING COMPONENTS OF THE EXTRACELLULAR MATRIX FBN1

Fibrillin-1

Marfan syndrome

MD+++, MEMA-T+++, SMCL+, EFF+++

COL3A1

Type 3 procollagen

Vascular Ehlers-Danlos syndrome

MD+, MEMA-T+

COL4A5

Type 4 procollagen

Alport syndrome

EFEMP2

Fibulin-4

Cutis laxa

GENES SPECIFYING COMPONENTS OF TGF-Β/BMP SIGNALING TGFBR1

TGF-β receptor-1

LDS

MD+++, MEMA-I+++, MEMAT+, EFF+++, EFD+

FTAAD TGFBR2

TGF-β receptor-2

LDS

MD+++, MEMA-I+++, MEMAT+, EFF+++, EFD+

FTAAD TGFB2

TGF-β2

FTAAD

SMAD3

SMAD3

FTAAD

GENES SPECIFYING COMPONENTS OF CONTRACTILITY OF VASCULAR SMOOTH MUSCLE CELLS ACTA2

α-actin

FTAAD

MYH11

Myosin heavy chain-11

FTAAD

MYLK

Myosin light chain kinase

FTAAD

PRKG1

cGMPdependent

FTAAD

MD++, EFF++

Receptor kinase (PKG I) FLNA

Filamin-A

Cerebral heterotopias/aortic aneurysm

TSC2

Tuberin

Tuberous sclerosis complex

GENES SPECIFYING COMPONENTS OF OTHER SIGNALING PATHWAYS JAG1

JAGGED-1

Alagille syndrome

NOTCH1

NOTCH-1

Bicuspid aortic valve/aortic aneurysm

SLC2A10

Glucose transporter 10

Arterial tortuosity syndrome

MD++, EFF+++

“+” to “+++” represents frequency of description in the literature. BMP, bone morphogenic protein; EFD, elastic fiber disorganization; EFF, elastic fiber fragmentation and/or loss; FTAAD, familial aortic aneurysm and dissection; LDS, LoeysDietz syndrome; MD, medial degeneration; MEMA-I, intralamellar mucoid extracellular matrix accumulation; MEMA-T, translamellar mucoid extracellular matrix accumulation; SMCL, smooth muscle cell nuclei loss; TGF, transforming growth factor. Reprinted from Ladich E, Yahagi K, Romero ME, et al. Vascular diseases: aortitis, aortic aneurysms, and vascular calcification. Cardiovasc Pathol. 2016;25(5):432-441. Copyright © 2016 Elsevier. With permission.

MARFAN SYNDROME Marfan syndrome is an autosomal dominant condition characterized by skeletal, ocular, and cardiovascular manifestations and is traditionally defined by clinical criteria (5). In most cases, there is a mutation in fibrillin-1 (on chromosome 15), a major component of isolated or elastin-associated microfibrils. Marfan patients with normal fibrillin-1 genotype may have mutations in the transforming growth factor-beta (TGF-β) receptor (6).

Cardiovascular manifestations include proximal aortic aneurysms, which may lead to aortic dissections and rupture, aortic incompetence; mitral valve prolapse; and peripheral artery aneurysms. Less than 10% of patients with ascending aortic aneurysms will have extracardiac clinical manifestations of Marfan syndrome, and about 3% to 5% of patients with ascending aortic dissections have Marfan syndrome. In a series of 513 patients with aortic root disease necessitating surgical repair, 32 patients had documented Marfan syndrome (7). Most aneurysms in patients with the full-blown syndrome are symptomatic before age 35. The characteristic histologic finding in Marfan syndrome is cystic medial degeneration (CMD), with or without features of acute or chronic dissection (Fig. 30.1). Medial degeneration (MD) is characterized by the loss of elastic lamellae and smooth muscle cells, with replacement by pools of proteoglycan matrix. Halushka et al, in a consensus document, have developed a synoptic grading system as a means of providing a standardized method for surgical pathology reporting and have replaced the term CMD with MD, which includes the presence of mucoid extracellular matrix accumulation (MEMA) further classified into intralamellar (MEMA-I) and translamellar (MEMA-T), without and with significant alterations in the arrangement of elastic lamellar units to varying degrees, including degeneration collapse and loss of elastic fibers, in the presence of smooth muscle loss (Fig. 30.2)(8,9).

FIGURE 30.1 Cystic medial degeneration. (A) There is a large area in the central media with loss of elastic layers and smooth muscle cells, with a pool of ground substance. (B) In this example, there is more diffuse loss and disorganization of elastic laminae, with increased proteoglycan matrix. The degree of cystic medial degeneration varies and in an individual patient does not allow the distinction between inherited forms of the aortic aneurysm without a clear genetic etiology.

FIGURE 30.2 Histologic images showing grades of medial degeneration. (A) Normal aorta. (B) Mild medial degeneration characterized by pooling of proteoglycan between elastic lamellae. (C) Moderate medial degeneration with focal loss of elastic lamellae and proteoglycan deposition. (D) Severe medial degeneration with marked loss of elastic lamellae, smooth muscle cells, and extensive proteoglycan deposition. All Movat pentachrome stain. Reprinted from Ladich E, Yahagi K, Romero ME, et al. Vascular diseases: aortitis, aortic aneurysms, and vascular calcification. Cardiovasc Pathol. 2016;25(5):432-441. Copyright © 2016 Elsevier. With permission.

There is variability in the degree of MD in patients with inherited aortic root disease; in only 25% is the degree severe, and in approximately 5% of patients, the aortic media is histologically normal (7). Therefore, histologic evaluation is not always helpful in assigning a genetic etiology for degenerative aortic root disease. The degree of histologic variability is likely in part a result of sampling, and the changes are patchy. Pregnancy is a predisposing factor for acute aortic dissection, although its occurrence during gestation, delivery, or postpartum is quite rare. FAMILIAL NON-MARFAN DISSECTIONS

Familial aortic dissections are about as common in patients without extravascular abnormalities as in patients with Marfan syndrome (7). The genetic basis is not known, although it has been shown that 5% of patients with non-Marfan familial dissection have mutations in the TGF-β receptor, as is the case of the recently described LDS (10). As with Marfan syndrome, the histologic characteristic is MD, although the degree is quite variable. EHLERS-DANLOS SYNDROME EDS type IV, the vascular type, results from mutations in the gene for type III procollagen (COL3A1). It is characterized by generalized joint hypermobility and related osteoarticular complications, dermal dysplasia varying from minor changes of skin texture to clinically relevant skin fragility and defective scarring and vascular and internal organ fragility. Affected patients are at risk for arterial, bowel, and uterine rupture (11). The diagnosis of EDS type IV should be considered in all patients under the age of 45 who present with arterial tearing or dissection. Most lesions occur in the thoracic or abdominal aorta, and/or its proximal branches like the renal, hepatic, splenic, and iliac arteries, and especially prone to aneurysms, dissections, and rupture. Other commonly affected locations include carotid, vertebral, and intracranial arteries, which may lead to stroke (12). The histologic features of EDS are not well characterized; indeed, in many cases, the media is histologically normal, and the pathologic features are that of rupture and pseudoaneurysm formation in an otherwise apparently normal vessel. In the Mayo Clinic series of aortic root resection, primarily for aneurysm, only 2 of 513 patients had evidence of EDS, less than 5% of hereditary cases. It is unclear whether MD is a typical finding of the disease, in contrast to Marfan syndrome and other non-Marfan familial dissections. AORTIC ROOT DISEASE IN PATIENTS WITH BICUSPID AORTIC VALVE Bicuspid aortic valve (BAV; or congenitally bicommissural valve) has an autosomal dominant inheritance (13) with prevalence at birth of 1% to 2%. There is a known association with aortic root aneurysm and dissection, the risk being 5 to 10 times that of the general population (14). Aortic dilatation is defined as an aortic diameter of greater than 40 mm irrespective of body surface area. In the Mayo Clinic series of surgically resected ascending aorta, more than 10% of patients (67 of 513) had a BAV. In this series, the mean age was 53 years, and the degree of medial necrosis was significantly less in patients with congenitally BAV as compared to other inherited connective tissue diseases; only 11% had severe MD (7) (Fig. 30.3).

However, it should be emphasized that in an individual case, histologic findings of the aortic media cannot distinguish the underlying etiology of MD. The mean growth rate of proximal ascending aortic aneurysms in patients with BAV and aortic stenosis is greater than that seen in patients with tricuspid aortic valves (1.9 vs. 1.3 mm/yr, respectively) (15). There is genetic evidence that there is a common gene that underlies BAV and aortic aneurysm and that the most dilated portion of the aorta is distal to the annulus (13). The genetic basis remains unknown, however, and is likely unrelated to fibrillin-1 because patients with Marfan syndrome have trileaflet aortic valves.

FIGURE 30.3 (A) Bicuspid aortic valve with intimal tear just above sinotubular junction. (B) Histologic section of acute dissection showing intima and media with blood in the dissection plane. (C) Histologic examination of the media showed cystic medial degeneration with increased proteoglycan matrix.

PSEUDOXANTHOMA ELASTICUM Pseudoxanthoma elasticum (PXE) is caused by mutations in the adenosine triphosphate (ATP)–binding cassette transporter C6 (ABCC6), also known as multidrug resistance-associated protein 6 (MRP6) gene. It is inherited as an autosomal recessive trait and has an incidence of 1 in 25,000 to 100,000. PXE is characterized by progressive calcification and fragmentation of elastic fibers in the skin, the retina, and the cardiovascular system (Fig. 30.4). Patients typically have a normal life span, the morbidity varies

based on the extent of extracutaneous involvement. The association between PXE and dissections is uncertain and based on single case reports (16).

FIGURE 30.4 Pseudoxanthoma elasticum. The internal elastic lamina of this coronary artery demonstrates duplication of the elastic layer, with focal calcifications. Movat pentachrome.

PRIMARILY HYPEROXALURIA Type I hyperoxaluria occurs in 1 per 120,000 live births and is transmitted as an autosomal recessive trait. It is caused by a deficiency of the peroxisomal liver-specific alanine: glyoxylate aminotransferase gene (i.e., AGT). Most patients develop renal failure in childhood. In the primary form, oxalate accumulation leads to the formation and the deposition of insoluble oxalate (CaOx) crystals in the kidney, leading to renal failure. Occasionally, extensive crystalline deposits within the walls of blood vessels (Fig. 30.5) may result in vasculopathy, clinically mimicking systemic vasculitis.

FIGURE 30.5 Primary hyperoxaluria. Note crystals of oxalate salt within the media of the renal artery.

OTHER NONINFLAMMATORY VASCULAR DISEASES ACQUIRED AORTIC ROOT DILATATION AND DISSECTION Most patients with degenerative aortic medial disease have no family history or evidence of inherited connective tissue disease. Many of the patients, who are on average 10 to 20 years older than patients with inherited diseases and BAV, have systemic hypertension. In approximately 25% of patients, however, there are no known predisposing factors. It is likely that these patients have a polygenetic predisposition to aortic dissection. The histologic features of idiopathic aortic root aneurysm, with or without dissection, are similar to those of inherited disease, although the degree of MD is typically mild. Again, the histologic features per se do not point to an underlying etiology. GENERAL APPROACH TO ASCENDING AORTIC ANEURYSM The surgical pathologist, when evaluating an aortic root aneurysm, should recognize the limitations of histologic assessment in assigning an etiology

(Table 30.2). Inflammatory lesions, such as idiopathic aortitis, should be excluded because there may be foci mimicking medial necrosis in the otherwise inflamed aortic media. In noninflammatory aortic aneurysms, the degree of MD is quite variable and does not point to a specific etiology. The number of aortic valve leaflets should be noted in cases with concomitant valve replacement, and the presence of acute dissection should be noted. In addition, any areas of healed dissection, characterized by elastic reduplication of the false lumen lining, should be described; these are readily detected by the use of elastic stains. Adequate sampling of the lesions (approximately one section per centimeter of aorta excised) aids in the identification of acute and healed dissections, aortitis, and the extent of MD. TABLE 30.2 Histopathologic Differential Diagnosis of Ascending Aortic Aneurysms Seen at Aortic Root Reconstruction with or without Aortic Valve Replacement Diagnosis

Approximate Relative Frequency

Histologic Features

Associated Histologic Findings

Associated Medical Conditions

Degenerative medial disease

95%

Medial degeneration (cystic medial necrosis): minimal to marked

Acute or healed dissection

Marfana Hypertensionb Bicuspid aortic valvec

Aortitis, necrotizing

67%

Exclude tangential cut

Dilated intercellular spaces, intercellular edema (spongiosis)

Exclude artifactual cell separation at edges of biopsy; less prominent with PPI use

Intraepithelial eosinophils

20-40

Intraepithelial neutrophils

10-30

Intraepithelial lymphocytes Mucosal erosion/ulceration

Other causes of low-grade epithelial insult including infectious esophagitis, pillinduced esophagitis, eosinophilic esophagitis “Squiggle cells”; exclude lymphocytic esophagitis

10-40

Inflammation of cardiac-type mucosa

Infectious esophagitis, pill-induced esophagitis Often mild with eosinophils and few neutrophils, exclude Helicobacter pylori gastritis

PPI, proton pump inhibitor.

Epithelial hyperplasia is manifested by expansion of the basal zone and elongation of lamina propria papillae (Fig. 31.2). Basal zone expansion is typically specified as a basal zone exceeding 15% of the total epithelial thickness; papillary elongation is defined as lengthening greater than two-thirds of the epithelial thickness. These

findings are indicative of increased epithelial proliferation and turnover and, since their description in 1970 (33), have garnered much attention as manifestations of mild reflux-induced injury (23,29,31,34,35). Most studies confirm that epithelial hyperplasia serves as a marker of reflux, but the sensitivity of this feature is debated, with reported values averaging 60% to 90% depending on cutoff and biopsy location (31). An additional issue is that accurate evaluation of epithelial hyperplasia requires well-oriented biopsies sectioned perpendicularly to the mucosal surface. Judging the basalzone thickness may also present problems because this layer is not obviously demarcated and is prone to observer variation (34). The basal layer consists of closely packed cells with round to oval nuclei, slightly basophilic cytoplasm, and such small amounts of cytoplasm that the distance between nuclei is less than the nuclear width (36). Dilation of intercellular spaces (spongiosis), originally described as an ultrastructural finding (37), is another reproducibly identifiable early marker of GERD (37-41).

FIGURE 31.2 Epithelial hyperplasia denoted by lengthening of the lamina propria papillae and expansion of the basal zone in an example of gastroesophageal reflux disease (GERD). Intraepithelial lymphocytes are focally prominent.

Intraepithelial eosinophils are an early indicator of GERD (Fig. 31.3) (35,42,43). Because specimen orientation is not crucial to its interpretation, this is a particularly useful feature when evaluating poorly oriented biopsies. The diagnostic value of intraepithelial eosinophils is limited by poor sensitivity; they are found in only 20% to 40% of cases of GERD, despite being the only histologic abnormality in 10% to 25% of patients (29,31,42,43). In addition, a

few eosinophils may occasionally appear in the epithelium of normal asymptomatic adult subjects with normal endoscopic findings and 24-hour pH monitoring (31). Although characteristic of GERD, intraepithelial eosinophils are not specific and can be found in other circumstances, including infectious esophagitis, pill-induced esophagitis, eosinophilic esophagitis, Crohn disease, collagen vascular disease, hypereosinophilic syndrome, and eosinophilic gastroenteritis, among others (29,44,45).

FIGURE 31.3 Numerous eosinophils with their distinctive bilobate nuclei are scattered among squamous cells.

Neutrophil infiltration denotes more severe degrees of epithelial injury, and is consequently an insensitive diagnostic marker, being noted in only 10% to 30% of cases (31,34,46). This infiltration is associated with other features of GERD, and is especially prominent in and around epithelial erosions and ulcerations of any cause. Other forms of active esophagitis, including infectious and pill-induced, thereby enter the differential diagnosis. Only present in about 40% of patients with GERD, mucosal erosion and ulceration represent the extreme end of the GERD spectrum (23). Biopsy specimens show the expected fibrinopurulent inflammatory exudate, necrotic slough,

and active granulation tissue, and sections should be carefully examined to exclude infectious agents or malignancy. In particular, the presence of inflammatory exudate strongly suggests mucosal destruction, even when it has not been directly sampled by the biopsy specimen, and the sections should then be studied for possible infectious organisms. The granulation tissue composing the ulcer bed can contain large, atypical mesenchymal cells that can be mistaken for carcinoma; immunohistochemical (IHC) stains for cytokeratins are helpful in excluding this possibility. Similarly, the squamous epithelium adjacent to the erosion or ulcer may demonstrate reactive atypia and prominent hyperplasia that can mimic squamous dysplasia or even invasive squamous cell carcinoma. Inflammatory changes in cardiac-type mucosa (“carditis”) located near the GEJ region (7,8,47-49) may be related to GERD or to Helicobacter pylori infection, although a minimal degree of mononuclear inflammation in this region is considered normal (5). The inflammation seen in GERD is characterized by eosinophil, neutrophil, and plasma cell infiltration of the lamina propria with variable degrees of active glandular injury (Fig. 31.4), which can be similar to H. pylori–associated carditis (47,48). Some studies have proposed that eosinophils predominate in GERD-associated carditis, whereas neutrophils and plasma cells predominate in H. pylori– associated carditis (50).

FIGURE 31.4 Inflammation of cardiac-type mucosa in a patient with gastroesophageal reflux disease (GERD). This mucosa was found in the distal esophagus, and stains for Helicobacter pylori were negative.

Other proposed indicators of epithelial injury include dilation and congestion of capillaries, balloon cells, and glycogenic acanthosis. Congestion or hemorrhage in the superficial papillae (Fig. 31.5) (51) corresponds to the red streaks noted at endoscopy. Unfortunately, this feature is of little use as an independent indicator because it is commonly accompanied by other markers of esophagitis; it is found in 10% to 30% of normal controls and in patients with esophageal varices; and in some cases, it probably represents an artifact of the biopsy procedure (29,34,51). Balloon cells are rounded, pale squamous cells swollen from leakage of extracellular fluid and proteins into the damaged cells (52). Balloon cells are common in patients with GERD, but are also seen in active esophagitis of other causes and in patients without GERD (29,52). Glycogenic acanthosis (53), as noted in the following texts, is characterized by enlarged, glycogen-laden squamous cells, but its association with GERD has not been generally accepted.

FIGURE 31.5 Dilated and congested vascular channel at the tip of a lamina propria papilla in a mucosal biopsy specimen.

Finally, intraepithelial lymphocytes (mostly T lymphocytes) are a normal attribute of the esophageal mucosa, but can be increased in number as part of the inflammatory reaction in GERD (54,55). The lymphocytes typically have irregular nuclear contours and appear to be entrapped between adjacent squamous cells, thereby resembling neutrophils. An increase in intraepithelial lymphocytes correlates with the presence of intraepithelial eosinophils and, thus, is neither an independent nor a sensitive histologic indicator of GERD.

LYMPHOCYTIC ESOPHAGITIS The presence of a marked increase in intraepithelial lymphocytes with associated epithelial injury and rare, or absent, granulocytes has been described as lymphocytic esophagitis (56-58). Endoscopic appearance of lymphocytic esophagitis is quite variable, ranging from completely normal mucosa to erosions, rings, linear furrows, or strictures (59). On the basis of the current literature, esophageal lymphocytosis is a common, nonspecific histologic finding, rather than a specific disease entity, that may be encountered in a variety of different situations. Because the range of intraepithelial lymphocytes can be considerably wide in healthy asymptomatic individuals, enumerating intraepithelial lymphocyte alone is of limited clinical value. Histologic criteria must include both intraepithelial lymphocytosis and evidence of epithelial injury (intercellular edema, basal zone expansion, and/or scattered necrotic keratinocytes), and reflect a mucosal abnormality that is not limited to the GEJ (59). In adults, lymphocytic esophagitis–pattern of injury is associated with GERD, infection, dysmotility, drug-induced injury, and immunemediated disorders/immunodeficiency, whereas in children, there is a strong association with Crohn disease (59,60). EOSINOPHILIC ESOPHAGITIS Eosinophilic esophagitis represents a chronic, local immunemediated esophageal disease, characterized clinically by symptoms related to esophageal dysfunction and histologically by eosinophilpredominant inflammation (61). Eosinophilic esophagitis occurs predominantly in children, but also in adults (62). Common presenting symptoms are feeding difficulties in infants and toddlers, nausea and vomiting in older children, and dysphagia and food impaction in adults (63,64). Many patients suffer from concomitant atopic disorders, including rhinitis, asthma, and eczema. Eosinophilic esophagitis is primarily non-immunoglobulin (Ig)E mediated, but associated with IgG4 (65,66). Endoscopic findings include esophageal rings, which may be fixed (“trachealization”) or transient (“felinization”), as well as longitudinal furrows, mucosal plaques, and

esophageal narrowing (62). Mucosal biopsies from the proximal and distal esophagus, as well as from any endoscopically visible lesions, should be submitted in separate jars. Histologic features encountered in eosinophilic esophagitis include 15 or more intraepithelial eosinophils in a high-power field of both proximal and distal mucosa as well as eosinophil microabscess formation (a cluster of at least four eosinophils), superficial layering of eosinophils (Fig. 31.6), eosinophil degranulation, and architectural changes including intercellular edema (spongiosis), hyperplasia of the basal zone, and elongation of papillae (67). Ultimately, the final diagnosis relies on clinicopathologic correlation, because none of the clinical symptoms, endoscopic findings, or histologic features in isolation is specific to eosinophilic esophagitis. Other systemic and local causes of esophageal eosinophilia should be excluded. Of note, response to PPI therapy does not exclude eosinophilic esophagitis, because some adult patients with eosinophilic esophagitis may achieve clinical and histologic remission on PPI therapy (61). It is also important to note that GERD is not defined as having fewer than 15 intraepithelial eosinophils in a high-power field, because more than 15 eosinophils may be found (68).

FIGURE 31.6 Eosinophilic esophagitis. Numerous eosinophils infiltrate the esophageal squamous epithelium. There is superficial layering of eosinophils and aggregates of four or more eosinophils forming microabscesses.

INFECTIOUS ESOPHAGITIS The esophagus is host to a few infections. Numerous organisms may involve the esophagus as part of a disseminated illness; in practice, however, most infectious esophagitis is caused by Candida species, herpes simplex virus, or cytomegalovirus (CMV). Affected patients often have some reason for impaired host response: immunosuppression (caused by congenital conditions, infection with human immunodeficiency virus (HIV), or cytotoxic and immunosuppressive therapy), malignant neoplasms, chronic debilitating disease, diabetes mellitus, or antibiotic therapy (69,70). Normal hosts, however, are not exempt from infectious esophagitis (71,72). Because effective therapy is often available, careful scrutiny for infectious agents is indicated, especially when biopsies show ulceration or exudate. The possibility of multiple infections should also be kept in mind. Multiple biopsies may be required to identify the causative organism (73). By sampling wide areas of the

esophagus, brush cytology can be an effective adjunct in identifying the causative organisms (74). Candida Esophagitis Candida species are the best known cause of infectious esophagitis. The usual culprit is Candida albicans, although sometimes C. tropicalis, C. krusei, or C. glabrata is also implicated. The gross appearance at endoscopy classically entails discrete or confluent white plaques issuing from erythematous, edematous, ulcerated, or diffusely friable mucosa of the mid or distal esophagus (75). These plaques are composed of the diagnostic fungal pseudohyphae and budding yeasts embedded in keratinous squamous material, fibrinous exudate, and necrotic debris (Fig. 31.7A). Special stains for fungi such as periodic acid-Schiff (PAS) or methenamine silver facilitate detection of the organism (Fig. 31.7B). The plaques overshadow the underlying epithelial changes of active esophagitis with variable neutrophilic inflammation, mucosal erosion, or ulceration with inflamed granulation tissue. Because Candida is a ubiquitous commensal of the gastrointestinal tract, fungal invasion into tissue or ulcer slough is required for a definite diagnosis. In rare cases, the organism can invade deeply into the esophageal wall and result in perforation and disseminated candidiasis. Chronic infection with this organism has been associated with esophageal strictures and intramural pseudodiverticulosis (76,77).

FIGURE 31.7 Candida esophagitis. (A) Luminal fibrinopurulent exudate attached to inflamed, reactive, and degenerating epithelium; the fungal organisms can barely be discerned on this hematoxylin and eosin stain. (B) The Grocott stain

clearly demonstrates the characteristic budding yeast-like cells and pseudohyphae of Candida.

Herpes Esophagitis Esophagitis due to herpes simplex virus is usually seen as an opportunistic infection in immunosuppressed patients, but it also can occur in otherwise healthy children and adults (72). Grossly, the mucosa exhibits small vesicles that evolve into discrete shallow ulcers; in severe cases, these coalesce into extensive zones of mucosal denudation (78). The ulcer bed is histologically undistinguished, consisting of necrotic debris and neutrophil-rich inflammatory exudate. The diagnostic herpetic inclusions are located in discohesive or multinucleated squamous cells at the margins of the ulcers (Fig. 31.8A). The characteristic inclusions include dense, eosinophilic, intranuclear (Cowdry type A) bodies, which are separated from a thickened nuclear membrane by a clear halo, and homogeneous ground-glass inclusions that fill the nuclei. Herpetic ulcers may be secondarily infected by fungi or bacteria. In healthy hosts, herpes simplex esophagitis is usually self-limited, but in patients with an underlying predisposing condition, the result may be esophageal perforation or disseminated infection.

FIGURE 31.8 Herpes esophagitis. (A) The distinctive intranuclear inclusions are found in multinucleated squamous cells present within an inflammatory and necrotic background. (B) Immunoreactivity for herpes simplex virus antigens confirms the diagnosis of herpes esophagitis.

Documenting herpes simplex esophagitis can be difficult. Biopsy sampling may miss the ulcer edges where the diagnostic changes

reside, or these changes may be obscured by necrosis and inflammation. The presence of ulceration in biopsies from susceptible patients should always raise suspicion, especially when prominent aggregates of large mononuclear cells are noted in the exudate (79). IHC methods can be helpful in confirming the presence of herpesvirus antigens in suspect or questionable cases (Fig. 31.8B), and may demonstrate infected cells before cytopathic effects are histologically recognizable. Cytomegalovirus Esophagitis CMV can infect the esophagus of immunocompromised patients (and only rarely normal individuals (71)), producing ulcers similar to those of herpes simplex esophagitis (80). The ulcer bed is the diagnostic focus: enlarged mesenchymal cells of the granulation tissue contain the heralded CMV inclusions—conspicuous intranuclear bodies surrounded by a clear halo together with, in many infected cells, coarse intracytoplasmic granules (Fig. 31.9). In patients under antiviral therapy, however, the inclusions may be less distinctive and the appearances mistaken for enlarged and reactive, but noninfected, cells. The presence of macrophage aggregates within the granulation tissue, particularly in a perivascular distribution, may be a diagnostic clue to CMV esophagitis (81). Immunostains are valuable in establishing the diagnosis when definitive inclusions are not seen.

FIGURE 31.9 Cytomegalovirus esophagitis. In the center of the field, a large infected cell shows a prominent intranuclear eosinophilic inclusion. Several smaller granular cytoplasmic inclusions can also be seen. Courtesy of Joel K. Greenson, MD, University of Michigan School of Medicine, Ann Arbor, MI.

Other Infections Rare examples of fungal esophagitis caused by Aspergillus and the Phycomycetes are reported. In addition, histoplasmosis, blastomycosis, and cryptococcosis may involve the esophagus, usually in association with either disseminated or mediastinal disease (82-86). Unusual examples of varicella-zoster virus esophageal infections have also been reported (87). Bacterial colonization of esophageal ulcers is a frequent observation but, in severely immunocompromised patients, bacteria can also become a primary opportunistic pathogen (88,89). Implicated bacteria include a range of gram-positive and gramnegative organisms, with a mixed population (chiefly representing oropharyngeal flora) commonly noted. Histologically, bacterial esophagitis is characterized by clusters of bacteria invading the esophageal wall and involving blood vessels with associated necrosis. Other unusual esophageal infections include bacillary

angiomatosis (90,91), tuberculosis (92-94), actinomycosis (95), syphilis, leishmaniasis (96,97), Whipple disease (98), and Chagas disease. Idiopathic esophageal ulcers are sometimes found in patients infected with HIV (99-101). Although HIV has been identified in these ulcers by IHC and electron microscopic studies, its presence probably does not reflect a pathogenic role, and other, as yet unidentified, agents may instead be responsible (102). The ulcers may heal with empiric therapy using corticosteroids. MISCELLANEOUS CAUSES OF ESOPHAGITIS Additional types of esophagitis are biopsied primarily to exclude infection and malignancy. Although histologic features may suggest the cause, the microscopic appearances are typically not specific, and the location of the lesions and the clinical data are the diagnostic attributes. Corrosive esophagitis is caused by the ingestion of caustic agents, including strong alkalis, acids, and nonphosphate detergents, either accidentally or with suicidal intent (103,104). These agents produce varying degrees of esophageal damage, ranging from mucosal erythema and edema to extensive ulceration, hemorrhage, and necrosis with thrombosis and secondary bacterial invasion. Potential complications include stricture formation, Barrett esophagus, and squamous carcinoma, although the risk of the latter is quite low. Biopsy specimens are seldom obtained from the acute phase of injury, but they may be employed to assess complications that occasionally develop. Pill-induced esophagitis refers to the esophageal injury resulting from direct contact of medication with mucosa. A wide range of drugs have been implicated; frequently cited agents include doxycycline and other antibiotics, aspirin and other nonsteroidal antiinflammatory drugs (NSAIDs), slow-release potassium preparations, ferrous salts, and alendronate (105-110). The local caustic action of these agents leads to discrete erosions or ulcers without distinguishing histologic features. Aside from ulcer and granulation

tissue, polarizable foreign material and multinucleated giant cells are often seen. This injury is commonly located in the mid-esophagus, the site of anatomic narrowing produced by the aortic arch or by an enlarged left atrium. Another cause of iatrogenic esophageal ulceration is the injection of sclerosing agents for treatment of esophageal varices. Varying degrees of localized necrosis and vascular thrombosis leading to ulceration and fibrosis are described, although biopsy specimens are seldom obtained (111). Other drugs can produce active esophageal injury through mechanisms other than direct mucosal contact. The best examples are cytotoxic chemotherapeutic agents, which can cause an active esophagitis with features ranging from mild inflammation to erosions, ulcerations, and strictures. Care must be taken in this setting to exclude infectious causes. Sloughing esophagitis is thought to be an uncommon degenerative disease of the squamous epithelium (112). The underlying pathogenesis of sloughing esophagitis is unknown. It usually occurs in elderly debilitated patients taking five or more medications, often including central nervous system depressants or medications known to cause esophageal injury (113). This is a clinicopathologic entity with endoscopically visible white plaques or membranes. The squamous epithelium has a two-toned appearance, including a superficial layer of necrotic and parakeratotic squamous cells with dark red cytoplasm and pyknotic nuclei, and a normalappearing viable deep layer (Fig. 31.10). The superficial necrotic layer is commonly separated from the deep viable layer (sloughing). A thin layer of purulent exudate may exist at the point of separation (112-114). In patients with extended survival despite their comorbid conditions, endoscopic and histologic resolution usually occurs (113,114).

FIGURE 31.10 Sloughing esophagitis. The squamous epithelium has a two-toned appearance, including a necrotic and parakeratotic superficial layer and a normalappearing viable deep layer. The superficial necrotic layer is commonly separated from the deep viable layer.

Radiation esophagitis develops as a complication of radiation therapy directed at neoplasms of the lung, the mediastinum, or the esophagus itself (115). The risk of radiation-induced injury increases at higher radiation doses and can be potentiated by concurrent administration of cytotoxic chemotherapy (116,117). Acute radiation damage, which occurs during the first few weeks after treatment, is manifest by mucosal necrosis and submucosal edema, but is seldom biopsied. Chronic radiation injury develops weeks to months after irradiation, usually when fractionated doses exceed about 60 Gy (118). The histologic changes are characterized by progressive submucosal fibrosis, capillary telangiectasia, thick-walled hyalinized arterioles, and atrophy of mucous glands, leading to persistent ulceration, strictures, or fistulae (119). Although mucosal biopsies usually show only nonspecific changes of active esophagitis, granulation tissue, and fibrosis, the presence of atypical fibroblasts

and “smudged” hyalinized collagen may suggest the correct diagnosis. Crohn disease rarely affects the esophagus. The pathologic features mirror those found elsewhere in the gastrointestinal (GI) tract: discrete ulcers, granulomas, fistulae, and transmural inflammation (120). The disease is usually associated with disease elsewhere in the GI tract (121), although it can present with isolated esophageal involvement (122). A definitive diagnosis requires a resected specimen, but aphthous ulcers and granulomas on mucosal biopsy may be suggestive in the proper setting. Acute graft-versus-host disease may cause cytoplasmic vacuolization of the basal cells and apoptosis of individual cells in the basal or lower middle layer of an uninflamed squamous epithelium, just as in cutaneous graft-versus-host disease (123). More prominent epithelial destruction and loss may occur in severe cases. Histologic changes are not helpful in distinguishing chronic graft-versus-host disease from acute graft-versus-host disease. Endoscopic or imaging evidence of esophageal webs remains the only uniformly accepted diagnostic feature of chronic graft-versushost disease within the GI tract (124). Diverse skin disorders, including lichen planus (125), pemphigus (126), bullous pemphigoid, epidermolysis bullosa, and Behçet syndrome (127), can also involve the esophagus. Rarely, examples of severe necrotizing esophagitis or esophageal ulceration that lack a clearly defined cause despite extensive diagnostic evaluation are recognized (128).

BARRETT ESOPHAGUS Barrett esophagus is a condition in which a metaplastic columnar mucosa that confers a predisposition to cancer replaces an esophageal squamous mucosa damaged by GERD (129). The known risk factors for the presence of Barrett esophagus include chronic (>5 years) GERD symptoms, advancing age (older than 50 years), male gender, tobacco usage, central obesity, and Caucasian

race (130). Diagnosis of Barrett esophagus is important because of the significantly increased risk for esophageal adenocarcinoma (130132). The general concept is that the normal squamous epithelium is damaged by chronic reflux, and subsequently reepithelialized by a columnar epithelium that is more resistant to the damaging effects of acid, pepsin, and bile. Regression of Barrett esophagus can be induced by acid suppression, antireflux surgery and ablative therapies, although relapse may occur (131,133-136). Barrett esophagus is noted in up to 14% of patients with symptomatic GERD who undergo endoscopy with biopsy (137,138). Although the prevalence varies with the population under study, it is estimated to affect 1.6% of individuals in the general population (138). Its prevalence increases with age; thus, it is primarily a disorder of adults, although cases in children are also clearly recognized (139-141). Men are affected about twice as often as women. The definition of Barrett esophagus has been refined over the past 20 years. In studies published in the 1970s and 1980s, Barrett esophagus was commonly defined by the presence of at least 2 to 3 cm of columnar epithelium in the lower esophagus. However, more recent studies have found that only those patients with intestinal metaplasia (defined by the presence of acid mucin–containing goblet cells) are at increased risk for developing dysplasia and adenocarcinoma. Accordingly, the definition of Barrett esophagus has now become dependent on the identification of goblet cells. The American College of Gastroenterology defines Barrett esophagus as an “extension of salmon-colored mucosa into the tubular esophagus extending greater than or equal to 1 cm proximal to the GEJ with biopsy confirmation of intestinal metaplasia” (130). Thus, both the endoscopic appearance and the presence of goblet cells are required for a diagnosis of Barrett esophagus. Segments less than 1 cm have been classified as “specialized intestinal metaplasia of the esophagogastric junction” instead of Barrett esophagus because of high interobserver variability (see later), and the low risk of esophageal adenocarcinoma.

THE ENDOSCOPIC AND HISTOLOGIC DIAGNOSIS OF BARRETT ESOPHAGUS The location of the GEJ has been defined as the anatomic region where the distal extent of the tubular esophagus is in contact with the proximal extent of the gastric folds. The location of the proximal extent of the gastric folds can be affected by respiration, air insufflation during endoscopy, and esophageal and gastric motility. Endoscopically, many patients with Barrett esophagus have a hiatal hernia, which makes identification of the GEJ more difficult. The requirement of at least 1 cm of endoscopically evident columnarlined tubular esophagus may decrease the interobserver variability in diagnosing Barrett esophagus. Specialized columnar epithelium is characterized by the presence of goblet cells (intestinal metaplasia), which have distended, mucinfilled cytoplasm, a barrel-shaped configuration, and indented nuclei (Figs. 31.11 and 31.12) (142). Histochemically, goblet cells contain both sialomucins and sulfated mucins that stain positively with Alcian blue at pH 2.5 (143) (Fig. 31.13). The presence of intestinal metaplasia distinguishes Barrett esophagus from several other types of glandular epithelium that may occur in the esophagus (Table 31.3). Goblet cells are not distributed uniformly in Barrett esophagus; their proportion varies considerably among patients and specimens (144). The density of goblet cells tends to be low in shortsegment Barrett esophagus (145). The columnar cells in between the goblet cells may resemble either gastric foveolar cells or intestinal absorptive cells (146). Pseudogoblet cells, which have a shape similar to that of goblet cells but contain neutral mucins (eosinophilic on hematoxylin and eosin stain), may be a diagnostic pitfall (Fig. 31.14). One clue is that pseudogoblet cells are often arranged in linear rows, whereas true goblet cells tend to be interspersed among non-goblet cells and separated from one another. The predominant form of intestinal metaplasia in Barrett esophagus—incomplete intestinal metaplasia—is composed of an admixture of goblet cells and foveolar-type cells that contain PASpositive neutral mucins. These foveolar-type cells may also contain

Alcian blue–positive acid mucins, although the staining intensity is less than that seen in the goblet cells, and the identification of these Alcian blue–positive columnar cells (so-called columnar blue cells) in the absence of goblet cells is insufficient for a definitive diagnosis of Barrett esophagus (147,148). Less commonly, the glandular component contains a variable number of goblet cells, welldeveloped intestinal absorptive cells, and sometimes even neuroendocrine cells and Paneth cells (complete intestinal metaplasia). In some cases, there is an admixture of incomplete and complete intestinal metaplasia. Pancreatic acinar metaplasia may also be found, but is of little diagnostic significance (149). Typically, the lamina propria demonstrates mild chronic inflammation and fibrosis, and the muscularis mucosae is often markedly thickened, splayed, or duplicated.

FIGURE 31.11 Barrett esophagus composed of a superficial, foveolar-like component (with a villiform surface) and underlying mucous glands, which have focally split the muscularis mucosae. The diagnostic goblet cells are evident even at low power.

FIGURE 31.12 High-magnification view of intestinal metaplastic epithelium of Barrett esophagus. Goblet cells are dispersed among columnar cells.

FIGURE 31.13 Alcian blue/periodic acid-Schiff (PAS) stain in Barrett esophagus with incomplete metaplasia. The goblet cells stain intensely blue with the Alcian blue portion of the stain because of the presence of acid mucins. The columnar

cells between the goblet cells stain with PAS, indicating the presence of neutral mucins.

FIGURE 31.14 Pseudogoblet cells. (A) Hematoxylin and eosin (H&E)–stained section of columnar epithelium with enlarged and distended pseudogoblet cells. Even on the routine stain, the tinctorial quality of the mucin differs from that seen in true goblet cells. (B) Alcian blue/periodic acid-Schiff (PAS) stain showing that all of

the enlarged and distended cells are PAS-positive and thus represent pseudogoblet cells. Reprinted from Goldblum JR. The significance and etiology of intestinal metaplasia of the esophagogastric junction. Ann Diagn Pathol. 2002;6(1):67-73. Copyright © 2002 Elsevier. With permission.

TABLE 31.3 Differential Diagnosis of Esophageal Glandular Epithelium Inadvertently sampled gastric mucosa Esophageal junctional mucosa Cardiac-type mucosa located in distal 1-2 cm Esophageal cardia-type glands Often located in the lamina propria of distal and proximal esophagus Heterotopic gastric fundic mucosa (gastric inlet patch) Noted in mid esophagus of 4%-10% of normal subjects Ciliated columnar epithelium Embryologic remnant found in infants or metaplastic Sebaceous glands Present endoscopically as small, yellow papules

Intestinal metaplasia is variably admixed with foci of cardiac-type or fundic-type epithelium, which to some degree resemble their normal counterparts in the stomach except for the presence of mucosal distortion, glandular atrophy, and mild inflammation (150) (Fig. 31.15). A biopsy specimen from the distal esophagus with either cardiac-type or fundic-type mucosa in the absence of intestinal metaplasia is not diagnostic of Barrett esophagus. However, if the endoscopic impression is clearly that of Barrett esophagus (i.e., at least 1 cm of columnar-lined esophagus), then the absence of goblet cells may simply be a function of sampling error (151).

FIGURE 31.15 Cardiac-type mucosa in a segment of Barrett esophagus. When compared to the normal gastric cardia, there is atrophy of cardiac-type glands and inflammation within the lamina propria. Intestinal metaplasia was seen in the adjacent mucosa.

INTESTINAL METAPLASIA OF THE GASTROESOPHAGEAL JUNCTION Intestinal metaplasia may be found in biopsy specimens taken near the GEJ in 9% to 36% of patients without an endoscopically recognizable segment of Barrett mucosa (152-155). However, given the difficulty in precisely localizing the GEJ by endoscopic techniques, it is not clear whether the intestinal metaplasia identified in these patients comes from just above or just below the GEJ, and intestinal metaplasia of the upper stomach and distal esophagus may be histologically indistinguishable. Regardless, incidental goblet cells seen in biopsies from the junction in the absence of an endoscopic lesion do not fulfill the criteria for a diagnosis of Barrett esophagus. Intestinal metaplasia of the gastric cardia is very common and described in up to 20% of asymptomatic subjects presenting for routine open access endoscopic examinations (156). Several studies support H. pylori as an etiologic factor for intestinal

metaplasia of the gastric cardia (47,48,157). In addition, although prospective data are relatively sparse, it has been suggested that the risk of progression to dysplasia and adenocarcinoma for cardia intestinal metaplasia is significantly lower than that for Barrett esophagus (158-161). Patients with intestinal metaplasia of the GEJ have not demonstrated an increase in the development of dysplasia or esophageal adenocarcinoma in large cohort studies after longterm follow-up, in contrast with patients with segments of intestinal metaplasia more than 1 cm (162). On basis of the current literature, the American College of Gastroenterology guideline does not recommend biopsy of a normal or slightly irregular GEJ (130). DYSPLASIA IN BARRETT ESOPHAGUS Barrett esophagus would remain an intriguing pathologic novelty except that it represents a premalignant condition predisposing to esophageal adenocarcinoma. Multiple meta-analyses demonstrate the incidence of adenocarcinoma ranging from 3.3/1000 patientyears in patients with no dysplasia to 65.8/1000 patient-years in patients with high-grade dysplasia (130,163-165). Although all patients with Barrett esophagus are at increased risk, certain patients are at higher risk than are others. As previously mentioned, there is evidence to support the contention that only those patients with goblet cells are at increased risk for developing adenocarcinoma (166-168). The presence of epithelial dysplasia, particularly high-grade dysplasia, is also a risk factor for synchronous or metachronous adenocarcinoma (169-173). Retrospective mapping studies have noted the frequent association of intestinal metaplasia and dysplasia to Barrett-related adenocarcinomas (166). Prospective studies have also documented a progression from intestinal metaplasia to dysplasia and eventually invasive adenocarcinoma (165,170). Although adenocarcinoma can arise in very short segments of Barrett esophagus (174), the risk of cancer is increased as the length of Barrett esophagus increases (175-179). Additional risk factors for the development of Barrett esophagus–associated dysplasia and adenocarcinoma are

advancing age, central obesity, tobacco usage, and male gender (130,165,179). In patients with known Barrett esophagus, a variety of medications, including PPIs, aspirin, nonsteroidal anti-inflammatory agents, and statins, have been associated with reduced risk of progression to dysplasia and/or esophageal adenocarcinoma (130). Endoscopically, dysplasia may appear as a nodule, erosion, or polyp, but, not uncommonly, dysplasia may also occur in mucosa that appears endoscopically normal, emphasizing the need for thorough sampling. In most institutions, patients are placed into a cancer surveillance program once a diagnosis of Barrett esophagus has been established, the goal of which is to identify epithelial dysplasia in a biopsy specimen before carcinoma has developed. Dysplasia surveillance includes four-quadrant biopsies taken at 2-cm intervals in patients without dysplasia and at 1-cm intervals in patients with prior dysplasia throughout the length of the Barrett segment. Mucosal abnormalities should be sampled separately, preferably with EMR (130). THE PATHOLOGIC DIAGNOSIS OF BARRETT ESOPHAGUS– RELATED DYSPLASIA Dysplasia can be defined as the presence of neoplastic epithelium confined within the basement membrane of the gland from which it arises (180). It is characterized by a combination of cytologic and architectural alterations and classified as either low-grade or highgrade depending on the degree of the abnormality present. Possible diagnoses include negative for dysplasia, low-grade or high-grade dysplasia, or changes indefinite for dysplasia. There are two distinct subtypes of dysplasia arising in Barrett esophagus: the wellestablished adenomatous or intestinal type and the recently described gastric foveolar type (181-184). Adenomatous-type dysplasia is discussed first. Low-grade dysplasia shows preservation or only minimal distortion of crypt architecture and is characterized cytologically by closely packed, overlapping, elongated basal nuclei with variable hyperchromasia, increased nucleus-to-cytoplasm ratio, and nuclear

stratification limited to basal half of cell cytoplasm (142,185,186) (Fig. 31.16). Goblet cell numbers are often reduced, and dystrophic goblet cells may be present. High-grade dysplasia shows more severe cytologic atypia and architectural complexity, although the distinction between low-grade and high-grade dysplasia may be somewhat arbitrary in some cases. High-grade dysplasia usually has loss of cell polarity and enlarged rounded nuclei with nuclear pleomorphism, irregular nuclear contours, and prominent mitotic figures, including atypical mitotic figures (Fig. 31.17). Apoptosis may also be increased. There is also more crypt complexity with branched, cribriform glands and a back-to-back pattern of closely packed glands. In some cases, the distinction between high-grade dysplasia and intramucosal adenocarcinoma (defined by the penetration of neoplastic cells through the basement membrane to infiltrate into the lamina propria or muscularis mucosae) may be difficult, particularly in a biopsy specimen (187,188) (Fig. 31.18). Biopsy diagnoses of “high-grade dysplasia with marked glandular distortion, cannot exclude intramucosal carcinoma” or “high-grade dysplasia suspicious for adenocarcinoma” are associated with a significantly higher likelihood of finding carcinoma in esophagectomy specimens than is a diagnosis of high-grade dysplasia alone and include any of the following histologic features: at least three dilated glands with intraluminal necrotic debris, glandular crowding with only an intervening fibroblast (back-to-back arrangement of glands), or ulcerated dysplastic epithelium (189,190).

FIGURE 31.16 Low-grade dysplasia in Barrett esophagus. (A) The glands are crowded, display focal branching, and are lined by dysplastic epithelium with enlarged, hyperchromatic, and partially stratified nuclei. Dystrophic goblet cells can be seen, but they are not specific for dysplasia. (B) In another example, a villiform configuration is present. The epithelium shows dysplastic features, including densely clumped chromatin, irregular nuclear contours, and occasional nucleoli.

FIGURE 31.17 High-grade dysplasia in Barrett esophagus. (A) Cytologic atypia is pronounced with an irregular chromatin pattern and loss of nuclear polarity. Mitotic figures are conspicuous, but they can also be noted in reactive epithelium. (B)

Another example demonstrates irregular and complex glandular profiles with gland crowding and prominent cytologic atypia extending to the luminal surface.

FIGURE 31.18 This complex glandular proliferation shows striking cytologic atypia and represents at least high-grade dysplasia in Barrett esophagus. In some areas, the glands are back-to-back with essentially no intervening lamina propria. The distinction of high-grade dysplasia from intramucosal adenocarcinoma is difficult in these types of cases.

Because of its metaplastic nature, the glands at the base of the Barrett mucosa show “baseline atypia” characterized by enlarged, slightly hyperchromatic cells with some stratification and increased mitotic activity. Thus, cytologic atypia involving the surface epithelium is a major diagnostic criterion for making a definitive diagnosis of dysplasia. That said, a diagnosis of dysplasia is prudent in cases with no definite surface involvement (191), but one should be very cautious when diagnosing dysplasia in this setting. Many patients with Barrett esophagus have ongoing GERD and active inflammation resulting in regenerative cytologic changes that may be difficult to distinguish from dysplasia. Thus, one should be cautious in making a diagnosis of dysplasia in the face of active inflammation. Cytologically, dysplastic epithelium shows variable nuclear

hyperchromasia and nuclear pleomorphism. Although these features may also be seen in reparative nuclei, the changes tend to be less severe and more uniform, with cells resembling their neighbors within the same gland or in adjacent glands. In contrast, there is usually an abrupt transition between dysplastic and adjacent nondysplastic mucosa. However, discrimination between the two processes is not always straightforward. In such cases, a diagnosis of “indefinite for dysplasia” is appropriate, signifying that the histologic changes are neither unequivocally neoplastic (“positive for dysplasia”) nor clearly reactive (“negative for dysplasia”). The “indefinite” category is a holding diagnosis and often employed when epithelial atypia is noted in a background of active neutrophilic inflammation and/or mucosal erosion; because the abnormalities could possibly represent florid regeneration, an express diagnosis of dysplasia is unsuitable. A diagnosis of indefinite may also be employed when the architectural or cytologic features are obscured by fixation, sectioning, or staining artifacts, or when the findings fall quantitatively or qualitatively short of a definitive diagnosis of dysplasia. For patients with indefinite for dysplasia, a repeat endoscopy with biopsies after optimization of acid suppressive medications for 3 to 6 months is recommended to confirm the diagnosis (130). Gastric foveolar-type dysplasia has basally oriented nuclei, which do not show stratification or loss of nuclear polarity (182). High-grade dysplasia is therefore identified by the presence of enlarged nuclei (three to four times the size of a lymphocyte), mild nuclear pleomorphism, prominent nucleoli, presence of eosinophilic cytoplasm, and crowded, irregular glandular architecture (182). Lowgrade dysplasia has monomorphic basally oriented nuclei, nuclear enlargement (two to three times the size of a lymphocyte), prominent nucleoli, foveolar mucinous cytoplasm, and increased number of glands occupying the full thickness of the mucosa (182). IHC for foveolar-type and intestinal-type mucins (i.e., MUC5AC and MUC2, respectively) may be helpful in distinguishing gastric foveolar-type dysplasia from intestinal-type dysplasia (183). Features helpful to distinguish gastric foveolar-type dysplasia from reactive epithelial

changes associated with GERD include the presence of fullthickness atypia and crowded glands in dysplasia and the presence of villiform, noncrowded architecture, surface nuclear stratification and atypia limited to the upper mucosa in GERD (184) (Fig. 31.19). Also, because gastric foveolar-type dysplasia arises in Barrett’s esophagus, goblet cells will be present, although they may be sparse.

FIGURE 31.19 This case of gastric foveolar-type dysplasia was originally diagnosed as reactive change. However, the presence of full-thickness atypia, crowded glands, and nuclear enlargement with mild pleomorphism and goblet cells in adjacent mucosa favors a diagnosis of gastric foveolar-type dysplasia.

Both sampling error and observer variation in the diagnosis of dysplasia make management of these patients difficult (192). Dysplasia can be diffusely distributed throughout a Barrett segment, but, in some cases, the dysplastic alterations are extremely focal (193). Thus, even when using a rigorous endoscopic sampling technique, small dysplastic foci can be left unsampled. Similarly,

12.7% to 17.6% of patients with a preoperative diagnosis of highgrade dysplasia are found to have adenocarcinoma in the esophagectomy specimen (194,195), which is still significant, although less than the 40% reported in earlier literature (196). Finally, there is significant interobserver variation in the diagnosis of Barrett-related dysplasia, particularly in the separation of “negative” versus “indefinite” versus “low-grade” dysplasia (185,197-200). This emphasizes the need to obtain multiple opinions on difficult cases. The American College of Gastroenterology guideline recommends that Barrett-related dysplasia of any grade should be reviewed by two pathologists, at least one of whom has specialized expertise in GI pathology (130). NATURAL HISTORY OF DYSPLASIA Multiple meta-analyses that have been published demonstrated the cancer risk in patients with Barrett esophagus based on the degree of dysplasia. One meta-analysis including 57 studies showed that the pooled annual incidence of esophageal adenocarcinoma was 0.33% in patients without dysplasia, lower than previously reported. In patients with short-segment Barrett esophagus reported from 16 studies, the annual cancer risk was 0.19% (163). For patients with Barrett-related low-grade dysplasia, the pooled annual incidence rate was 0.54% for esophageal adenocarcinoma alone, and 1.73% for esophageal adenocarcinoma and/or high-grade dysplasia. However, substantial heterogeneity was observed, which could be explained by stratifying based on low-grade dysplasia/Barrett esophagus ratio as a surrogate for quality of pathology; the pooled annual incidence rate of esophageal adenocarcinoma was 0.76% in studies with a low-grade dysplasia/Barrett esophagus ratio less than 0.15 and 0.32% in studies with a ratio greater than 0.15 (201). These results suggest that the risk of progression increases when the diagnosis of low-grade dysplasia is made more stringently. A meta-analysis reported the weighted annual incidence of esophageal adenocarcinoma in patients with high-grade dysplasia to be 6.58% (164). Several prospective randomized trials reported

much higher yearly progression rates of 15% to 19% in patients with high-grade dysplasia (202,203). One of the trials required confirmation of high-grade dysplasia by a second expert pathologist, again suggesting that stringent diagnosis likely predicts the subsequent esophageal adenocarcinoma risk. Patients with Barrett esophagus predominantly die of causes other than esophageal adenocarcinoma. A meta-analysis reported a pooled incidence rate of fatal esophageal adenocarcinoma of 3 per 1000 person-years. Among patients who died during surveillance, 7% of deaths were from esophageal adenocarcinoma and 93% because of other causes, with cardiovascular disease being the most common cause of death (204). SURROGATE BIOMARKERS Given the limitations of light microscopy described earlier, numerous adjunctive techniques have been proposed as diagnostic or prognostic markers for patients with Barrett esophagus (205,206). For an ancillary test to be helpful in the clinical care of patients with Barrett esophagus, the test should aid in the diagnosis of dysplasia by improving interobserver reproducibility, and/or stratify the risk of disease progression by identifying high-risk patients and therefore guiding surveillance. Despite the tremendous breadth of work in studying molecular advances, the ideal biomarker for Barrett esophagus has not yet been acquired (206). On the basis of the current literature, morphology remains the gold standard for the diagnosis of Barrett esophagus and Barrett-related dysplasia (205). Immunohistochemistry for p53 is the most extensively studied marker, and is used clinically by some pathologists to assess Barrettrelated dysplasia; however, this is not a widely accepted routine practice, because there is no evidence at this time that a p53 result should modify the morphologic diagnosis (205). Although p53 appears to be a promising marker for identifying high-risk patients with Barrett esophagus, existing data are insufficient to recommend p53 staining for routine use as a prognostic marker (205).

NEUROMUSCULAR DISORDERS Assorted disorders of the esophagus are manifested by abnormalities in motility and sphincter function, and are diagnosed by clinical, radiographic, and manometric findings. Pathologic correlates have generally been studied only in end-stage autopsy and rarely in resection specimens; consequently, the nature and evolution of the early changes are relatively inaccessible and poorly understood. The surgical pathologist is therefore largely limited to evaluating the complications of disordered motility, notably refluxassociated esophagitis, Barrett esophagus, and carcinoma. Aside from achalasia, the morphologic features of these conditions are only briefly sketched. Idiopathic muscular hypertrophy is characterized by marked thickening of the muscularis propria, particularly the inner circular layer of the distal esophagus (207,208). This is the pathologic counterpart of diffuse esophageal spasm, a rare condition in older men manifesting with dysphagia and angina-like chest pain. Functional evidence suggests that the primary abnormality is in the neural control of muscular contraction, but a neuropathologic lesion is not well documented; however, lymphocytic infiltration of the myenteric plexus is sometimes noted. This condition should be differentiated from leiomyomatosis, in which multiple confluent nodules of disorganized smooth muscle expand the lower esophageal and proximal gastric musculature (209). Leiomyomatosis primarily affects female adolescents and young adults, and may be associated with vulvar leiomyomas (210,211) and Alport syndrome (212). Interestingly, this disease has been found in patients with rectal muscular hypertrophy, causing chronic constipation, thereby mimicking Hirschsprung disease (213). Systemic sclerosis (scleroderma) commonly involves the esophagus, resulting in atrophy and fibrous replacement of the smooth muscle of the muscularis propria, chiefly the inner circular layer (214). Varying degrees of submucosal fibrosis, nonspecific inflammatory infiltration, and proliferative changes of smaller arteries

may also be noted. These changes can lead to gastroesophageal reflux with the subsequent development of esophagitis, Barrett esophagus, and an increased risk of esophageal adenocarcinoma. Atrophy and fibrosis of the muscularis propria can also be seen in other conditions, including systemic lupus erythematosus, Sjögren syndrome, primary visceral myopathies, and cricopharyngeal dysphagia. Achalasia is an esophageal motor disorder characterized by failure of relaxation of the lower esophageal sphincter and lack of progressive peristalsis in the esophageal body. This condition may occur secondary to a variety of conditions, including Chagas disease, sarcoidosis, or a host of intrathoracic malignancies or inflammatory conditions (secondary achalasia or “pseudoachalasia”) (215-217). However, most cases are idiopathic and not associated with an underlying condition (primary achalasia). The cardinal morphologic alteration is the depletion or complete absence of myenteric ganglion cells associated with a myenteric inflammatory infiltrate composed predominantly of cytotoxic T cells (218-221) (Figs. 31.20 to 31.23). These changes may be seen not only in esophagectomy specimens but also in biopsies of the muscularis propria taken at the time of myotomy. There are also a number of secondary changes that can be identified in esophagectomy specimens resected for end-stage achalasia that involve all layers of the esophageal wall (218,221,222). Patients with achalasia have a significantly increased risk of esophageal squamous cell carcinoma (223-224), which is presumably secondary to a multistep process beginning with marked squamous hyperplasia to squamous dysplasia and carcinoma similar to that seen in sporadic esophageal squamous cell carcinoma (225). Treatment of achalasia by esophagomyotomy decreases the lower esophageal sphincter muscle tone, which is often combined with an antireflux fundoplication to reduce the risk of GERD. Other complications may include peptic ulceration, fibrous stricture, and, in some cases, Barrett esophagus, and adenocarcinoma (226).

FIGURE 31.20 Esophagectomy specimen from a patient with end-stage achalasia. The mucosal surface has a cerebriform appearance secondary to marked squamous hyperplasia. The muscularis propria is markedly thickened, and there is constriction of the distal esophagus. Courtesy of Henry D. Appelman, MD, University of Michigan School of Medicine, Ann Arbor, MI.

FIGURE 31.21 High-magnification view of the myenteric plexus from a patient with achalasia. A marked lymphocytic infiltrate is present, including infiltration into ganglion cell cytoplasm (ganglionitis). Reprinted from Goldblum JR, Rice TW, Richter JE. Histopathologic features in esophagomyotomy specimens from patients with achalasia. Gastroenterology. 1996;111(3):648-654. Copyright © 1996 Elsevier. With permission.

FIGURE 31.22 Myenteric plexus from a patient with achalasia showing a scarred myenteric nerve with a minimal amount of lymphocytic inflammation. Ganglion cells are not seen. Reprinted from Goldblum JR, Rice TW, Richter JE. Histopathologic features in esophagomyotomy specimens from patients with achalasia. Gastroenterology. 1996;111(3):648-654. Copyright © 1996 Elsevier. With permission.

FIGURE 31.23 Myenteric plexus in a patient with achalasia stained with an antibody for CD8. Most of the cells surrounding this scarred myenteric nerve are CD8-positive lymphocytes.

BENIGN TUMORS AND TUMORLIKE LESIONS A diversity of benign tumors and nonneoplastic masses can be seen in the esophagus. They are, however, mostly uncommon lesions, small, and asymptomatic, whose importance lies in their distinction from malignant tumors. Most clinically apparent tumors grow or protrude into the lumen, and thus appear as polyps at endoscopy. The differential diagnosis of polypoid esophageal lesions is summarized in Table 31.4.

TABLE 31.4 Differential Diagnosis of Polypoid Esophageal Tumors Lesion

Distinguishing Features

BENIGN Squamous papilloma

Bland epithelium covering fibrovascular cores

Fibrovascular polyp

Pedunculated mass of fibrous (± adipose) tissue, found in upper esophagus

Inflammatory fibroid polyp

Vascular fibroblastic tissue with mixed inflammation

Submucosal tumors

Leiomyoma and granular cell tumor most common

Inflammatory esophagogastric polyp

Granulation tissue and edema

MALIGNANT Squamous cell carcinoma

Especially verrucous variant

Adenocarcinoma

Infrequently polypoid

Sarcomatoid carcinoma

Biphasic, atypical spindle cells, stroma predominates

Malignant melanoma

Melanin pigmentation, junctional changes

Sarcomas

Rare, distinguish from carcinoma with spindle cell component

Metastatic tumors

Occasionally produce polypoid mass

SQUAMOUS PAPILLOMA Squamous papillomas are the most common benign esophageal epithelial neoplasm (227,228). Most are small (50% mucinous component), poorly cohesive carcinoma, signet-ring cell carcinoma and other variants, micropapillary carcinoma, medullary carcinoma with lymphoid stroma, hepatoid adenocarcinoma, parietal cell carcinoma, mucoepidermoid carcinoma, Paneth cell carcinoma, and mixed adenocarcinoma (340). Grading is best applied for tubular and papillary adenocarcinomas. Adenocarcinoma may be graded into well differentiated (>95% of tumor composed of glands), moderately differentiated (50%-95% composed of glands), and poorly differentiated (49% or less composed of glands); however, a two-tier system is preferred in order to improved reproducibility of grading (340). Using a twotier system, low grade encompasses well-differentiated and moderately differentiated carcinomas, whereas high grade encompasses poorly differentiated carcinomas. Although the WHO classification scheme does not distinguish between intestinal and diffuse types of gastric carcinoma, it does not preclude addition or combination of other systems. The Lauren classification (343) identifies three histologic types: intestinal, diffuse, and mixed. Intestinal adenocarcinoma of the stomach closely resembles a colon cancer (Fig. 32.36). Grossly, it tends to be nodular, polypoid, or ulcerated and is well demarcated. Histologically, it is characterized predominantly by a glandular pattern, which may have solid or papillary areas. The individual cells are columnar or cuboidal, with a basally located nucleus. Cells with readily apparent intracytoplasmic mucin droplets are uncommon, although moderate quantities of mucin are present within gland lumina. In contrast, diffuse carcinoma (Fig. 32.37) is more likely to have a plaque-like surface component and an ill-defined, widely infiltrating growth, which is composed of individual cells or small groups and cords of cells. Between these is a fibrous or mucoid stroma. The cells of diffuse carcinoma may contain mucin droplets, sometimes producing a signet-ring configuration (Fig. 32.38). Most cases of linitis plastica (“leather-bottle stomach”) will be classified as diffuse carcinomas. Mucinous carcinomas can be intestinal or diffuse, depending on their gross and microscopic configuration. As with all schemes, Lauren classification is not perfect; interobserver agreement is moderate at most, partly because of indeterminate and mixed types. Some authors have tended to equate the terms intestinal and diffuse with well-differentiated and

poorly differentiated. However, this is misleading because some poorly differentiated carcinomas may be sharply circumscribed and intestinal in type. Diffuse carcinomas are typically poorly differentiated with regard to gland formation, but some may have low-grade nuclear features.

FIGURE 32.36 Intestinal adenocarcinoma of the stomach.

FIGURE 32.37 Diffuse carcinoma of the stomach showing submucosal infiltration by single cells and small strings of cells.

FIGURE 32.38 Diffuse carcinoma showing multiple signet-ring cells.

The Cancer Genome Atlas (TCGA) has proposed a molecular classification of gastric cancer based on four molecular subtypes: EBV associated, microsatellite instability high (MSI-H), genomically stable (GS), and chromosomal instability (CIN) (344). The CIN subtype accounts for almost 50% of all gastric carcinomas and is associated with intestinal-type cancer (tubular, papillary, mucinous), TP53 mutations, and RTK-RAS activation (Fig. 32.36). Approximately 25% of these tumors are also associated with HER2 amplification. A subset of poorly cohesive carcinomas also exhibits this molecular phenotype. CIN gastric cancer is slightly more common in men and occurs most commonly in the gastric cardia/GEJ, followed by the body and antrum. The prognosis is variable, but these tumors respond better to adjuvant chemotherapy than the other TCGA subtypes, and they may also respond to HER2targeted therapy (345). The GS subtype is seen in about 20% of gastric carcinomas and tends to be diagnosed at an earlier age (median, 59 years). It occurs in all regions of the stomach and exhibits a relatively equal gender distribution. GS gastric cancers are associated with the poorly cohesive (diffuse-type) subtype of carcinoma, including those with signet-ring cell differentiation (Figs. 32.37 and 32.38). They harbor mutations in CDH1 and RHOA as well as CLDN18-ARHGAP fusions. These tumors have a poor prognosis with limited response to chemotherapy compared to other subtypes (345). The MSI-H (mismatch repair protein deficient) subtype accounts for 10% to 20% of all gastric carcinomas, affects women more than men, and tends to present at an older age (median, 2 years). This molecular subtype occurs predominantly in the antrum and exhibits intestinal-type (tubular, papillary, mucinous) histology (Fig. 32.39). A subset is associated with prominent lymphoid stroma. This subtype may be associated with Helicobacter infection. In most cases, MSI-H is due to sporadic MLH1 promoter hypermethylation with subsequent loss of MLH1 and PMS2 (346). Rarely, this tumor may occur in association with Lynch syndrome. Mutations in PIKC3A (42%) and ERBB3 (26%) may also be seen. There is a suggestion that these tumors have a relatively better prognosis than other molecular subtypes. Moreover, these tumors may respond to PD-1 blockade immunotherapy.

FIGURE 32.39 (A,B) Gastric adenocarcinoma with prominent lymphoid stroma. The tumor cells exhibit loss of expression for (C) MLH1 and (D) PMS2 due to promoter hypermethylation of MLH1.

EBV-associated cancer represents 5% to 10% of all gastric carcinomas (Fig. 32.40). This subtype occurs predominantly in men (2:1), with mean age of 65 years. The gastric cardia, fundus, and body are preferentially affected (compared to the antrum) (347). These tumors, which also occur in gastric stumps, account for 80% of gastric carcinomas with lymphoid stroma (348). This subtype is associated with high levels of DNA methylation, PIK3CA mutations (80%), CDKN2A silencing, and amplification of PD-L1 and PD-L1 overexpression by IHC (subset). EBV-associated cancer has a better prognosis relative to other molecular subtypes. In addition, there is a potential role for PD-1/PD-L1 immunotherapy for those tumors that demonstrate a more aggressive clinical course (349).

FIGURE 32.40 (A) Gastric carcinoma with lymphoid stroma (Epstein-Barr virus [EBV]–positive gastric carcinoma). (B) In situ hybridization targeting EBV-encoded small RNA (EBER) demonstrates strong, diffuse positivity in the nuclei of the carcinoma cells. The infiltrating lymphocytes are negative.

Gastric cancer is also subdivided by location, as those occurring at the cardia, those present in the distal stomach, and those present diffusely throughout the stomach (these are almost invariably of the diffuse histologic type). The most important change in recent years for the classification of gastric cancers has been for those neoplasms that straddle the GEJ, which may have arisen either in the gastric cardia or in Barrett esophagus (350). For the purpose of staging tumors that involve the GEJ, the latest American Joint Committee on Cancer (AJCC) Cancer Staging Manual (351) defines tumors whose epicenter is located within less than or equal to 2 cm of the GEJ as esophageal. Tumors that involve the GEJ, but with an epicenter less than 2 cm into the proximal stomach, as well as tumors of the cardia/proximal stomach whose epicenters are less than or equal to 2 cm into the proximal stomach, but which do not involve the GEJ, are staged as gastric cancers. From a practical perspective, the AJCC definition provides an easily applicable scheme. EPIDEMIOLOGY OF GASTRIC CANCER AND PRECURSOR LESIONS Adenocarcinoma of the stomach is primarily a disease of older individuals; it is rare under the age of 30 years. Intestinal carcinoma involving the distal stomach is now seen much less frequently in persons born in North America or Western Europe than it is in persons born in Asia, Africa, or South America. There is a statistical association between this type of cancer and intestinal metaplasia (particularly incomplete metaplasia) and MAG (352). Both intestinal and diffuse types of gastric cancer are strongly associated with H. pylori infection (80,353), but the mechanism of carcinogenesis may differ (354-356). Diffuse cancer is not confined to the elderly and may occur in adults of all ages, as well as occasionally affecting children (357). The incidence of gastric cancer is increased in the setting of dominantly inherited cancer syndromes, namely, hereditary diffuse gastric cancer (HDGC) (358), FAP syndrome (359,360), Lynch syndrome (361,362), GAPPS (277), Li-Fraumeni syndrome (363), and juvenile polyposis syndrome (364). Another major risk for the development of gastric carcinoma is a prior partial gastrectomy that has been present for 20 years or more (215,365,366). Such individuals are three to five times more likely to develop cancer than are age-matched controls. The mechanisms underlying this neoplastic potential are poorly understood, and patients who had a peptic duodenal ulcer without gastrectomy do not carry this increased cancer risk. Rarer and less

profound risk factors for the development of gastric cancer include pernicious anemia (367), Ménétrier disease (368), and gastric polyps, particularly adenomas (369-371). HEREDITARY DIFFUSE GASTRIC CANCER HDGC is defined on clinical grounds as an autosomal dominant cancer susceptibility syndrome characterized by signet-ring cell (diffuse) gastric cancer (358,372-377). About onethird of HDGC are associated with a germline mutation in the E-cadherin gene (CDH1). Tumor cells may show absent or reduced expression for E-cadherin, but retained expression may also occur. There is a striking geographical variation in the frequency of CDH1 germline mutations, ranging from 20% in countries with a high incidence of gastric cancer to 40% in countries with a low incidence (373). In the remaining two-thirds of HDGC cases, no CDH1 germline mutations have been identified, and large studies searching for a genetic cause have so far failed to identify further susceptibility genes. Total gastrectomy is now recommended for at-risk family members with a CDH1 mutation (358,374-376). Early-stage HDGC is often characterized by multiple foci of in situ signet-ring cells as well as pagetoid-like spread of signet-ring cells within the pits and early infiltration of the lamina propria (Figs. 32.34 and 32.41) (372,373,378). These changes, which predominantly involve the proximal stomach, but may involve the entire stomach, are found in up to 96% of asymptomatic CDH1 mutation carriers, requiring complete histologic examination of gastrectomy specimens (378). Because a subset of these patients are also at risk for lobular carcinoma of the breast, metastatic carcinoma should be excluded, particularly if the patient is female (379).

FIGURE 32.41 (A) Discontinuous foci of in situ signet-ring cells within the lamina propria. (B) Typical appearance of early diffuse signet-ring cell carcinoma associated with germline CDH1 mutation.

PROGNOSTIC FACTORS The overall prognosis of gastric cancer is poor, with an average of only 10% to 15% 5-year survival, even in patients who receive a “curative” resection. Adverse prognostic factors include age older than 70 years, tumor location (proximal is worse than distal), venous/lymphatic invasion, carcinoembryonic antigen (CEA) more than 10 ng per mL, and CA19-9 more than 37 μg per mL (380,381). Independent prognostic significance has not been demonstrated for gross tumor configuration and tumor size. Survival is highest in individuals with intestinal-type carcinoma because they generally present earlier with less

advanced disease. When matched stage for stage, there is no difference in survival between the tumor types (382). The only exception to this appears to be gastric cancer with an invasive micropapillary pattern (“inside-out pattern”), which has been reported to be a variant with poor prognosis and rapid progression (383). Three modes of tumor spread may occur: via lymphatics, via the bloodstream, or via the transperitoneal route. Lymphatic metastases to lymph nodes along the greater and lesser curvatures are present in more than 70% of resection specimens. Later, there is spread to porta hepatis and paraaortic nodes. Occasionally, there may be early spread via the thoracic duct to a left supraclavicular node (the node of Virchow). Spread via the bloodstream results initially in liver metastases. Later, the lungs and other distant sites are affected. Spread transperitoneally may involve any intraabdominal site, but particularly the pelvis—the ovary being a favored location. Krukenberg tumor is ovarian metastases of a signet-ring carcinoma, which, in most cases, originates in the stomach. The most powerful determinant of prognosis is the pathologic stage. The tumor-nodemetastasis (TNM) system is widely used, and it is recommended that staging data be included in all surgical pathology reports of resection specimens (Table 32.5) (384). T1 lesions are subdivided into (a) and (b) to allow for a more precise coding of depth of invasion (Table 32.5). Lymphatic and venous vessel invasion are both adverse prognostic factors and, particularly in early gastric cancer, are predictive of lymph node metastases (385-390). The number of lymph nodes involved by gastric cancer is directly correlated to the 5-year survival rate, with 46% of patients alive with 1 to 6 lymph nodes involved in comparison to 30% of patients with involvement in 7 to 15 perigastric lymph nodes (389). Accordingly, surgical lymphadenectomy is divided into limited (D1/D0) and aggressive (D2) lymph node dissection, with an overall 5-year survival for curative gastrectomy of 23% versus 50% (390). TABLE 32.5 TNM Staging of Gastric Carcinoma TX

Primary tumor cannot be assessed

T0

No evidence of primary tumor

Tis

Carcinoma in situ: intraepithelial tumor without invasion of the lamina propria, high-grade dysplasia

T1

Tumor invades lamina propria, muscularis mucosae, or submucosa

T1a

Tumor invades lamina propria or muscularis mucosae

T1b

Tumor invades submucosa

T2

Tumor invades muscularis propriaa

T3

Tumor penetrates subserosa connective tissue without invasion of the visceral peritoneum or adjacent structuresb,c

T4

Tumor invades serosa (visceral peritoneum) or adjacent structuresb,c

T4a

Tumor invades serosa (visceral peritoneum)

T4b

Tumor invades adjacent structures/organs

NX

Regional lymph node(s) cannot be assessed

N0

No regional node metastasis

N1

Metastasis in one or two regional lymph nodes

N2

Metastasis in three to six regional lymph nodes

N3

Metastasis in seven or more regional lymph nodes

N3a

Metastasis in seven to 15 regional lymph nodes

N3b

Metastasis in 16 or more regional lymph nodes

M0

No distant metastasis

M1

Distant metastasis

aA

tumor may penetrate the muscularis propria with extension into the gastrocolic or gastrohepatic ligaments, or into the greater or lesser omentum, without perforation of the visceral peritoneum covering these structures. In this case, the tumor is classified as T3. If there is perforation of the visceral peritoneum covering the gastric ligaments or the omentum, the tumor is classified as T4. bThe adjacent structures of the stomach include the spleen, transverse colon, liver, diaphragm, pancreas, abdominal wall, adrenal gland, kidney, small intestine, and retroperitoneum. cIntramural

extension to the duodenum or esophagus is not considered invasion of an adjacent structure, but is classified using the depth of the greatest invasion in any of these sites. From Amin MB, Edge SB, Greene FL, et al., eds. AJCC Cancer Staging Manual. 8th ed. Springer; 2017;211; adapted with permission of SNCSC.

Human epidermal growth factor receptor 2 (HER2) gain of function in gastric cancer patients is associated with increased cell motility, invasiveness, angiogenesis, resistance to apoptosis, and metastatic potential (391). Overexpression of HER2 in gastric cancer, most commonly as a result of amplification of the 17q12-q21 region, is detectable by IHC techniques and occurs in 15% to 25% of gastric cancers, a meta-analysis showing an overall prevalence of 19% among gastric cancers (392). Although HER2 amplification is associated with decreased overall survival in gastric cancer patients (393), the multinational ToGA trial demonstrated increased progression-free and overall survival in gastric cancer patients with HER2-overexpressing tumors when targeted therapy to HER2 was added to conventional treatment (394). Most laboratories perform IHC for HER as a first-line assay, which has been validated and shows high concordance with cytogenetic studies in the assessment of HER2 overexpression (395). Similar to the HER2 assessment in breast carcinoma, equivocal (2+) cases are referred for fluorescence in situ hybridization (FISH) or chromogen in situ hybridization (CISH) (396). Challenges in interpretation of slide-based IHC techniques include intratumoral heterogeneity in HER2 amplification (397) and differing criteria than those used in breast cancer (398-400); the main differences are (a) gastric cancer often does not show complete membranous staining (staining is often basolateral) and (b) only five cells in a biopsy specimen are required to show immunoreactivity in order to be considered a positive result. Patients with recurrent locally advanced or metastatic gastroesophageal adenocarcinoma whose tumors express PD-L1 may be considered for treatment with an immune checkpoint inhibitor that targets PD-1, a receptor for PD-L1 on immune cells (401). The scoring scheme in the evaluation of PD-L1 expression in gastric cancers is based on a combined positive score (CPS). CPS is based on the number of PD-L1 staining cells (tumor cells, lymphocytes, macrophages) relative to all viable tumor cells as follows: CPS = # of PD-L1–positive cells/total # of tumor cells × 100. PD-L1 expression is defined as CPS greater than or equal

to 1. No expression is defined as PD-L1 less than 1. A minimum of 100 viable tumor cells in the specimen is required to be considered adequate for PD-L1 evaluation. A recent study indicates digital image analysis performs as well as pathologist interpretation (402). GROSS APPEARANCE, BIOPSY, AND CYTOLOGIC DIAGNOSIS Intestinal carcinomas may show a variety of gross appearances and can be described as polypoid (Fig. 32.42), fungating (Fig. 32.43), ulcerated (Fig. 32.44), or diffusely infiltrating (Fig. 32.45). Diffuse cancers are generally infiltrative in type. These appearances carry no prognostic significance, independent of stage. Ulcerated carcinomas may be distinguished from gastric peptic ulcer by being bigger, more irregular, and having a heaped-up or rolled edge. Any endoscopically suggestive lesion in the stomach should have multiple biopsies taken for full pathologic evaluation.

FIGURE 32.42 Polypoid gastric cancer is exophytic and sharply demarcated from the surrounding mucosa.

FIGURE 32.43 Ulcerative gastric cancer features central ulcer with raised margins surrounded by a thickened gastric wall with well-demarcated margins.

FIGURE 32.44 Infiltrative ulcerated gastric cancer with infiltration into surrounding gastric mucosa.

FIGURE 32.45 Diffuse infiltrative gastric cancer without marked ulceration or raised margins. The gastric mucosa is thickened and indurated.

Special problems may arise in some cases of diffuse carcinoma because, occasionally, the intramucosal component may be small in comparison to an extensive submucosal and mural involvement. Signet-ring cells may have nuclei that, on first examination, appear deceptively bland. If a diagnostic problem is encountered, it is recommended that, first, the presence of cytoplasmic mucus be confirmed by a combined AB/PAS stain, and second, that an immunostain for pankeratin or CEA be utilized to distinguish histiocytes from epithelial cancer cells. Occasionally, the diagnosis can only be established surgically. With adequate biopsy material, the diagnostic accuracy of gastric biopsies performed for a suspicion of cancer is 83%. Brush cytology of these lesions, when used by itself, is 85% accurate; however, when used in conjunction with biopsy, a combined accuracy of 96% is achieved (403,404). More and more preoperative staging of gastric cancer patients is performed by endoscopic ultrasound (EUS) to help distinguish the different wall layers of the stomach; determine the depth of invasion of neoplasms; and, if necessary, perform EUS-

guided fine-needle aspiration (EUS-FNA) of either the lesion itself or perigastric/regional lymph nodes to determine the presence of distant metastases (405,406). The cytomorphology of normal gastric surface epithelial cells may appear as regular cell sheets with a honeycomb appearance, and the individual cells have rounded nuclei containing an even distribution of chromatin. Occasional individual cells can be seen to be columnar (tapered at one end, squat at the other). The cytology of inflamed gastric mucosa is similar, but the cells become more cuboidal and have reduced amounts of cytoplasm. They also have mildly enlarged nuclei, with prominent single or double nucleoli. Cells showing intestinal metaplasia can be confused with signet-ring cancer. They have a globule of cytoplasmic mucus displacing (and sometimes indenting) the nucleus, but the nuclei themselves are bland and show inflammatory features only (Fig. 32.46). In contrast, gastric adenocarcinoma is characterized by increased cellularity, abnormal cellular arrangement ranging from either isolated cells, and haphazard crowded arrangement to gland formation with distorted hyperchromatic nuclei that contain multiple or giant nucleoli (Fig. 32.47).

FIGURE 32.46 Brush cytology specimen of stomach showing intestinal metaplasia (goblet cells) and cells with inflammatory changes (nuclear enlargement and prominent nucleoli) (Papanicolaou stain).

FIGURE 32.47 Brush cytology specimen of intestinal carcinoma. Note the high nuclear-to-cytoplasmic ratio, the irregular nuclear outlines, and the uneven distribution of chromatin.

IHC stains show that, overall, more than 95% of gastric adenocarcinomas are positive for AE1/AE3, pankeratin, and CAM 5.2; 60% to 90% are positive for EMA, CK20, CA 19-9, CK7, and polyclonal CEA; 40% are positive for p53; less than 25% are positive for Bcl-2, S-100, and prostate-specific antigen (PSA); and close to 0% are positive for chromogranin, synaptophysin, and Melan-A. EARLY GASTRIC CANCER This term was originally used in the Japanese literature to describe infiltrating adenocarcinomas in which the primary growth is confined to the mucosa or submucosa of the stomach. Early gastric cancer is not the same as carcinoma in situ or gastric dysplasia— conditions in which tumor cells have not penetrated the basement membrane and have no metastatic potential. Some cases of early gastric cancer may have isolated local lymph node metastases or even hepatic metastases, but most cases are still potentially curable by surgery. A subclassification of the gross appearances of early gastric cancer was devised by the Japanese Gastroenterological Endoscopy Society. Lesions may be categorized as flat, elevated, protruded, depressed, excavated, or combined forms (407). Although this terminology may be useful for the accurate gross descriptions of lesions, the correlation with microscopic appearances and prognosis is imperfect (408). Its chief value is as an aid to the endoscopist in detecting the subtle surface features of early cancer. As would be expected, early gastric cancer is mainly identified in the distal stomach, particularly along the lesser curve (409). The incidence of multicentricity has been estimated at 10%. Most tumors are 2 cm or less in diameter, although cases as large as 8 cm have been described (409). The histology of early gastric cancer is similar to that of advanced cancer, with intestinal, diffuse, and mixed forms described. The importance of correctly identifying early gastric cancer lies in the excellent results of interventional treatment. For intramucosal cancer, the cure rate is quoted as 93% when no regional lymph node metastases are present and 91% when they are present. For early cancers with submucosal involvement, the overall cure rate is 89%, which decreases to 80% in cases with lymph node metastases (388). Early gastric cancers are very amendable to interventional endoscopy, and both EMR and ESD have allowed for successful endoscopic resection of lesions (410,411).

GASTROBLASTOMA Gastroblastoma is a rare biphasic tumor arising in the muscularis propria that consists of varying proportions of uniform epithelial cell and uniform spindle cells arranged in nests (412,413). The epithelial cells have scant, pale cytoplasm; uniform round nuclei; and small, inconspicuous nucleoli (Fig. 32.48). The spindle cells are elongated and may be set within a myxoid matrix. The epithelioid cells are positive for cytokeratin, whereas the spindle cells express CD10 and CD56. The cells are negative for CD117, ANO1/DOG1, CD34, desmin, SMA, synaptophysin, chromogranin, and S100 protein. The diagnosis is confirmed by the identification of a MALAT1-GLI1 fusion in the tumor cells (414). Affected patients range in age from 9 to 79 years (median, 27 years) (415). Too few cases are reported with sufficient clinical follow-up to predict prognosis, but some tumors recur and/or metastasize.

FIGURE 32.48 Gastroblastoma. (A) Biphasic tumor arising in the muscularis propria consists of gland-forming epithelial cell and uniform epithelioid to spindles cells arranged in nests. (B) Spindled cells are uniform in appearance.

NEUROENDOCRINE TUMORS Formerly, it was thought that gastric NETs were rare, but it is now apparent that they account for 11% to 41% of all GI endocrine neoplasms (416,417). In fact, the incidence of NETs has increased incrementally over the past three decades (418). Three major clinicopathologic types of gastric NET are recognized (416,419). Type I tumors are the most common (74%). These arise in a background of corpus atrophic gastritis, usually resulting from pernicious anemia. The average age of onset is 63 years (range 15-88 years), and the male-to-female ratio is 1:2.5. Typically, they are multiple, with 57% of cases having more than two tumors. Type I tumors occur in association with achlorhydria, antral Gcell hyperplasia, and hypergastrinemia (420,421). Gastrin is trophic for ECL cells, which proliferate, resulting initially in simple hyperplasia, then later in nodular hyperplasia (called dysplasia by some), and ultimately in neoplasia (185). Nodules greater than 5 mm in diameter or those that invade the submucosa are considered to be neoplasms, not nodular hyperplasia (dysplasia) (Fig. 32.49). Tumor development, which occurs very slowly, results in the formation of multiple small mucosal and submucosal nodules seldom more than 1 cm in diameter. These are present in the body of the stomach. Histologically, these NETs appear as small clusters or ribbons of cells at the base of the mucosa. Individual cells are regular, with rounded nuclei having a diffuse chromatin pattern. The cytoplasm is grayish and not obviously granular. They are strongly positive for chromogranin and synaptophysin. Metastatic spread to lymph nodes is exceedingly rare and generally occurs only in tumors greater than 1 cm in diameter with involvement of the muscularis propria or angioinvasion. Despite the rare lymph node metastases, no deaths due to type I tumors have been reported. Removing the gastrin stimulation by antrectomy may cause tumor regression (422). No systemic hormonal effects are attributable to type I tumors.

FIGURE 32.49 (A) Multiple mucosal polypoid nodules. (B) Extreme endocrine cell hyperplasia is present, resulting in microcarcinoid nodules in the gastric mucosa. Note background corpus atrophic gastritis and intestinal metaplasia. (C) Gastric neuroendocrine tumor in this setting is clinically benign.

Type II tumors arise in individuals with ZES (6% of gastric endocrine tumors). The sex ratio is equal, and mean age of occurrence is 50 years (range 28-67 years). They arise from ECL cells in the body mucosa in a background of ECL hyperplasia secondary to a gastrinproducing duodenal carcinoid (423). As with type I carcinoids, hypergastrinemia appears to be the growth trigger for type II carcinoids, although the risk for gastric tumor development is at least 100 times greater in MEN1-associated ZES carcinoids than it is in sporadic ZES (425). It appears probable that mutation of the suppressor MEN1 oncogene on chromosome 11q13 acts as an additional etiologic factor. Morphologically, type II tumors are generally similar to type I tumors but may occasionally become large (Fig. 32.50) and metastasize to regional nodes (424).

FIGURE 32.50 (A) Type II gastric neuroendocrine tumor arising in association with Zollinger-Ellison syndrome. (B) Multiple nodules are present.

Type III gastric endocrine tumors are second in order of frequency (13% of gastric endocrine neoplasms). They occur predominantly in men (male-to-female ratio 2.8:1), with a mean age of 55 years (range 21-38 years). These tumors are sporadic, solitary, and not accompanied by hypergastrinemia, atrophic gastritis, or pernicious anemia (425). They may present clinically with hemorrhage or obstruction. In rare examples, there is evidence of production of histamine or 5-hydroxytryptophan (producing cutaneous flushing in the absence of diarrhea). Type III tumors may occur anywhere in the stomach but are most common in the corpus. They may be derived from ECL, EC, or G cells. G-cell tumors are generally located in the antrum and may give rise to gastrin production. Type III tumors may behave in a malignant manner if they are larger than 2 cm in diameter or show angioinvasion or deep muscle invasion (425). Lymph node metastases occur in 71% of cases, with a death rate of 27% and a mean survival of 28 months. Type III tumors are graded (Table 32.5). Complete excision is recommended for these tumors. Other clinicopathologic subtypes of gastric NET include somatostatin-producing D-cell NET, gastrin-producing G-cell NET, and serotonin-producing EC cell NET. Gastric neuroendocrine carcinomas are poorly differentiated malignancies (6% of gastric endocrine tumors), with a slight male predominance (male-to-female ratio 2:1). Morphologically, they are generally typical small cell carcinomas, large cell carcinomas, or a mixture of small and intermediate cells (426,427). The prognosis is uniformly poor. A subset of gastric adenocarcinomas shows a minor component of neuroendocrine differentiation; however, the definition of a true gastric mixed neuroendocrine-nonneuroendocrine neoplasm (MiNEN) requires a combined neuroendocrine and nonneuroendocrine component each of which is morphologically and immunohistologically recognizable as a distinct component and constitutes greater than or equal to 30% of the neoplasm (428). Gastric neuroendocrine neoplasms are classified as either well differentiated or poorly differentiated based on morphology and Ki-67 and/or mitotic index (Table 32.6) (429). The tumor-node-metastasis staging of gastric neuroendocrine tumors is provided in Table 32.7 (384). TABLE 32.6 World Health Organization Classification of Gastrointestinal Neuroendocrine Tumors Ki-67a Index (%)

Mitotic Index/10 HPFb

WELL-DIFFERENTIATED NEUROENDOCRINE NEOPLASMS (NENS) NET grade 1 (G1)

20

POORLY DIFFERENTIATED NEUROENDOCRINE NEOPLASMS (NENS) Neuroendocrine carcinoma

>20

>20

Small cell Large cell MIXED NEUROENDOCRINE-NONNEUROENDOCRINE NEOPLASMS (MINEN) Mixed adenocarcinoma NET

0-20

0-20

Mixed adenocarcinoma NEC (mixed adenoneuroendocrine carcinoma)

>20

>20

HPF, high-power field; NET, neuroendocrine tumor. Adapted from La Rosa S, Rindi G, Solcia E, et al. Gastric neuroendocrine neoplasms. In: WHO Classification of Tumours Editorial Board, ed. Digestive System Tumours, WHO Classification of Tumours Series. 5th ed. Vol 1. International Agency for Research on Cancer; 2019:104-109. With permission. aMIB1 antibody % positive of 500-2000 tumor cells in area of highest labeling. b10

HPF=2mm2. At least 50 HPF (at 40×) must be evaluated in area of highest mitotic index.

TABLE 32.7 TNM Staging of Gastric Neuroendocrine Tumors TX

Primary tumor cannot be assessed

T0

No evidence of primary tumor

T1*

Invades lamina propria or submucosa and less than or equal to 1 cm in size

T2*

Invades muscularis propria or greater than 1 cm I size

T3*

Invades through the muscularis propria into subserosal tissue without penetration of overlying serosa

T4*

Invades visceral peritoneum (serosal) or other organs or adjacent structures

NX

Regional lymph node(s) cannot be assessed

N0

No regional node metastasis

N1

Regional lymph node metastases

M0

No distant metastasis

M1

Distant metastasis

M1a

Metastases confined to liver

M1b

Metastases in at least one extrahepatic site (lung, ovary, nonregional lymph node, peritoneum, bone)

M1c

Both hepatic and extrahepatic metastases

*Note: For any T, add (m) for multiple tumors [TX(#) or TX(m)], where X = 1 − 4 and # = number of primary tumors identified. For multiple tumors with different Ts, use the highest. Adapted from Amin MB, Edge SB, Greene FL, et al, eds. AJCC Cancer Staging Manual. 8th ed. Springer; 2017.

GASTRIC LYMPHOMAS The stomach is one of the most common sites of extranodal lymphomas, with primary lymphomas accounting for up to 10% of malignancies at this site and apparently increasing in frequency (430,431). They occur worldwide with equal sex incidence, most commonly in the over-50 age group. It is very important to distinguish between primary gastric lymphomas and nodal lymphomas that are disseminated with secondary involvement of the stomach. A primary gastric lymphoma is defined as a neoplasm where, at the time of diagnosis, the main bulk of tumor is present in the stomach. Positive mesenteric lymph nodes and bone marrow involvement are regarded as localized metastases and do not invalidate a diagnosis of primary gastric lymphoma. Although rare examples of Hodgkin disease (432) and T-cell lymphoma (433) may be encountered, the vast majority of gastric lymphomas are of B-cell origin. The term pseudolymphoma is obsolete. It is now realized that most of the cases previously described as pseudolymphoma are, in fact, examples of mucosa-associated lymphoid tissue (MALT) lymphoma (434). Nevertheless, lymphoid hyperplasia may occasionally involve the stomach, particularly in association with peptic ulcers. On light microscopic appearances alone, it may be impossible to separate hyperplasia from low-grade lymphoma. It is, therefore, strongly recommended that, to facilitate this distinction, tissue should be obtained for IHC analysis and gene rearrangement studies. EXTRANODAL MARGINAL ZONE LYMPHOMA OF MUCOSA-ASSOCIATED LYMPHOID TISSUE The majority of primary gastric lymphomas are thought to arise from the extranodal marginal zone of follicles comprising MALT. These lymphomas have a unique appearance (Fig. 32.51) and behavior (430). They arise in a background of chronic gastritis, typically caused by infection with H. pylori. H. pylori antigens do not directly stimulate MALT lymphoma cells but cause secretion of IL-2 from adjacent T cells and induce IL-2 receptors on the tumor cells themselves (435). Approximately 75% of MALT lymphomas respond to antibiotic therapy directed against H. pylori (436) and, following treatment, may become reduced in size or regress entirely. Whether the clinical response represents “cure” or suppression remains unclear at present. MALT lymphoma is a low-grade lymphoma (430), but it may transform to a high-grade lymphoma, namely, diffuse large B-cell lymphoma (DLBCL) (430).

FIGURE 32.51 Extranodal marginal zone lymphoma. (A) A uniform dense infiltration by small to intermediate lymphocytes and small cleaved follicle center cells is present. (B) CD20 highlights the neoplastic lymphoid cells.

The gross appearance of MALT lymphoma may vary: some cases resemble peptic ulcers, whereas others have enlarged mucosal folds, but in many instances, the mucosa is flat and either hyperemic or normal. There are five cardinal histologic features characteristic of MALT lymphomas: (a) an infiltrate of small lymphocytes and small cleaved follicle center (centrocyte like, or CCL) cells (Fig. 32.51), (b) lymphoid follicles, (c) neoplastic plasma cells, (d) lymphoepithelial lesions (Fig. 32.52), and (e) Dutcher bodies (PAS-positive intranuclear inclusions).

FIGURE 32.52 Lymphoepithelial lesion from a case of mucosa-associated lymphoid tissue lymphoma.

The CCL cells may have a variety of cytologic appearances. In most instances, they are of intermediate size (slightly bigger than a small lymphocyte), with an irregular, dense nucleus. The cytoplasm is clear, and there is a well-defined nuclear membrane. In some cases, a few, or even many, larger cells may be present. These resemble noncleaved, small lymphocytes (centroblasts). In other cases, the neoplastic cells can be extremely inconspicuous and show only subtle differences from small lymphocytes. Most MALT lymphomas contain mixtures of these cell types. Typically, CCL cells are present in a perifollicular location and arise from the marginal zone. As the disease progresses, these cells may begin to infiltrate and destroy the

follicles themselves (follicular colonization). Frequently, MALT lymphoma cells appear reactive in nature, and in about one-third of cases, plasma cell differentiation is present, particularly in the zone of lamina propria immediately beneath the surface epithelium. Because these cells are rarely bizarre and are easily recognizable as neoplastic, polymerase chain reaction (PCR) testing may be required to demonstrate clonality (430,436). Lymphocytes infiltrating the epithelium of the stomach are highly characteristic of MALT lymphomas but can also be present in lymphocytic gastritis. To be suggestive of lymphoma, the infiltrate must be present as a lymphoepithelial lesion (a discrete cluster of three or more lymphocytes) (430). These distort or displace adjacent epithelial cells, partially destroying the gland. In lymphocytic gastritis, the lymphocytes (which are T cells rather than B cells) are usually present as single cells within the epithelium. A characteristic immunophenotype has been detected in MALT lymphomas and is constant for tumors with different proportions of cell types. They are immunopositive for CD20 and immunonegative for CD5, CD10, Bcl-6, and cyclin D1 (430). In particular, CD5 negativity is considered very useful in distinguishing MALT lymphomas from other small cell lymphomas. Commonly, MALT lymphomas contain CD3-positive T cells. These are not part of the neoplasm and are present secondary to stimulation by antigens from H. pylori organisms. CD21-positive cells may also be intermingled with lymphoma cells and are considered to represent residual follicular dendritic cells from destroyed lymphoid follicles (430). A t(11;18)(q21;q21) site is present in about 25% of MALT lymphomas (437) and is associated with failure of lymphoma regression after H. pylori elimination and a poor response to treatment with oral alkylating agents (430). DIFFUSE LARGE B-CELL LYMPHOMAS Most high-grade lymphomas of the stomach are DLBCLs. On morphologic grounds, it is generally not possible to determine whether these arise from a transformed MALT lymphoma (431). Occasionally, however, components of MALT lymphoma and DLBCL are found together. A major diagnostic challenge with biopsy material from suspected DLBCL is to separate lymphoma from a poorly differentiated carcinoma. Lymphoma cells tend to infiltrate widely in the lamina propria in a sheetlike manner, but they often spare existing gastric pits and glands (Fig. 32.53). In contrast to MALT lymphoma, epithelial infiltration (lymphoepithelial lesion) is not a common finding. Carcinomas tend to destroy mucosal structures as they infiltrate. The cells of a lymphoma are totally noncohesive, with no tendency to form clumps and cords. The nuclei of DLBCLs are characteristically vesicular, with prominent nucleoli and nuclear membranes (Fig. 32.54). Additional help in separating lymphoma from carcinoma can be obtained by special staining. Obviously, the presence of intracytoplasmic mucus virtually confirms the diagnosis of adenocarcinoma, although negative stains do not exclude it. IHC for CD20 and cytokeratin may be performed on fixed tissue. Gastric large cell lymphomas are characteristically negative for cytokeratin and positive with CD20. Signet-ring cells have been identified rarely in gastric lymphomas, where their appearance is usually considered to be the result of retained immunoglobulin (438).

FIGURE 32.53 Gastric lymphoma infiltrating the mucosa in a sheetlike manner with relative sparing of epithelial elements.

FIGURE 32.54 Diffuse large B-cell lymphoma of the stomach.

The coincidental occurrence of MALT lymphoma with intestinal gastric carcinoma has been noted by several authors, and perhaps this is not surprising considering that both diseases are related to H. pylori infection. OTHER B-CELL LYMPHOMAS Other primary gastric lymphomas (non-MALT type) include mantle cell lymphoma, follicular lymphoma, and Burkitt lymphoma. Mantle cell lymphomas are multicentric and commonly present as polyposis involving multiple sites in the GI tract (439,440). Histologically, they contain uniform small- or medium-sized lymphocytes with round or cleaved nuclei and no admixture of blast cells. The immunophenotype is typically IgM+, IgD+, CD5+, CD43+, and CD23−. Most cases are also CD10− and have Bcl-1 rearrangements. Burkitt lymphoma (441) and follicular center cell lymphoma (439) of the stomach are rare. Their morphology is identical to that encountered at other sites.

MESENCHYMAL TUMORS The classification of gastric mesenchymal tissue tumors is given in Table 32.8. Previously, the term gastrointestinal stromal tumor (GIST) was applied to mesenchymal tumors of all types. However, this diagnosis should be restricted to neoplasms arising from the interstitial cells of Cajal (GI pacemaker cells). Tumors arising from smooth muscle should be called leiomyoma or leiomyosarcoma, and those arising from nerves should be called schwannoma or malignant peripheral nerve sheath tumor. TABLE 32.8 Gastric Mesenchymal Neoplasms Benign Calcifying fibrous tumor Gastrointestinal stromal tumor (spindle, epithelioid, or mixed) Hemangioma Inflammatory fibroid polyp Leiomyoma Lipoma Lymphangioma Plexiform fibromyxoma Schwannoma Low (Intermediate) Malignant Potential Desmoid fibromatosis Glomus tumor (most are benign) Granular cell tumor Inflammatory myofibroblastic tumor Perivascular epithelioid cell tumor (PEComa) Solitary fibrous tumor Malignant Angiosarcoma Follicular dendritic cell sarcoma Gastrointestinal stromal tumor (spindle, epithelioid, or mixed) Kaposi sarcoma Malignant gastrointestinal neuroectodermal tumor (clear cell sarcoma) Malignant peripheral nerve sheath tumor Leiomyosarcoma Liposarcoma Primitive neuroectodermal tumor Rhabdomyosarcoma Synovial sarcoma Undifferentiated pleomorphic sarcoma

GASTROINTESTINAL STROMAL TUMORS The interstitial cells of Cajal, which are intercalated between the autonomic nerves and smooth muscle cells, control the peristaltic action of the intestinal tract. Ultrastructurally, they show features similar to GISTs (442), including incomplete myoid differentiation with interdigitating cytoplasmic processes, desmosome-like gap junctions, and synapse-like cell contacts. Normal interstitial cells of Cajal express CD117 (443,444). Almost 60% of all GISTs arise in the stomach. Small so-called micro-GISTs are also common at this site (445-447).

Approximately 95% of gastric GISTs have gain-of-function mutations in the KIT gene and are also positive for CD117. The most common mutations (in-frame deletions, point mutations, duplications, and insertions) are at the juxtamembrane domain (exon 11). Less commonly, mutations occur at the extracellular domain (exon 9) or at exons 13 and 17. Most of the remaining 5% of gastric GISTs have mutations in the PDGFRA gene (juxtamembrane exon 12 or tyrosine kinase domains at exon 18 or 14). A very small number of tumors have no detectable mutation (wild type) (444). Occasional GISTs harbor mutations in BRAF (448). Detecting individual mutations is useful in predicting the prognosis of a GIST and determining whether it will respond to imatinib therapy. Also, PDGFRA D842V-mutant GISTs may respond to PDGFRA-targeted therapy (449). GISTs account for approximately 2% of all malignant gastric tumors. The incidence is between 14.4 and 11 cases per 1 million (444). The sex incidence is equal, and the peak incidence is between the fifth and eighth decades of life. Clinical symptoms vary, and many tumors are discovered incidentally. The usual complaint is vague upper abdominal discomfort or pain. Anorexia, nausea, vomiting, and weight loss are uncommon. Larger tumors may ulcerate, giving rise to frank upper or lower GI hemorrhage or anemia (450). Rarely, tumors may bleed into the peritoneal cavity. Grossly, most GISTs are solitary rounded or lobulated lesions with a clearly defined margin. They primarily involve the muscularis propria and submucosa. Tumors bulge into the gastric lumen and have attenuated mucosa covering their surface (Fig. 32.55). Subserosal extension may occur, and the peritoneal covering may be interrupted. The cut surface displays a flat, whorled appearance, with the tumor substance usually firm with foci of necrosis or hemorrhage. Large tumors may be cystic.

FIGURE 32.55 Gastric gastrointestinal stromal tumor forms exophytic polypoid mass that protrudes into the lumen.

Spindle cell GIST accounts for only 20% of all clinically significant gastric stromal neoplasms. Eighty percent are found in the corpus, 13% at the cardia, and only 8% in the antrum. The recognized subtypes include sclerosing spindle cell, palisading vacuolated, hypercellular, and sarcomatous (443). Sclerosing spindle cell type is the most common to occur in the stomach; it is composed of interlacing fascicles and whorls of uniform elongated

cells, with cigar-shaped vesicular nuclei and eosinophilic cytoplasm (Fig. 32.56). Paranuclear vacuoles are common. The palisading variant may have a pattern so prominent that there is a close resemblance to a schwannoma (Fig. 32.57). The hypercellular subtype contains tightly packed uniform spindle cells without significant atypia or mitotic activity (Fig. 32.58). The sarcomatous subtype has significant mitotic activity and pleomorphism; it is quite uncommon, and before diagnosing such a tumor as a GIST, other diagnostic entities should be excluded.

FIGURE 32.56 Spindle cell gastrointestinal stromal tumor is composed of interlacing fascicles of cells with ovoid nuclei.

FIGURE 32.57 neurilemmoma.

Spindle cell gastrointestinal stromal tumor with palisaded appearance resembles a

FIGURE 32.58 Cellular spindle cell gastrointestinal stromal tumor.

Epithelioid GIST involves the corpus and antrum in approximately equal numbers. The sclerosing variant has a syncytial pattern and is composed of rounded cells with a clear or lightly eosinophilic cytoplasm (Fig. 32.59). Cytoplasmic clearing appears to be the result of a fixation artifact because frozen sections do not show this feature. Close examination of formalin-fixed sections reveals condensed, retracted eosinophilic cytoplasm surrounding the nuclei (Fig. 32.60). Sometimes, the vacuolation is eccentric, giving a false impression of a fatcontaining neoplasm or signet-ring cells (Fig. 32.61) (451). Typically, the sclerosing epithelioid GISTs have a low mitotic rate, but focal atypia and multinuclear cells are common. The discohesive variant comprises epithelioid cells surrounded by a lacunar space and has sharply defined cell borders. Nuclear pleomorphism is common. The hypercellular and sarcomatous variants parallel the spindle cell tumors, except, of course, that the cells are epithelioid in type (443).

FIGURE 32.59 Epithelioid gastrointestinal stromal tumor with rounded nuclei and a clear cytoplasm.

FIGURE 32.60 Epithelioid gastrointestinal stromal tumor of the stomach. Formalin-fixed sections reveal condensed, retracted eosinophilic cytoplasm surrounding the nuclei with rounded nuclei and clear cytoplasm.

FIGURE 32.61 Epithelioid gastrointestinal stromal tumor of the stomach. Eccentric vacuolation imparts a signet-ring cell appearance.

Ninety-five percent of GISTs are positive for CD117 (Fig. 32.62). However, this marker is not unique for GIST and may be encountered in mast cell tumors, seminomas, small cell carcinomas, granulocytic sarcomas, melanomas, angiosarcomas, and even some adenocarcinomas (444). ANO1 (formerly DOG-1 [discovered on GIST]), which detects a chloride channel protein, is very sensitive and specific for GIST (Fig. 32.63) and is of particular diagnostic value in the setting of CD117-negative GISTs (452,453). In general, a panel of IHC stains should be utilized in the diagnosis of gastric GIST. For example, 65% to 70% of gastric GISTs are positive for CD34, 70% are positive for h-caldesmon, 35% are positive for smooth muscle actin, 5% are positive for desmin, and less than 5% are positive for S-100 (444). A panel will help to exclude other neoplasms that, on routine H&E sections,

might be mistaken for GIST. These include smooth muscle tumors, nerve sheath tumors, solitary fibrous tumor, desmoid fibromatosis, inflammatory myofibroblastic tumor, melanoma, perivascular epithelioid cell tumor (PEComa), and spindle cell carcinoma.

FIGURE 32.62 Gastrointestinal stromal tumor with strong, diffuse expression for CD117.

FIGURE 32.63 Gastrointestinal stromal tumor with strong, diffuse expression for DOG-1/ANO1.

Multiple gastric GISTs (Fig. 32.64) can be encountered in four scenarios:

FIGURE 32.64 Multiple gastric gastrointestinal stromal tumors often arise in association with SDH deficiency.

1. Carney syndrome consists of GIST, pulmonary chondroma, and functioning extraadrenal paraganglioma. Patients with this condition tend to be young females (average age at presentation is 16.5 years; male-to-female ratio, 1:12), and the gastric neoplasms tend to be multicentric. Although the GISTs in this syndrome are typically epithelioid tumors, they tend to demonstrate relatively indolent behavior (454). 2. Carney-Stratakis syndrome, which is an autosomal dominant disorder, usually affects young adults and, in contrast to Carney triad, is characterized by GIST and (familial) paraganglioma (Fig. 32.65). Patients with this subtype have germline mutations in one of the succinate dehydrogenase genes (SDHB, SDHC, or SDHD). KIT and PDGFRA mutations are absent in these tumors, even though they express CD117 (455-457). Tumors in this setting also demonstrate relatively indolent behavior. 3. Sporadic succinate dehydrogenase subunit B (SDHB)–deficient GIST tumors are almost exclusively encountered in the pediatric population and appear to be limited to the antrum of the stomach (458-460), although very occasional adult cases have been reported (461). Both the sporadic and the syndrome succinate dehydrogenase–deficient GISTs show a high propensity for lymph node metastasis but overall have a very protracted clinical course. From a day-to-day perspective, it is important to highlight that immunostains for CD117 (c-kit) and DOG-1 are positive in more than 90% of SDHB-deficient GISTs (462). 4. Familial GIST is associated with germline mutations in KIT or PDGFRA. Affected patients may develop multiple tumors that may demonstrate clinically malignant behavior. Other manifestations of KIT activation, such as cutaneous pigmentation and mastocytosis, can also occur (463).

FIGURE 32.65 (A) Multiple gastric gastrointestinal stromal tumors associated with SDH deficiency. (B) Lymph node involvement is not uncommon. (C) These tumors express CD117 but harbor no underlying CD117 mutation. (D) SDH-B expression is absent.

GIST occurs with increased frequency in neurofibromatosis type I, although most examples involve the duodenum and not the stomach (464,465). The tumors are generally indolent and may be accompanied by diffuse hyperplasia of Cajal cells. Typically, KIT and PDGFRA mutations are not detected. Prognosis of Gastric Gastrointestinal Stromal Tumors Two major factors enter into the determination of malignant potential of gastric GIST: gross size and mitotic activity per 50 hpf (444,466,467). Fifty high-power field equate to roughly an area of 5 mm2. In this context, it is important to note that most modern microscopes are equipped with wider ×40 lenses, and as such, only about 20 hpf are required to cover an area of 5 mm2 (468). As such, one should determine the actual number of fields required on one’s own individual microscope before embarking on counting hpf. Studies by Miettinen et al. (444,468) have provided exact measurements of metastatic potential for these categories (Table 53.8). Mitotic rate and size are not completely independent variables, however; and it is unusual to find that small tumors are mitotically active. Larger tumors that may initially be assessed as benign will often have more mitotic figures present when multiple areas of the lesion are sampled.

TABLE 32.8 Prognosis of Gastric GIST Based on Mitotic Index and Tumor Size Mitotic Index (Mitoses/5 mm2)

Tumor Size (cm)

% Progressive Diseasea

≤5

≤2

0

>2-≤5

1.9

>5-≤10

3.6

>10

12

≤2

0

>2-≤5

16

>5-≤10

55

>10

86

>5

aDefined

as metastasis or death due to disease. GIST, gastrointestinal stromal tumor. Data based on Miettinen M, Lasota J. Gastrointestinal stromal tumors: pathology and prognosis at different sites. Semin Diagn Pathol. 2006;23:70-83.

Introduction of the drug imatinib has revolutionized the treatment and prognosis of metastatic malignant GISTs. Patients on treatment can expect, at least initially, that the disease stabilizes or only progresses slowly. The drug acts by selectively inhibiting ABL, BCR-ABL, KIT, and platelet-derived growth factor receptor (PDGFR) tyrosine kinases. The extent of clinical response will vary with the exact KIT or PDGFRA mutation present (469,470). SCHWANNOMA By far, the most common of these is schwannoma, which accounts for about 5% of all mesenchymal tumors. Histologically, they are similar to schwannomas at other sites but are difficult to distinguish from GISTs by light microscopy. Schwannomas occur at an average age of 58 years. They are spheroid or ovoid in shape, with an average diameter of 3 cm (range 0.5-7 cm), and are well demarcated. Small numbers of lymphocytes may be scattered throughout the tumor, and lymphoid aggregates may be present at the periphery (471). A distinctive reticular/microcystic variant occurs somewhat preferentially in the stomach (Fig. 32.66) (472,473). Schwannomas show strong diffuse immunopositivity for S-100, with negative results for CD117, desmin, and muscle actin staining. CD34 positivity is rare. There is no association with neurofibromatosis.

FIGURE 32.66 Gastric schwannoma, reticular/microcystic variant. (A) The tumor arises in the gastric wall. (B,C) Reticular, microcystic appearance is typical for this tumor. (D). Despite the unusual appearance, reticular/microcystic schwannomas are positive for S100 protein.

INFLAMMATORY FIBROID POLYP Inflammatory fibroid polyps may occur anywhere in the GI tract (474,475), but about 35% of recorded cases have been in the stomach, particularly the antrum. The sex incidence is similar, and the mean age at presentation is 60 years. They may present with colicky abdominal pain, early satiety, and pyloric obstruction. Vomiting, diarrhea, and bleeding are uncommon. In the stomach, inflammatory fibroid polyps tend to be sessile rather than pedunculated. As would be expected, polyps discovered incidentally are small (mean size 1.7 cm), whereas symptomatic lesions may be several centimeters in diameter. One recorded polyp measured 9 cm in length and 6 cm in diameter. They have now been demonstrated to be mesenchymal neoplasms that arise secondary to mutations in exons 12, 14, and 18 of the platelet-derived growth factor receptor α (PDGFRA) gene (475,476). Histologically (Fig. 32.67), they consist of an overgrowth of loose connective tissue in the submucosa that initially stretches and later ulcerates mucosa at the polyp tip. This tissue may also infiltrate and partly replace the muscularis propria. These polyps contain a variable inflammatory cell infiltrate of eosinophils, lymphocytes, and plasma cells (Fig. 32.68). Numerous small-caliber, thin-walled vessels are also present, which are commonly surrounded by a hypocellular zone of stroma (Fig. 32.68). Mitotic activity is usually not a feature, but multinucleated giant cells may rarely be seen. In some polyps, eosinophils are particularly abundant (up to 20 per hpf). All polyps are positive for vimentin, and a majority

(86%) are positive for CD34 (477). Muscle-specific actin (HHF35), α-smooth muscle actin, calponin, histiocytic markers, fascin, and cyclin D1 may also be positive (477,478), with the latter two markers found to be positive in 100% of inflammatory fibroid polyps reported in one series.

FIGURE 32.67 Inflammatory fibroid polyp consisting of cellular connective tissue with a variable inflammatory component.

FIGURE 32.68 Inflammatory fibroid polyp demonstrating (A) prominent eosinophilic infiltrate and (B) a perivascular hypocellular zone.

PLEXIFORM FIBROMYXOMA Plexiform fibromyxoma is a rare benign neoplasm that occurs in the gastric antrum, but may extend into extragastric soft tissue or into the duodenal bulb. It is characterized by a distinctive plexiform intramural growth with multiple micronodules and a paucicellular to moderately cellular myxoid to collagenous and fibromyxoid stroma (479) (Fig. 32.69). The constituent cells are positive for α-smooth muscle actin and variably for CD10, but negative for CD117, DOG1, CD34, desmin, and S100 protein. It is associated with activation of the Sonic Hedgehog signaling pathway due to a MALAT1-GLI1 oncogenic fusion with upregulation of GLI1 (480).

FIGURE 32.69 Plexiform fibromyxoma. (A,B) Distinctive plexiform intramural growth with micronodules. (C) Paucicellular myxoid or fibromyxoid stroma is characteristic of this tumor. (D) SMA highlights the tumor cells.

Other benign stromal tumors of the stomach include leiomyomas (481), lipomas (482), glomus tumors (483) (Fig. 32.70), granular cell tumors (484), and benign calcifying fibrous tumors (Fig. 32.71) (485). Small spindle cell leiomyomas are a relatively common incidental finding, with lesions less than 5 mm in diameter being present in 16% of all resected stomachs if a thorough search is conducted (486). These are of little clinical significance. Large leiomyomas are rare. They are typically positive for desmin and smooth muscle actin but negative for CD117 (481). Occasional cases are associated with EBV (458).

FIGURE 32.70 Glomus tumor. The cells are round, sharply demarcated, and uniform in appearance.

FIGURE 32.71 Gastric calcifying fibrous tumor. Dense hyaline fibrosis contains scattered psammomatous calcifications. Scattered lymphocytes may also be present.

GASTRIC SARCOMAS Leiomyosarcoma is rare in the stomach, and criteria for distinguishing clinically benign from clinically malignant smooth muscle gastric tumors are not well defined; however, a recent multicenter study indicated that nonesophageal smooth muscle tumors measuring greater than 10 cm and/or showing greater than or equal to 3 mitoses per 5 mm2 may behave aggressively (481). In addition, there are occasional sarcomas that are recognizable as specific entities, including rhabdomyosarcoma (487), undifferentiated pleomorphic sarcoma (488), liposarcoma (489), clear cell sarcoma/malignant GI neuroectodermal tumor (490,491), angiosarcoma, Kaposi sarcoma, and follicular dendritic cell sarcoma (492). MALIGNANT GASTROINTESTINAL NEUROECTODERMAL TUMOR Malignant GI neuroectodermal tumor (clear cell sarcoma) tends to occur in young adults (median age, 33 years) but may also occur in older patients. Following the small bowel, the stomach is the second most common site to be affected (491,493). The tumors vary in size from 2 to 15 cm. These tumors are typically composed of uniform epithelioid cells with eosinophilic or clear cytoplasm arranged in a nested, sheetlike, pseudoalveolar, or pseudopapillary pattern (Fig. 32.72); a more solid spindled cell pattern may also be seen. The tumor cells are positive for S100 protein and SOX10, but negative for other melanocytic markers. CD56 and synaptophysin expression may also be seen. Most of these tumors harbor EWSR1 fusions with ATF1 or CREB1.

FIGURE 32.72 Malignant gastrointestinal neuroectodermal tumor (clear cell sarcoma). (A) The tumor is composed of spindled cells arranged in nested and short fascicular patterns. (B) The cells are uniform and epithelioid with eosinophilic cytoplasm. (C) The tumor is positive for S100 protein. (D) SOX10 is also positive.

VASCULAR LESIONS The most important vascular lesion of the stomach is gastric antral vascular ectasia (GAVE), also called the watermelon stomach (494-496). This lesion is identified endoscopically by the presence of red patches, spots, or mucosal red stripes that radiate from the gastric pylorus. Biopsy of the reddened area shows dilated capillaries in the superficial lamina propria immediately below the surface epithelium. These capillaries may contain fibrin thrombi (Fig. 32.73) and may be surrounded by eosinophilic homogeneous material (fibrohyalinosis). There may also be fibromuscular hyperplasia of the lamina propria with foveolar hyperplasia, resembling chemical gastropathy. The cause of GAVE is unknown, but there is some evidence that it is related to abnormal gastric motility with mucosal prolapse (492). About 30% of patients will also have cirrhosis.

FIGURE 32.73 Gastric antral vascular ectasia, characterized by dilated capillaries beneath the surface epithelium.

Vascular ectasia may also occur in the proximal stomach of patients with portal hypertension. This lesion has been termed portal hypertensive gastropathy (PHG) (497,498). It occurs in 61% of patients with cirrhosis but may also be seen in noncirrhotic portal hypertension. Endoscopically, four types of lesion may be encountered: a mosaiclike mucosal pattern described as being akin to snake skin, red point lesions, cherry red spots, and blackbrown spots (496). Naturally, these lesions are rarely biopsied but appear to consist of a relatively mild degree of mucosal capillary dilatation that is not accompanied by fibrin thrombi, fibrohyalinosis, or fibromuscular hyperplasia of the lamina propria. A Dieulafoy lesion (caliber persistent artery of the stomach) is an uncommon lesion, but it has a high mortality (60%) (499,500). It is characterized by the presence of an unusually large diameter artery in the gastric submucosa, generally located on the lesser curvature within a few centimeters of the pylorus. The vessel is normal histologically but is attached to the deep surface of the muscularis mucosae. As this vessel compresses the mucosa, there is gradual erosion, ultimately resulting in bleeding both from the artery and its accompanying vein (500). Other vascular lesions of the stomach include arteriovenous malformations, which consist of tortuous vessels distributed through all layers of the gastric wall. Isolated hemangiomas and lymphangiomas are uncommon, although angiomatosis may occur as part of the OslerWeber-Rendu, Maffucci, and Klippel-Trenaunay syndromes. Rarely, pyogenic granulomas may occur (501). Vascular neoplasms of the stomach include Kaposi sarcoma (502) and, rarely, angiosarcoma (503). These tumors have the same histologic features in the stomach as they do at other sites.

METABOLIC DISEASES INVOLVING THE STOMACH GI involvement is common in amyloidosis. In the stomach, amyloid deposits may be present in the walls of submucosal arteries and within the muscularis mucosae and muscularis propria (504). Rarely, amyloidosis is more widely distributed in the lamina propria and is a cause of enlarged gastric folds. In addition to the usual causes of amyloidosis, patients on long-term hemodialysis for renal failure may develop β-2-microglobulin amyloidosis (505).

Uremia may produce gastric petechial hemorrhages or even large areas of extravasated blood. Slow oozing, rather than rapid loss, is usual. This is presumed to occur because of a functional platelet abnormality. Gastric erosions and ulcers may also be present (506), probably produced by a direct toxic effect of urea on the mucosa. Diabetes mellitus may result in gastroparesis (dilatation and delayed emptying) and even lead to atony. This disturbed motility is likely the result of an autonomic neuropathy (507). Gastric mucosal calcinosis is rare. It is usually secondary to metastatic calcification (hypercalcemia and hyperphosphatemia) but may also result from dystrophic calcification where it may be secondary to atrophic gastritis, hypervitaminosis A, organ transplantation, and aluminum-containing antacids (508).

ACKNOWLEDGMENTS This chapter was adapted from the sixth edition chapter co-authored by Dr. David F. Schaefer and Dr. David A. Owen. While we have made extensive modifications to incorporate recent advances in gastric pathology, as testament to the significant contributions by these two distinguished pathologists, the backbone of the chapter remains largely intact. We acknowledge them and thank them for use of their material for the current chapter.

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33

Nonneoplastic Intestinal Diseases Shyam Sampath Raghavan ■ Scott R. Owens

EVALUATION OF SMALL INTESTINAL MUCOSAL BIOPSY SPECIMENS SPECIMEN PROCUREMENT AND PROCESSING Small intestinal mucosal biopsy examination remains one of the most important steps in evaluating patients with malabsorption (1,2). An endoscope is used to obtain duodenal specimens: at least one from the duodenal bulb and multiple from the distal duodenum (1). Adequate specimen size is necessary to accurately evaluate mucosal disease, and, because the procedure is performed under direct vision, many specimens can safely be obtained. Recognition of the orientation of the specimen is important, although it may not be necessary to achieve perfect orientation for interpretation. Histotechnologists experienced with gastrointestinal specimens can often achieve good results by carefully embedding specimens on edge, perpendicular to the mucosal surface. Proper specimen evaluation is best achieved by examination of optimally oriented intestinal villi obtained from the central region of the biopsy specimen. As such, it is crucial not to let tangential sectioning interfere with histologic examination. A standard approach is to examine multiple step-leveled sections for each biopsy specimen under hematoxylin and eosin staining (i.e. obtaining ribbons of sections from at least three levels). Occasionally, examination with Alcian blue–periodic acidSchiff (PAS) stain with a hematoxylin counterstain can be used if there is suspicion of Whipple disease and/or Mycobacterium avium-intracellulare complex (MAC) infection. This stain can also be used to detect foveolar metaplasia in villous epithelium. A trichrome stain can be a useful adjunct for confirming the collagen deposition seen in ischemia or collagenous sprue.

NORMAL SMALL INTESTINAL HISTOLOGY Small intestinal mucosa is composed of an epithelial component, a lamina propria, and muscularis mucosae (Fig. 33.1). The surface epithelium forms projections called villi with intervening invaginations called the crypts of Lieberkühn. There are two forms of mucosal epithelium in the small intestine with similar appearances but somewhat distinct functions (3). Villous epithelium is the primary component, with basally placed rounded nuclei and abundant cytoplasm (Fig. 33.2). The apical surface contains a brush border for absorption, and lymphocytes are scattered among the individual epithelial cells in the ratio of one lymphocyte for every 4 or 5 cells in the proximal small intestine (3). The normal villous to crypt length ratio approximates 3:1 to 5:1 (3). The crypt epithelium functions primarily in epithelial cell renewal. There are numerous goblet cells along with stem cells and scattered endocrine cells (3). Beneath the epithelium, filling the core of the villi, and surrounding the crypts lies the lamina propria, which contains numerous inflammatory cells, including a rich population of plasma cells. As a result, the lamina propria plays a large role in intestinal immunity. Defining the base of the mucosa, the muscularis mucosae is a thin muscular layer that separates the overlying mucosa from the underlying submucosa.

FIGURE 33.1 Section of small intestine mucosa obtained near the center of the biopsy specimen. The villi are long and slender, and the ratio of the villous to crypt length is approximately 5:1. The lamina propria contains mixed inflammatory cells.

FIGURE 33.2 Normal small intestine. The brush border is discernible, and some inflammatory cells, including scattered plasma cells, are seen in the lamina propria. Enterocyte nuclei are basally located and relatively evenly aligned. An occasional intraepithelial lymphocyte is present.

Brunner glands are located in the submucosa of the proximal duodenum and empty into the intestinal lumen or into the crypts (4). They are lined by cuboidal or columnar cells with pale cytoplasm (3), and their primary role is to secrete a viscoelastic mucous as well as bicarbonate to protect the mucosal epithelium (4). They have an inconsistent effect on villous architecture (3). Normal-length villi may be encountered overlying Brunner glands, but usually the villi are distorted and appear short. Similarly, villi are often short and distorted next to or overlying lymphoid aggregates. This type of appearance in association with Brunner glands and/or lymphoid aggregates should not be considered evidence of celiac sprue (CS). Artifactually shortened villi can also be seen in biopsies without muscularis mucosae; when this component is not present, the mucosa can spread laterally, giving the impression of shortened villi (3). In general, identifying four villi in a row indicates that the villous architecture of the whole biopsy specimen is probably normal (3,5-7). This does not mean that biopsy specimens with less than four aligned normal villi should be considered inadequate for evaluation, because even one normal villus in a proximal small intestine biopsy specimen rules out fully developed CS. Conversely, finding four normal villi in a row does not necessarily rule out focal lesions. Accumulations of various pigments can be seen in the small intestines of apparently normal individuals. The term pseudomelanosis duodeni has been used to describe the accumulation of brownblack pigment in the lamina propria macrophages of the proximal duodenum (3,8,9). Iron and sulfur,

usually in the form of ferrous sulfide, are the principal components of these benign acquired deposits. The condition does not impair the function of the small intestine. Associations include chronic heart failure, chronic renal failure, hypertension, diabetes, gastrointestinal bleeding, and oral iron intake (8,9). PATTERNS OF ABNORMAL SMALL INTESTINE ARCHITECTURE The responses to injury in the small intestinal mucosa are limited, and recognition of a response pattern can be useful in formulating a differential diagnosis (Table 33.1). In this chapter, the term severe villous abnormality describes a flat intestinal mucosa in which no villi are seen. Usually, this change is diffuse and is accompanied by intraepithelial lymphocytosis (at least 30-40 intraepithelial lymphocytes/100 enterocytes) and associated with crypt hyperplasia, characterized by numerous mitotic figures. The terms severe villous abnormality and flat intestinal mucosa are preferred to villous atrophy because the mucosa in the forms associated with crypt hyperplasia is actually of normal thickness. The term variable villous abnormality describes specimens in which the villi are either only focally flat or are less than flat (mild or moderate villous shortening). Many specimens in this category also show increased intraepithelial lymphocytes. These changes may be associated with features that suggest a specific diagnosis (e.g., numerous eosinophils, granulomas, parasites), or they may be nonspecific. TABLE 33.1 Patterns of Abnormal Small-Bowel Architecture Entities usually associated with a diffuse severe villous abnormality and crypt hyperplasia Celiac sprue (CS) Refractory or unclassified sprue Other protein allergies Lymphocytic enterocolitis Entities usually associated with a variable villous abnormality and crypt hypoplasia Kwashiorkor, malnutrition Megaloblastic anemia Radiation and chemotherapeutic effect Microvillous inclusion disease End-stage refractory or unclassified sprue Entities usually associated with a nonspecific variable villous abnormality, usually not flat Changes associated with dermatitis herpetiformis Partially treated or partially developed CS Infection Stasis Tropical sprue Zollinger-Ellison syndrome Mastocytosis Nonspecific duodenitis Autoimmune enteropathy Immune disorders Inflammatory bowel disease (i.e., Crohn disease) Drug effect (e.g., nonsteroidal anti-inflammatory drugs [NSAIDs], olmesartan) Lymphocytic enterocolitis Helicobacter pylori infection Small bowel bacterial overgrowth Peptic duodenitis Ischemia Radiation enteritis Entities associated with variable villous abnormalities illustrating specific diagnostic changes Collagenous sprue Common variable immunodeficiency Whipple disease Mycobacterium avium-intracellulare complex (MAIC) infection Eosinophilic gastroenteritis Parasitic infestation Waldenström macroglobulinemia Lymphangiectasia Enteropathy-associated T-cell lymphoma Abetalipoproteinemia Acrodermatitis enteropathica Tufting enteropathy

Entities Associated With Diffuse Severe Villous Abnormality and Crypt Hyperplasia Celiac Sprue. Celiac sprue (CS), which is also known as gluten-induced enteropathy, gluten-sensitive enteropathy, and nontropical sprue, is a major cause of malabsorption (1,6,7,10-14). Although the adage “all that flattens is not sprue” is true (15), almost all adult patients in North America with a severe villous abnormality and crypt hyperplasia do have CS. The pathogenesis of CS involves immunologic injury to the enterocyte associated with the ingestion of gluten—the toxic component of cereal grains such as wheat (gliadins), rye (secalins), and barley (hordeins). In susceptible individuals, exposure to these grain peptides leads to a cascade of changes in the surface epithelium as well as the lamina propria that ultimately lead to epithelial injury via both the innate and the adaptive immune system (13). CS is a human leukocyte antigen (HLA)–associated condition that is associated primarily with the major histocompatibility complex class II alleles DQA1*0501 and DQB1*0201, which make up the DQ2 heterodimer (14). This HLA-DQ2 allelic combination is found in more than 90% of patients with CS, with the remaining patients carrying the HLA-DQ8 phenotype (DQA1*03-DQB*0302) (1,14,16). HLA testing is not routinely performed for the initial diagnosis of CS because approximately 30% of the apparently healthy white population carry these alleles; however, testing negative for both HLA-DQ types makes the diagnosis of CS extremely unlikely (12,13,17). Patients with CS usually show a rapid and dramatic clinical, serologic, and histologic improvement with the removal of gluten from the diet, and they quickly relapse following its reintroduction (1,7,11,16,18). The principal features of CS are crypt hyperplasia, loss of villous height, chronic inflammatory cell infiltration, and surface intraepithelial lymphocytosis (greater than 40 per 100 enterocytes). The flat mucosa of CS is associated with increased lymphocytes, plasma cells, and occasional eosinophils in the lamina propria (Figs. 33.3 and 33.4) (10). About 70% of the intraepithelial lymphocytes express CD8 and about 10% CD4; the remaining 20% are CD3-positive, CD4-negative, and CD8-negative. Moreover, increased numbers of lymphocytes express the γ/δ T-cell receptor (16,19). With inflammation, the enterocyte nuclei lose their basilar alignment and become stratified. Neutrophils may be present but are usually not prominent. The histologic abnormalities are most severe in the proximal intestinal mucosa, gradually lessening distally. With gluten withdrawal, the abnormalities recede from distal to cephalad. Thus, proximal small intestine biopsy specimens may remain abnormal for quite some time, even in patients showing marked clinical improvement.

FIGURE 33.3 Severe villous abnormality typical of celiac sprue. The mucosa is flat and the villi are absent. There is lymphoplasmacytosis in the lamina propria and numerous lymphocytes in the epithelium.

FIGURE 33.4 High-magnification view of small intestine mucosa in celiac sprue. In addition to the expansion of the lamina propria by lymphocytes and plasma cells, numerous intraepithelial lymphocytes are present.

A pathologist does not make the diagnosis of CS in isolation, and is only able to indicate that the constellation of histologic findings is compatible with a diagnosis of CS. Definitive diagnosis depends on the demonstration of a suitable clinical presentation, compatible serologic tests (e.g., immunoglobulin [Ig] A antiendomysial antibodies, IgA antitissue transglutaminase antibodies), appropriate small intestine histology, and clinical and serologic response to gluten withdrawal (1,14,16,17). Subsequent gluten challenge and repeat biopsy to assess for relapse are no longer recommended. Mucosal lesions of CS can be classified into five types using the Marsh-Oberhuber scheme (Table 33.2) (1,10,16,20,21). Type 0, the preinfiltrative lesion, is essentially normal, with under 40 lymphocytes per 100 enterocytes and without crypt hyperplasia or villous blunting. Type 1, the infiltrative lesion, is characterized by intraepithelial lymphocytosis (at least 40/100 enterocytes) without crypt hyperplasia or villous blunting. The type 2 lesion, which is also known as the hyperplastic lesion, shows a mild variable villous abnormality with epithelial lymphocytosis. The type 3 lesion, also known as the destructive lesion, represents the classic CS lesion described earlier with the triad of intraepithelial lymphocytosis, crypt hyperplasia, and villous blunting. Some investigators subdivide the type 3 lesion into a type 3a with partial villous shortening, a type 3b with a more severe villous change, and a type 3c lesion that is essentially flat (severe blunting) (10,20). The hypoplastic type 4 lesion is considered an atrophic end-stage lesion that is seen in a minority of patients unresponsive to gluten withdrawal; it includes the lesion of collagenous sprue (discussed in the section “Collagenous Sprue”). TABLE 33.2 Marsh Classification Type 0 (Preinfiltrative)

Type 1 (Infiltrative)

Type 2 (InfiltrativeHyperplastic)

Type 3a (Destructive)

Type 3b (Destructive)

Type 3C (Destructive)

Type 4 (Hypop

Intraepithelial lymphocytes (#/100 enterocytes)

Normal (40

>40

>40

>40

>40

>40

Crypts

Normal

Normal

Hyperplastic

Hyperplastic

Hyperplastic

Hyperplastic

Atrophic

Villi

Normal

Normal

Normal

Mild blunting

Moderate blunting

Severe blunting (flat)

Severe blunti (flat)

The histologic differential diagnosis includes all entities that may cause at least a focal severe villous abnormality, including common variable immunodeficiency (CVID), protein allergies other than to gluten,

some cases of infectious gastroenteritis (22), rare cases of tropical sprue (23), Zollinger-Ellison syndrome (5), chronic ischemia, inflammatory bowel disease (IBD) including Crohn disease (CD) (10,24), stasis with bacterial overgrowth (25), ischemia, peptic duodenitis (10), and nonspecific duodenitis. Clinicopathologic correlation is essential for proper diagnosis. Helicobacter pylori can also be associated with an increase in intraepithelial lymphocytes, generally without any changes in villous architecture (26). All biopsy specimens should be carefully evaluated for plasma cells because their absence in selective IgA immunodeficiency and CVID is easy to overlook. Numerous neutrophils, cryptitis, and crypt abscess formation are usually not part of CS; in such cases, entities such as infectious gastroenteritis, IBD including CD, nonspecific duodenitis, and stasis syndromes should therefore be considered. The most common cause of unresponsiveness after implementing a gluten-free diet is that the diet is not completely gluten free (1,16). Furthermore, wheat is commonly used as an extender in processed foods, and it is occasionally present in seemingly noncereal grain products such as ice cream, cocoa mixes, instant coffee, and salad dressings. Medications, vitamins, and mineral supplements may also contain gluten (27). If dietary indiscretions are ruled out, patients may have refractory or unclassified sprue, which may respond to the administration of corticosteroids, azathioprine, cyclosporin, or mesalamine (1,13,14,28). Refractory sprue can also be associated with cavitation of mesenteric lymph nodes and hyposplenism (29). Persistent symptoms despite gluten withdrawal with histologic improvement should be a clue to search for comorbidities that may cause persistent diarrhea, such as pancreatic insufficiency, secondary lactase deficiency, bacterial overgrowth, coexisting IBD, or collagenous or lymphocytic colitis (see the section “Lymphocytic Enterocolitis”) (14,16,30). Lymphoma must also be considered in nonresponsive patients, and this should prompt a re-review of biopsy specimens or rebiopsy. Furthermore, abnormal intraepithelial T-cell immunophenotypes and T-cell receptor gene rearrangements in some patients with refractory sprue suggest that many refractory patients could, in fact, have low-grade T-cell lymphomas (28,31). Enteropathy-associated T-cell lymphoma is discussed in detail in the chapter on neoplastic intestinal pathology. OTHER PROTEIN ALLERGIES Patients with allergic reactions to chicken, soy protein, milk, eggs, and tuna fish have been reported to show a flat mucosa similar to that seen in CS (25,32-34). The definitive diagnosis depends on identifying the offending protein, showing a response to its withdrawal from the diet, and demonstrating recrudescence of symptoms and pathology with its reintroduction. LYMPHOCYTIC ENTEROCOLITIS CS and other sprue-like lesions may be associated with a colonic epithelial lymphocytosis (35-37) with or without gastric epithelial lymphocytosis (38). A recent study showed that 4.3% of patients with CS had concurrent evidence of a colonic inflammation identical to lymphocytic colitis (38). This finding was more prevalent in older patients who had more severe villous abnormality. Similarly, a study showed that 15% of patients with lymphocytic colitis also had CS-like histology on a small intestine biopsy (39). Colonic microscopic abnormalities in patients with CS occur after experimental exposure to wheat or gliadin enemas (40), suggesting that the entire intestinal tract may be susceptible to gluten-induced injury. In some patients with true CS (responsive to gluten withdrawal), one possibility is that occult dietary gluten actually reaches the colon and induces the histologic changes of lymphocytic colitis. However, approximately half of the patients with spruelike small-bowel lesions and lymphocytic colitis have not responded to gluten withdrawal. The term lymphocytic enterocolitis has been coined to describe this refractory sprue–like condition associated with colonic mucosal abnormalities (35). Entities Associated With Variable Villous Abnormality and Crypt Hypoplasia Marasmus and/or Kwashiorkor. Biopsy specimens from malnourished patients with marasmus (i.e., severe calorie and protein deficiency) may be normal, but patchy areas of decreased villous height without increased mitotic activity have been reported (41). In kwashiorkor (i.e., low protein but adequate caloric intake), 6 of 10 patients reported by Brunser et al. (42) had lesions indistinguishable from those

of CS. Some cases have shown a variable villous abnormality associated with increased intraepithelial lymphocytes (43). Megaloblastic Anemia. Nutritional deficiency of folate and vitamin B12 may result in impaired epithelial cell replacement because of decreased DNA synthesis. Consequently, a variable villous abnormality with or without megaloblastic epithelial changes can be seen (44). Differentiating folate and vitamin B12 deficiency from CS is usually straightforward because the degree of villous blunting is not as severe and is associated with a decrease in crypt apoptosis. In addition, an increase in intraepithelial lymphocytes is typically not present (45,46). It should be noted that vitamin B12 deficiency is relatively common in CS, with a prevalence of 12% in one study (47). Radiation and Chemotherapy Effect. Because radiation therapy and chemotherapeutic agents inhibit DNA synthesis, the intestinal mucosal changes can be similar to those in folate and vitamin B12 deficiency and are associated with decreased mitotic activity in the crypts. Chemotherapy and radiation may also cause focal necrosis of epithelial cells (apoptosis), markedly regenerative epithelial changes, and increased numbers of chronic inflammatory cells within the mucosa and submucosa (48-50). Microvillous Inclusion Disease. Microvillous inclusion disease (MVID) is an autosomal recessive disease characterized by severe secretory diarrhea typically within the first week of life (51). Studies have shown an association with mutations in the gene MYO5B, which encodes myosin Vb, a protein responsible for organelle transport and endosome recycling (52-54). Diarrhea persists despite total parenteral nutrition, and patients rarely survive beyond the age of 2 years without small intestine transplantation (52). Biopsy specimens usually have severe villous abnormality, mild crypt hyperplasia, and a variable degree of inflammation in the lamina propria. Small intestine biopsy specimens contain a severe villous abnormality with crypt hypoplasia (55). On electron microscopy, enterocytes have an absence of membranous microvilli, with an accumulation of “inclusions” of microvilli within the cytoplasm (56). These intracytoplasmic aggregates are apparent on periodic acid–Schiff stain with diastase (PASD) and CD10, which also highlight an absent brush border. Immunostaining for Rab11, which is a protein in the Ras family of monomeric G-proteins, can also be used for diagnosis, as cases of MVID have diffuse apical staining of surface enterocytes (57). This finding is not present in patients without this disorder. Entities Associated With a Nonspecific Variable Villous Abnormality Many diseases are associated with nonspecific variable villous abnormalities that are usually characterized by mild blunting. Some patients have intraepithelial lymphocytosis with essentially normal mucosal architecture. This has been shown to occur in patients with Hashimoto thyroiditis, Graves disease, rheumatoid arthritis, psoriasis, multiple sclerosis, chronic nonsteroidal anti-inflammatory drugs (NSAID) use, bacterial overgrowth, H. pylori infection, and Crohn disease (58,59). Anywhere from 10% to 20% of these biopsy specimens are from patients with clinically latent or partially treated CS (Fig. 33.5) (58). Other etiologies in the differential diagnosis can be seen in Table 33.1.

FIGURE 33.5 Small intestine biopsy from a patient with celiac sprue after several weeks of a gluten-free diet. The villous abnormality is now variable, with blunted villi that vary from nearly flat (right) to a villous:crypt ratio of around 1:1 (left). The lamina propria and epithelium continue to have inflammation.

DERMATITIS HERPETIFORMIS Dermatitis herpetiformis (DH) is an itchy, blistering skin condition that typically appears on the elbows, knees, and buttocks and is characterized by granular IgA deposition at the dermal-epidermal junction (60,61). Greater than 90% of patients with DH have associated changes of gluten-sensitive enteropathy on endoscopic and histologic examination, although only approximately 10% exhibit symptoms of malabsorption (59-62). Classically, patients have histologic changes compatible with Marsh type 1 and type 2 lesions (63), and approximately 75% of patients with DH have clinical improvement of their skin lesions when they are on a gluten-free diet (61,64). TROPICAL SPRUE/ENVIRONMENTAL ENTEROPATHY Tropical sprue (TS) is a chronic diarrheal and intestinal malabsorption syndrome of presumed infectious etiology. The disease is seen in patients from South and Southeast Asia; West Africa; parts of Mexico, Puerto Rico, and the Caribbean islands; and some areas of Central and South America (5,23,65-67). Affected patients usually live in these regions for years, but symptoms may be seen in people who have been in affected areas for as little as a few months. Patients most often respond dramatically to the administration of folate, vitamin B12, and tetracycline or other broad-spectrum antibiotics, implying that an infectious agent may be important in the etiology. The histologic findings in tropical sprue are very similar to those of CS (65). There is an increase in intraepithelial lymphocytes with crypt hyperplasia and mild to moderate villous blunting (Fig. 33.6). A completely flat small intestine biopsy is very uncommon, although on occasion, a severe histologic lesion indistinguishable from CS may be seen. The terminal ileum is often the most intensely involved site in TS, whereas the duodenum bears the brunt of the damage in CS.

FIGURE 33.6 Variable villous abnormality of tropical sprue. There is moderate villous shortening with increased intraepithelial lymphocytes.

INFECTIOUS GASTROENTERITIS There are a variety of bacterial, viral, fungal, and parasitic organisms that can lead to gastroenteritis. Most infections are self-limited, and patients who undergo endoscopy and biopsy typically have chronic issues or are immunocompromised. Common viral etiologies include norovirus, rotavirus, cytomegalovirus (CMV), adenovirus, and herpes simplex virus (HSV) (68). In some cases, such as CMV, adenovirus, and HSV, diagnosis can usually be made on identifying a characteristic viral inclusion, or with the aid of immunohistochemical staining. The most common causes of bacterial gastroenteritis include E. coli species, as well as Salmonella, Shigella, Campylobacter, Vibrio, Yersinia and C. difficile, among others. Most often, biopsy specimens contain mucosal injury and variable degrees of inflammation that are etiologically nonspecific. Jejunal mucosal lesions in infectious gastroenteritis are typically patchy with variable villous abnormalities; rarely, the villous abnormality can be severe, closely mimicking CS (5,22,69,70). In addition to increased chronic inflammatory cells, acute inflammatory cells are often seen both within epithelial cells and in the lamina propria. The acute onset and temporary nature of the symptoms, coupled with the acute inflammatory changes in biopsy specimens, usually help in the distinction from CS. SMALL INTESTINAL BACTERIAL OVERGROWTH SYNDROME (BLIND-LOOP SYNDROME, STASIS SYNDROME) Small intestinal bacterial overgrowth (SIBO) syndrome is characterized by the presence of excessive bacteria in the small intestine and is believed to be a potential source for so-called “irritable bowel syndrome” (71). The etiology is believed to be secondary to gut stasis, leading to overgrowth and, ultimately, malabsorption, nutrient deficiencies, and weight loss (72). Although SIBO can occur in the absence of an anatomic predisposition, it can occur in surgical blind loops, small-intestinal diverticulosis, intestinal pseudo-obstruction, and during episodes of bowel obstruction. In periods of gut stasis, growth of bacteria is not inhibited by normal regulatory factors such as mixing with gastric juice and peristalsis and leads to bacterial overgrowth. This causes mucosal injury, which can include villous blunting, and may be associated with intraepithelial lymphocytosis with occasional neutrophilic inflammation, similar to CS (25). Ultimately, epithelial damage leads to malabsorption of fat and vitamins (72). MASTOCYTOSIS Systemic mastocytosis (SM) is a rare, clonal myeloproliferative disorder characterized by infiltration of mast cells in tissues that can include the skin (urticaria pigmentosa), bones, lymph nodes, and other parenchymal organs (73). Over 90% of cases have a D816V KIT mutation (74,75). SM is one manifestation of the so-called “mast cell activation disorders,” which also include other conditions in which mast cell mediators play a role in causing symptoms, but which do not always have histologic findings of abnormal mast cells (76,77).

The major criterion for SM diagnosis is the finding of mast cell infiltrates, consisting of at least 15 cells in aggregate, in the bone marrow or extracutaneous sites (75). Minor diagnostic criteria include (1) atypical or spindle-shaped morphology affecting more than 25% of the mast cells, (2) detection of the D816V KIT mutation, (3) aberrant expression of CD2 and/or CD25 on mast cells, and (4) a serum tryptase level greater than 20 ng per mL. Diagnosis requires the major and one minor criteria or three minor criteria (75). Gastrointestinal involvement is common in SM and may result in abdominal pain, diarrhea, and/or malabsorption (78-80). Rare patients have had histologic and clinical features of CS (80-82). Gastrointestinal endoscopic findings in the small intestine or colon can include mucosal nodules or more general nodularity, erosions and friability, or thickening or loss of folds (74). Identification of abnormal mast cells in tissue biopsy is notoriously difficult and mast cells can be mistaken for fibroblasts or other types of inflammatory cells (83). Mast cell aggregates tend to be between the crypts and/or just below the luminal surface, and the majority are round or ovoid with centrally placed nuclei and pale eosinophilic or clear cytoplasm. They can also be spindled, elongated, or large. Most gastrointestinal involvement by SM contains a marked infiltrate of eosinophils that can obscure the abnormal mast cells and possibly be confused with eosinophilic gastroenteritis (74) but that can also be a clue to the diagnosis. Immunohistochemistry (IHC) for CD117 and mast cell tryptase can be used to identify the mast cells and to highlight their abnormal distribution and morphology. Some cases are negative for CD117, so performing both stains together is prudent. Aberrant coexpression of CD2 and/or CD25 is also a helpful feature. Of note, a recent study identified a group of patients who met the diagnostic criteria for gastrointestinal involvement by SM based on symptoms and the finding of abnormal mast cells in the tissue but who had no evidence of disease elsewhere and who all had spontaneous remission of their symptoms (84). In patients with established SM, the presence of a D816V KIT mutation predicts nonresponse to imatinib. These patients can be treated with other therapy, including antihistamines, prednisolone, interferon alpha, cladribine, and investigational drugs (85). Some observers believe that a finding of more than 20 normal-appearing mast cells per high-power microscopic field is indicative of mastocytic enterocolitis and should be treated with antihistamines and/or mast cell stabilizers (86). This putative entity has been postulated to explain symptoms in patients with intractable diarrhea similar to that of the diarrhea-predominant form of irritable bowel syndrome, and normal endoscopic findings. This diagnosis, however, is not well characterized, and additional study has not established a reliable tissue mast cell count associated with these symptoms. Thus, most experts do not routinely stain for or count normal-appearing mast cells in practice (83). PEPTIC DUODENITIS AND PEPTIC ULCER DISEASE Peptic duodenitis and peptic ulcer disease encompass a spectrum of chronic injury to the duodenum. Peptic ulcers, in particular, refer to a mucosal break with exposure of acidic intraluminal contents to the submucosa. This usually occurs in the stomach or the proximal duodenum, and the most common etiologies include infection with H. pylori and chronic use of aspirin or other NSAIDs (87). Normal proximal duodenal (bulb) mucosa typically has a mild decrease in villous height and increased crypt mitoses when compared to the distal duodenum or jejunum (88,89). Peptic duodenitis may represent an exaggeration of this response and is characterized by more extreme villous blunting, increased plasma cell infiltration, and neutrophilic inflammation (89-92). Gastric foveolar metaplasia and Brunner gland hyperplasia are also common findings but are not necessary for a diagnosis. Care should be taken not to interpret gastric heterotopia as foveolar metaplasia; the former should contain specialized gastric oxyntic glands. Active duodenitis is generally limited to the first part of the duodenum, and it can have erosions, a variable villous abnormality associated with increased acute and chronic inflammatory cells, and gastric foveolar metaplasia of villous epithelium (Fig. 33.7) (89-92).

FIGURE 33.7 Gastric foveolar metaplasia of the duodenum. The metaplastic epithelium is composed of columnar cells with basilar-placed nuclei and clear cytoplasm identical to those of the gastric pits and contrasts with the absorptive and goblet cells of the normal duodenum on the right.

The differential diagnosis of peptic duodenitis/peptic ulcer disease includes H. pylori infection/gastritis, NSAID injury, Crohn disease, celiac disease, and other infections. A thorough search of a concurrent antral biopsy, possibly with the aid of immunohistochemical stains, may aid in the evaluation of H. pylori infection. AUTOIMMUNE ENTEROPATHY Autoimmune enteropathy (AIE) is a condition characterized by intractable diarrhea, failed response to dietary changes, villous abnormality of the small intestine, and the presence of autoantibodies (93). Although the disease often affects infants within the first six months of life, it is becoming increasingly recognized in adults (94). Patients with AIE often have variable immunodeficiency and other autoimmune issues such as juvenile-onset diabetes mellitus, rheumatoid arthritis, and hemolytic anemia (95-97). IPEX syndrome is an X-linked recessive disorder that manifests with intractable diarrhea, endocrinopathies (i.e. diabetes, thyroiditis) and dermatitis (98). It is caused by loss-of-function mutations in FOXP3 gene, which encodes for a transcription factor critical for normal development of regulatory T-cells, resulting in hyperactivation of immune cells (98). AIE can also be seen with APECED (autoimmune phenomenon, polyendocrinopathy, candidiasis, and ectodermal dystrophy) syndrome, which is an autosomal recessive disease that presents in early childhood or adolescence caused by mutations in the AIRE gene (93,98). Lastly, AIE can also be seen in association with CVID, as discussed later in the chapter. There are no specific histologic features for AIE. There is usually some combination of villous blunting, crypt apoptosis and lymphocytosis, and robust lamina propria expansion by inflammatory cells (98). Surface and crypt epithelial degenerative and regenerative changes occur, but many illustrated cases have few intraepithelial lymphocytes—a feature that may distinguish AIE from CS. Many cases of AIE have absence of, or a decrease in, Paneth cells, goblet cells, or both, which can occur in both adults and children (98). Patients with AIE can also develop colitis. In some cases, the associated colitis has resembled lymphocytic colitis, whereas in others, the endoscopic and histologic picture is similar to ulcerative colitis (UC) (94,99,100). In some cases, there can be considerable overlap with olmesartaninduced GI disease, and hence history of use of this medication is critical in making the correct diagnosis. Treatment of AIE can be quite challenging and often requires total parenteral nutrition in combination with systemic steroids (prednisone) or other immunosuppressive agents such as azathioprine, methotrexate, tacrolimus, cyclosporine, or mycophenolate mofetil (98). Biologic agents such as infliximab, rituximab, and abatacept can also be used. Most children with IPEX receive hematopoietic stem cell transplant (98). Entities Associated With Variable Villous Abnormalities Illustrating Specific Diagnostic Changes

Collagenous Sprue. Collagenous sprue is a rare form of enteropathy characterized by diarrhea and malabsorption unresponsive to a gluten-free diet (101-103). The pathophysiology is not fully understood, although potential etiologies include medication-induced cases, a form of end stage CS, immunemediated small intestine disease, and abnormalities in fibrosis/fibrogenesis (103,104). There is an association with collagenous gastritis and colitis, with one study having rates of these entities of 46.7% and 57.1%, respectively (103). Diagnostic criteria are varied, but most patients have had a maximum subepithelial collagen deposit exceeding 10 μm, and one study showed a range of 10-300 μm (102,103,105). This finding can be patchy, and multiple biopsies may be needed for a diagnosis. Patients typically require a combination of steroids/immunosuppressive agents along with gluten avoidance for clinical control (101,102,105,106). Further, collagenous sprue should be considered in patients with refractory or unclassified sprue who are unresponsive to a gluten-free diet (105,106). Interestingly, some patients with collagenous sprue have T-cell receptor gene rearrangements, suggesting that at least a proportion of these cases are actually lymphoproliferative disorders (105). The occurrence of overt lymphoma has been rare. Recently, there have been reports of the antihypertensive drug olmesartan, along with some related angiotensin II receptor blocking (ARB) agents, causing CS- or collagenous sprue-like changes as well as lymphocytic and collagenous gastritis and colitis (104). Discontinuation of the drug led to histologic and symptomatic improvement. Immunodeficiency Syndromes Excluding Acquired Immunodeficiency Syndrome. There are many primary immunodeficiency syndromes that manifest with gastrointestinal symptoms, usually identified in childhood. Among these are selective IgA deficiency, X-linked agammaglobulinemia, Xlinked hyper-IgM syndrome, CVID, severe combined immunodeficiency (SCID), IPEX (see earlier), interleukin-10-receptor deficiency, DiGeorge syndrome, chronic granulomatous disease and WiskottAldrich syndrome (107). The gastrointestinal pathology of the two most common forms, selective IgA deficiency and CVID, will be considered in more detail. Selective IgA deficiency is the most common primary antibody deficiency and is defined by a serum IgA level of less than 50 μg per mL in individuals older than 4 years with the presence of normal IgG and IgM levels; other causes of hypogammaglobulinemia and T-cell dysfunction must be excluded (108,109). Normal villous morphology can be seen on routine light microscopy in selective IgA deficiency, although nodular lymphoid hyperplasia may be present (107,108). There is often a reduction in plasma cells, particularly of the IgA subtype. There is a higher incidence of certain infections like giardiasis, similar to patients with CVID. There is also an association between selective IgA deficiency and CS; identifying the absence of plasma cells or a reduction in plasma cells can aid in the distinction. CVID is an uncommon primary immunodeficiency disorder characterized by recurrent bacterial and parasitic infections. Patients with CVID typically have chronic diarrhea, malabsorption, and recurrent gastrointestinal giardiasis and can often be misdiagnosed as CS, because the findings on small intestinal biopsy can be very similar (26,107,110-112). Although histologic findings are variable, a recent study showed the most frequent findings to be intraepithelial lymphocytosis, reduction in or absence of plasma cells, and lymphoid hyperplasia (large lymphoid aggregates in the lamina propria and submucosa) (113). There is usually mild or no villous blunting. In contrast to CS, where they are increased, plasma cells in CVID are usually markedly decreased and IgA-containing plasma cells are typically absent. With routine light microscopy, nodular lymphoid hyperplasia of the gastrointestinal (GI) tract can be difficult to distinguish from lymphoproliferative disorders. Furthermore, lymphoma associated with nodular lymphoid hyperplasia has been described in patients with CVID (112-115). Whipple Disease. Whipple disease is a rare systemic illness characterized by diarrhea, weight loss, and arthralgias (116). The disease is caused by Tropheryma whippeli, a rod-shaped microorganism (116-118). Although a definitive mode of transmission is not known, the disease is associated with contact with contaminated soil, poor living conditions with exposure to sewage, or close contact with individuals with infection (116). The most common features of Whipple disease include arthralgia; arthritis and fever; and gastrointestinal symptoms such as diarrhea, abdominal pain, and weight loss (116,119). The diagnosis can usually be made from duodenal biopsies, in which there are large numbers of foamy macrophages within the lamina propria containing diastase-resistant, PAS positive material. In

ambiguous or histologically challenging cases, the diagnosis can be confirmed by polymerase chain reaction (PCR) assay for the bacterial 16S ribosomal RNA, electron microscopy, or IHC (116,120-122). Whipple disease responds dramatically to antibiotic therapy, although there can be relapses and prolonged treatment is necessary. Without adequate treatment, Whipple disease can be fatal (119). In addition to affecting the small intestine, the bacteria can affect the mesenteric lymph nodes, the cardiac valves (endocarditis), and the central nervous system (encephalitis) (121,123). Endoscopically, the mucosal folds appear thickened with a pale-yellow surface; erythema may be present. Detailed histologic examination shows mild to moderate villous blunting with massive expansion of the lamina propria by foamy macrophages (Fig. 33.8) containing the characteristic diastase-resistant PAS-positive material (Fig. 33.9). The number of overlying intraepithelial lymphocytes is not typically increased. With antibiotic treatment, the pattern of mucosal infiltration can become patchy (124). The macrophages recede to the muscularis mucosae, submucosa, and basilar portions of the lamina propria, and the cytoplasmic inclusions become “tissue paper–like” with PAS stain, reminiscent of the inclusions of Gaucher cells. PAS-positive and Whipple IHC-positive macrophages can persist for years after treatment, although PCR assays on intestinal tissues usually convert to negative in less than 12 months (121,124).

FIGURE 33.8 Whipple disease. The lamina propria contains pale macrophages that broaden and flatten the intestinal villi. Note the scattered clear spaces, some of which may have contained lipid and others of which are dilated lacteals.

FIGURE 33.9 Whipple disease. The cytoplasm of the macrophages in Whipple disease contains coarsely granular, intensely PAS-positive material.

The histologic differential diagnosis of Whipple disease includes other intracellular organisms such as histoplasmosis (125), MAC infection (126), and Rhodococcus equi. The macrophages in disseminated histoplasmosis contain dot-like inclusions that are usually surrounded by a clear halo; PAS and silver

stains demonstrate budding yeast forms that are distinct from the organisms in Whipple disease. Although both MAC and Whipple disease contain an abundance of foamy macrophages that are diastase resistant, PAS positive, T. whippeli organisms are not acid-fast and thus will not be highlighted on a Ziehl-Neelsen stain. Eosinophilic Gastroenteritis. Eosinophilic gastroenteritis describes a collection of clinical syndromes usually seen in children or young adults characterized by eosinophilic inflammation of the GI tract in the absence of secondary causes of eosinophilia (127-130). The inflammation can involve any part of the GI tract, from the esophagus to the rectum, and often manifests in distinct clinical findings. For example, “eosinophilic esophagitis” is associated with dysphagia and an endoscopic appearing of furrows or rings (129). Mucosal involvement of the stomach and small intestine is associated with diarrhea and malabsorption, whereas involvement of the submucosa and muscularis propria is associated with intestinal obstruction (128,130). When the inflammation involves the subserosa, eosinophilic ascites can be present. As a whole, these diseases are rare, with eosinophilic esophagitis being the most common, predominantly in the pediatric population (128). The histologic diagnosis of eosinophilic gastroenteritis is challenging, because there are no standard guidelines. There must be a high degree of clinical suspicion and exclusion of other sources of eosinophilia. Infiltration of the submucosa, muscularis propria, and subserosal connective tissue by eosinophils is always abnormal and, when it is corroborated clinically, is diagnostic of eosinophilic gastroenteritis. The diagnosis of the mucosal pattern of eosinophilic gastroenteritis in biopsy specimens can be particularly challenging to the pathologist, because, with the exception of the esophagus, scattered intramucosal eosinophils are normal in the GI tract, but collections of eosinophils not associated with other inflammatory cells, groups of eosinophils associated with focal mucosal architectural distortion or injury (e.g., cryptitis, crypt abscesses), and extensive infiltration of the muscularis mucosae by eosinophils are all abnormal, although the last item is relatively subjective. Similarly, in a corroborative clinical setting, these may be diagnostic of eosinophilic gastroenteritis. It should be noted that mucosal involvement in eosinophilic gastroenteritis is notoriously patchy. If there is high clinical suspicion, multiple or additional biopsy specimens should be obtained. Numerous intramucosal eosinophils can be seen in other conditions, as well. A search for parasites should always be conducted when eosinophils are prominent (i.e., Ascaris, Trichuris, Schistosomiasis, Ancylostoma, Enterobius, Giardia, Anisakis, and Trichinella). Other entities in the differential diagnosis include food and drug allergies, hypereosinophilic syndrome, SM, and connective tissue disease (130). Eosinophils can also be associated with chronic inflammatory conditions such as Crohn disease, UC, and celiac disease, as well as primary intestinal lymphoma, especially T-cell lymphomas (130). Given all of these settings where eosinophilia in the luminal GI tract can be found, clinical correlation is essential when invoking the diagnosis of bona fide eosinophilic gastroenteritis. Parasitic Infestations. Although a large number of parasites may infect the GI tract, this discussion concentrates on those that are common and often associated with intestinal malabsorption. Infection with Cryptosporidium species, Microsporidia species, and Cystisospora species are discussed later (see section “Interpretation of Endoscopic Biopsy Specimens from Immunosuppressed Patients, Including Patients with Human Immunodeficiency Virus and Acquired Immunodeficiency Syndrome”). G G ( R G. G. ) I . Infection by G. lamblia is common, especially in underdeveloped countries or in areas where the water supply is contaminated by fecal material (131-134). It is also the most common protozoal disease in the United States. The first signs of infection usually appear after 6 to 15 days and include explosive, foul-smelling watery diarrhea, nausea, epigastric pain, and weight loss. Endoscopic examination is typically unremarkable, and histologic changes in small intestinal biopsies can be subtle and challenging to identify. Diagnosis can be aided with the use of stool examination, enzyme-linked immunosorbent assays (ELISA), direct immunofluorescent antibody microscopy on stool samples, or PCR (21,132,133). Although pathologists usually encounter Giardia in duodenal biopsy specimens, the organism has been found in the gastric antrum, ileal specimens, and even colonic biopsy specimens (135,136). In fact, a recent study showed that in patients whose duodenal biopsies had histologic

features suggesting the possibility of Giardia but in which identifiable organisms were absent, they could actually be found in ileal biopsies (136). Although most biopsies in patients with giardiasis are essentially unremarkable apart from the presence of the organisms, they can occasionally have mild villous blunting with inflammatory expansion of the lamina propria (Fig. 33.10) (131,133,135). The histologic diagnosis rests on the demonstration of Giardia trophozoites along the surface of epithelial cells in biopsy specimens (Fig. 33.11). Morphologically, the trophozoite has a “pear-shaped” appearance with two prominent nuclei. They often appear as if they are “falling off” the mucosa-like leaves and, for this reason, may be overlooked under the assumption that they are simply mucus from the goblet cells on the villi. Special stains such as Giemsa or trichrome can assist in the diagnosis, although H&E preparations typically suffice. When trophozoites are identified, the lamina propria should carefully be examined for plasma cells because Giardia is commonly seen in patients with CVID.

FIGURE 33.10 Giardiasis. The mucosal histology in giardiasis is variable; in this case, it shows a nearly normal villous morphology. The organisms are in a cluster to the right of the villus in the middle.

FIGURE 33.11 Giardiasis. Seen en face, Giardia lamblia trophozoites are pear-shaped, with prominent paired nuclei and a pointed caudal end.

S (S I ). Strongyloides stercoralis is a soil transmitted intestinal nematode that is endemic to the tropics and subtropics. Most infections are asymptomatic, although fatal cases have been documented in immunocompromised hosts (131,137140). Acute infection is usually associated with larval migration to the small intestine via the lungs and esophagus. Infected individuals may have irritation at the site of skin penetration with localized edema or urticaria (137). In the intestines, the female lives and lays eggs, continuing the life cycle within the new host. Gastrointestinal symptoms, such as diarrhea, constipation, abdominal pain, or anorexia, occur secondary to the organisms’ life cycle within the mucosa of the duodenum and jejunum (137). The female can continue to lay eggs and produce more rhabditiform larvae, which pass in the stool. The rhabditiform larvae may also develop into infective filariform larvae in the gut, and these may invade the intestinal mucosa or the perianal skin and set up a cycle referred to as autoinfection or hyperinfection syndrome with dissemination (139). The diagnosis is usually made by demonstrating larvae in the stools. A number of immunoassays such as ELISA can be used to improve diagnostic sensitivity (137). Endoscopically, the lesions associated with mucosal invasion appear erythematous, edematous and/or ulcerated, and may be seen anywhere in the GI tract from the stomach to the colon. Histologic examination reveals larvae or worms within the intestinal crypts; adult worms have characteristic tails (140,141). With autoinfection, the infective filariform larvae may also be identified in fresh stool, colonic mucosal biopsy specimens, or in the intestinal wall, where they are sometimes accompanied by an eosinophilic and/or granulomatous inflammatory reaction (131,140). Patients with long-standing infection may develop a chronic colitis resembling UC. The presence of eosinophilic and/or granulomatous inflammation and an absence of crypt abscesses should alert one to the possibility of strongyloidiasis (142). C A (C ) I . Nematodes of the species Aonchotheca philippinensis can infect humans and can cause a protein-losing enteropathy that may be fatal (143). Infections have been described in patients from the Philippines, Thailand, Iran, Korea, and Egypt and are associated with consumption of raw fish (131,143,144). The larvae infest the jejunum and upper ileum and have only rarely been described in biopsy specimens. The adult worm and

eggs bear a morphologic resemblance to those of trichuriasis. The diagnosis is usually made by identifying eggs, larvae, or adult worms in the stool. Waldenström Macroglobulinemia. Intestinal involvement by Waldenström macroglobulinemia is rare and is often characterized by lymphangiectasia, which is found in 1% to 3% of patients with the disease (131,132,145). Endoscopically, the mucosal surface may be white, nodular, granular, or mottled. Grossly, the serosa has small white nodules and occasionally distended serosal lymphatics. Histologic sections contain marked mucosal and submucosal lymphangiectasia with short, broad villi. A coarse and sometimes fragmented eosinophilic material is present in the dilated lymphatics, the lamina propria, and within macrophages; this material is composed of immunoglobulin (classically IgM) and can be confirmed with IHC and a negative Congo red stain. The material will also stain with PAS. The B-cell lymphoproliferative disorder associated with Waldenström macroglobulinemia (lymphoplasmacytic lymphoma) has a high frequency of MYD88 L265P somatic mutation (146). Intestinal Lymphangiectasia. Some forms of intestinal lymphangiectasia (IL) can be an important cause of protein losing enteropathy. This condition is characterized by a dilation of the intestinal lymphatics, ultimately leading to loss of fluid and nutrients into the GI tract (147-149). In extreme situations, this can lead to hypoproteinemia, edema, lymphocytopenia, hypogammaglobulinemia, and other significant nutritional deficiencies. Histologically, biopsies have focal or diffuse dilation of the mucosal, submucosal, and subserosal lymphatics. Not all types of IL, however, are this clinically significant, and incidental cases are not uncommonly encountered during upper endoscopy in asymptomatic individuals, where they can appear as pinpoint white dots, whitish discoloration at the tips of folds, or whitish macules or papules (148). Clinically consequential cases of IL occur in a primary or secondary form. The primary form is a congenital disorder leading to leakage of protein-rich chyle into the intestinal lumen (150,151). Secondary lymphangiectasia is associated with many diseases, including retroperitoneal fibrosis, pancreatitis, constrictive pericarditis, primary myocardial disease, intestinal Behçet disease, intestinal malignancy, Waldenström macroglobulinemia, and sarcoidosis (149,151,152). In both forms, the histology in mucosal biopsy specimens is identical—dilated lymphatics located in otherwise normal tissue (Fig. 33.12). Therapy includes the treatment of underlying conditions, dietary manipulation, and, in some localized forms of lymphangiectasia, segmental resection (149).

FIGURE 33.12 Intestinal lymphangiectasia. The primary and secondary forms have an identical histologic appearance, with dilated lymphatics/lacteals located in otherwise normal mucosa and submucosa.

Abetalipoproteinemia. Abetalipoproteinemia, hypobetalipoproteinemia and chylomicron retention disease make up the familial hypocholesterolemias, rare genetic diseases that cause malnutrition, failure to thrive, growth failure, and vitamin E deficiency (153). Abetalipoproteinemia, hypobetalipoproteinemia, and chylomicron retention disease are caused by mutations in the APOB gene, microsomal triglyceride transfer protein gene, and SAR1B gene, respectively (153). These all ultimately lead to the inability to form chylomicrons for fat transport, resulting in fat accumulation in absorptive cells; the changes are most prominent at the tips of the villi (153,154). The enterocytes have prominent vacuolization by lipid droplets, which is characteristic, but not pathognomonic, because similar changes can be seen in megaloblastic anemia, CS, and tropical sprue (155). This appearance is also occasionally observed in patients without any apparent disease process. Acrodermatitis Enteropathica. Acrodermatitis enteropathica is an autosomal recessive form of zinc deficiency, conferred by mutations in the SLC39A4 gene (156,157). Patients present with eczematous, pink, scaly plaques, which can become vesicular, bullous, pustular, or desquamative (158). Patients can also develop angular cheilitis and paronychia, similar to the changes seen in dietary zinc deficiency. From a gastrointestinal perspective, patients present with diarrhea and features of malabsorption. Small intestinal histology varies, and some investigators have reported a severe villous abnormality similar to that in CS, whereas others normal or only minimally abnormal small intestinal mucosa by routine light microscopy. Ultrastructural changes that consist of rodlike, fibrillar inclusions in Paneth cells are considered diagnostic for acrodermatitis enteropathica (159). Tufting Enteropathy. The term tufting enteropathy has been applied to a sometimes-familial, intractable diarrhea syndrome in children (160-162), which is caused by mutations of EpCAM (162). The symptoms usually begin in the neonatal period, when affected patients require total parenteral nutrition. Small intestinal biopsy specimens have a variable villous abnormality with epithelial crowding, disorganization, and focal tufting. There is usually no increase in intraepithelial lymphocytes.

NONNEOPLASTIC POLYPS AND NODULES OF THE SMALL INTESTINE HETEROTOPIAS, ADENOMYOMA, AND MYOEPITHELIAL HAMARTOMA The most common nonneoplastic polyps of the small intestine are nodules of gastric and pancreatic heterotopia (84,163). Although congenital rests of gastric mucosa may be seen throughout the GI tract, the most common site in the small intestine is the duodenal bulb. Nodules of gastric heterotopia are composed of architecturally normal oxyntic glands composed of chief cells and parietal cells, with overlying foveolar epithelium. Small nodules (15 intraepithelial lymphocytes/100 enterocytes vs. normal of 6) is abnormal and could be a manifestation of CS, or it may be seen in patients with lymphocytic enterocolitis (296,298).

INTERPRETATION OF ENDOSCOPIC BIOPSY SPECIMENS FROM IMMUNOSUPPRESSED PATIENTS, INCLUDING PATIENTS WITH HUMAN IMMUNODEFICIENCY VIRUS AND ACQUIRED IMMUNODEFICIENCY SYNDROME Endoscopy and histologic examination play a vital role in the diagnosis and management of patients with immunodeficiency (302). These patients are at increased risk for opportunistic infections as well as neoplasia, and the lesions can often be subtle. In the last two decades, profound changes have occurred in the medical management of human immunodeficiency virus (HIV) and AIDS with highly active antiretroviral therapy (HAART). This therapy has markedly reduced the incidence of opportunistic infection and is invariably the first-line approach not only for HIV and AIDS but also for the complicating infections. However, the interpretation of intestinal biopsy specimens from patients with untreated HIV and AIDS remains one of the premier challenges in surgical pathology. In addition to infection with microbiologic pathogens, these patients can develop so-called “HIV enterocolopathy” characterized by villous blunting, crypt hyperplasia, apoptosis, and mild intraepithelial lymphocytosis. These findings have been attributed to the effect of HIV on enterocytes, although a variety of viruses have also been detected in the stool of affected patients (302). COMMON INFECTIOUS AGENTS Bacterial Infection Immunocompromised patients can become infected with the same bacterial pathogens that affect other patients. These include C. difficile, E. coli, and Yersinia, Salmonella, and Shigella species, among others. Diagnosis can be made by identifying the organism in stool culture. When patients undergo biopsy, the specimens often contain the spectrum of changes associated with infectious colitis or acute self-limited colitis. In addition to common pathogens, immunocompromised patients can be infected with a variety of unusual pathogens that are described in a subsequent section. Parasitic Infestations G. lamblia can infect immunocompromised individuals in addition to otherwise healthy children and adults. It is seen in high frequency in men who have sex with men (MSM) and in patients with HIV/AIDS (303,304). Although it may be a nonpathogenic commensal in MSM, it can rarely cause fulminant colitis in patients with AIDS (304). Histologically, it looks similar to its counterpart in immunocompetent patients. Viral Infections CMV is the most common opportunistic viral pathogen in HIV, and patients with CD4 cell counts of less than 100 per µL can have infection in the GI tract (303). Infections cause both superficial and deep ulcers but can also cause more severe pathology, including pseudomembranes, perforation, and pneumatosis intestinalis (303). Endothelial and stromal cells are preferentially infected and have the characteristic histologic finding of “owl-eye” inclusion in the nucleus as well as granular-appearing,

eosinophilic cytoplasmic inclusions. Macrophages and epithelium can also be infected. IHC can be used for detection in challenging or ambiguous cases. HSV is associated with painful discrete ulcers, vesicles, or pustular lesions in the distal rectum or perianal skin as well as the esophagus (303-305). HSV1 is typically found in the esophagus, whereas HSV2 is more commonly found in the anorectal region, although each virus can affect either location. The virus typically infects squamous epithelium, causing a characteristic eosinophilic “ground-glass” nuclear inclusion (the so-called Cowdry type A inclusion). Infected cells are often multinucleated with nuclear molding (302). In addition to the viral cytopathic changes, the inflammatory pattern in the rectum shows (a) ulceration with neutrophils within the lamina propria, (b) cryptitis, and (c) crypt abscesses. HSV inclusions are not seen in the colon or rectum but are found in the anal transition zone epithelium or perianal skin. It is easily cultured and immunostained. Acute gastrointestinal infection with adenovirus is usually self-limited in immunocompetent patients, but immunocompromised patients may suffer from a more serious or protracted course (302-304). Endoscopy may reveal normal or mildly friable and erythematous mucosa, and biopsy samples may have only mild changes such as blunted epithelium and an increase in apoptotic bodies. An infected cell may have a smudgy, amphophilic nuclear inclusion that can be verified with IHC (302). Fungal Infections The GI tract, the main reservoir for Candida spp. and intestinal and disseminated infections, can occur in the setting of immunosuppression. C. albicans is a major source of esophagitis, producing characteristic white plaques (302). Seeing this organism in the small intestine or colon is unusual unless disseminated candidiasis has occurred (303). Histoplasmosis, discussed earlier as a differential diagnosis for Whipple disease, can also affect the GI tract in patients who are immunocompromised. UNUSUAL INFECTIOUS AGENTS COMMONLY FOUND IN PATIENTS WITH ACQUIRED IMMUNODEFICIENCY SYNDROME Bacterial Infections Intestinal spirochetosis, caused by Brachyspira aalborgi or Brachyspira pilosicoli, can be seen in patients with HIV or AIDS, as well as in children and in patients with other disorders who do not have HIV infection (303,306). Infection is often asymptomatic but children and immunocompromised patients (including those with HIV/AIDS) can present with diarrhea and abdominal pain (315). The endoscopic appearance is usually unremarkable (303), and the microscopic appearance is very subtle, with a thickened and “fuzzy” appearance to colonic mucosa on the luminal surface, which is caused by the spirochete organisms embedding themselves into the luminal border of the absorptive cells (Fig. 33.25). They do not attach to goblet cells. Identification of intestinal spirochetosis can be enhanced by the use of the Warthin-Starry stain or by electron microscopy. An immunostain for Treponema species will also highlight the organisms.

FIGURE 33.25 Intestinal spirochetosis from a patient with HIV/AIDS. The spirochetes appear as a “fuzzy” thickening or accentuation of the apical cell borders that stains with hematoxylin.

Mycobacterium tuberculosis (TB) and avium complex (MAC) are well-documented pathogens, particularly in HIV-affected areas in Africa (303). M. tuberculosis can often be identified in the GI tract in patients with active pulmonary disease. Gastrointestinal tuberculosis most often affects the ileocecum but can affect any location of the GI tract. Endoscopically, infected areas have extensive ulceration with subsequent stricture formation (303). Histologically, these areas have well-formed, caseating granulomas in immunocompetent patients, and neutrophil-rich histiocytic inflammation in those who are immunocompromised (303). Distinguishing TB infection from Crohn disease can be challenging. Helpful clues suggesting TB include large granulomas with caseous necrosis, abundant histiocyte palisading, and lack of chronic enteritis in surrounding areas. GI tract involvement by MAC is usually part of disseminated infection and can affect any portion of the GI tract. Histologically, there is infiltration of the lamina propria by foamy macrophages that are PASpositive; as such, the histology can closely mimic Whipple disease (Fig. 33.26A) (126,307). However, MAC is typically patchy and lacks the large lipid vacuoles of Whipple’s disease, and the organisms are acid-fast positive (Fig. 33.26B). The infiltrative macrophages may be subtle, and some recommend that an acid-fast stain be done on all mucosal biopsy specimens from patients with HIV or AIDS.

FIGURE 33.26 Mycobacterium avium-intracellulare complex (MAC) infection. (A) The lamina propria is infiltrated by foamy macrophages, and the appearance closely mimics Whipple disease (see Fig. 33.8). In contrast to Whipple disease, however, the “fat vacuoles” are absent. (B) The periodic acid–Schiff (PAS) stain in MAC reveals faintly positive bacillary forms, in contrast to the coarsely granular, intensely PAS-positive inclusions of Whipple disease (see Fig. 33.9). The more intense positivity is in goblet cells. These organisms can also be highlighted with acid-fast stains such as Ziehl-Neelsen.

HIV-associated infection with Chlamydia trachomatis and Neisseria gonorrhoeae is also well documented and is most commonly associated with proctitis in MSM (303). Infection with syphilis (Treponema pallidum) and lymphogranuloma venerum is also common in the distal colon and rectum and can strongly mimic IBD with a robust lymphoplasmacytic infiltrate, architectural changes, and active inflammation. Diagnosis of syphilis often relies on serum testing or special studies performed on rectal tissue, because silver and immunohistochemical staining are not sensitive (308).

Diarrheagenic bacterial enterocolitis has been described in patients with HIV or AIDS (309). Colonic biopsy specimens demonstrate surface epithelial inflammation and degeneration associated with Gramnegative bacteria adherent to the surface of enterocytes (Fig. 33.27). The histology resembles that seen in EPEC infection (310).

FIGURE 33.27 Diarrheogenic bacterial colitis in acquired immunodeficiency syndrome. Note the surface epithelial injury and the adherent rod-shaped bacteria on the luminal surface.

Parasitic Infestations Gastrointestinal parasitic infections are common in immunocompromised patients. Before HAART therapy was developed, Cryptosporidium parvum was the most common parasitic infection, typically affecting the proximal small intestine, although involvement of the colorectum was frequently present (303). In immunocompromised patients, it causes severe, explosive, watery diarrhea that is resistant to most forms of therapy (132,304). On endoscopy, the findings are often minimal or nonspecific. Histologically, there is variable villous abnormality, crypt hyperplasia, and a mixed infiltrate with numerous eosinophils (302,303). The diagnosis is readily made with a stool sample or PCR on infected specimens (133) or by identifying the organism on histology, where 2 to 5 µm basophilic spheres can be seen protruding below the apical membrane of the epithelium. These stain with acid-fast, Giemsa, Gram, and Warthin-Starry stains and can also be highlighted by immunofluorescence (Fig. 33.28) (303).

FIGURE 33.28 Cryptosporidiosis in a small intestinal biopsy. The organism appears as basophilic dots measuring approximately 3 μm that attach to the luminal border of epithelial cells.

Cystisospora belli (formerly, Isospora belli) is a less common parasite that similarly involves the small intestine but can also involve the colorectum (303). The endoscopic and histologic features are very

similar to Cryptosporidium, although the organisms are 10 to 30 µm, are located within the enterocytes, and are paranuclear (Fig. 33.29) (302,303). They stain with PAS, Giemsa, and methenamine silver stains.

FIGURE 33.29 Cystisospora species infection in proximal small intestine. Ovoid developmental forms are seen in the enterocyte cytoplasm near the villous tip. Courtesy of Audrey J. Lazenby, MD, University of Nebraska, Omaha, Nebraska.

Microsporidia infections are caused by Enterocytozoon bieneusi and Encephalitozoon intestinalis (formerly known as Septata intestinalis) (303). They have been recognized in up to 50% of HIV or AIDS patients with chronic unexplained diarrhea (133,309,311,312). The parasites infect the small intestine and can easily be missed on routine histologic sections, because they are small and intracellular (Fig. 33.30). Mature spores measuring 1.5 μm are an inconsistent finding; these stain Gram-positive and can be seen as a cluster of small dots in the apical cytoplasm of the enterocyte (309,313). The nucleated sporont is a larger (3-5 μm), rounded, basophilic structure often surrounded by a halo and is found in surface enterocytes near the villous tips or in degenerating cells. This form frequently causes a cup-shaped indentation of the enterocyte nucleus that can be an important clue in identification on H&Estained sections. Examination with polarized light can occasionally accentuate the organisms’ polar filament (303).

FIGURE 33.30 Enterocytozoon bieneusi infection of small intestine. Mature spores appear as tiny (1.5 µm) dots, whereas the larger nucleated sporonts are rounded, more basophilic structures often surrounded by a halo in the enterocyte cytoplasm.

Stool studies with special stains or PCR-based assays can make the diagnosis (133,302). Genus and species identification may be important because albendazole is effective against E. intestinalis (314), whereas fumagillin is used for E. bieneusi (315). One important difference between E. bieneusi and E. intestinalis is the propensity for the latter to infect lamina propria macrophages, fibroblasts, and endothelial cells as well as enterocytes (316,317). In contrast, E. bieneusi infects only enterocytes. Electron microscopy and immunofluorescent/enzyme-linked assays can also be used for identification (318). Cyclospora cayetanensis is one of the more recently described parasites and has features very similar to Cryptosporidium and Cystisospora (133,303). The organisms are usually located in a parasitefilled vacuole in the upper portion of the enterocyte. They are acid-fast and auramine positive but negative for PAS, Gram, Giemsa, and silver stains (302,303). Cases are usually diagnosed on stool examination or with the aid of a PCR-based test (319,320). Strongyloides stercoralis is a nematode endemic to the tropic and subtropic regions and the southeastern United States. Interestingly, immunocompromised patients on chronic steroid therapy and with a history of solid organ transplantation are at a higher risk for infection than those with HIV/AIDS, which does not appear to be a significant risk factor (302). Transmission occurs when filariform larvae, which exist in soil, penetrate exposed skin and migrate to the duodenum and jejunum, where females lay eggs. Patients can be infected for decades with only mild symptoms. Hyperinfection occurs in immunocompromised patients, and symptoms include diarrhea, vomiting, intestinal obstruction, and protein losing enteropathy. Endoscopy is nonspecific and histology mixed inflammation with large curved organisms with pointed tails and basophilic ova, within intestinal crypts (302). Identification of larvae or eggs in stool or tissue leads to a diagnosis; one can also use serologic detection of Strongyloides immunoglobulins. OTHER LESIONS IN HUMAN IMMUNODEFICIENCY VIRUS OR ACQUIRED IMMUNODEFICIENCY SYNDROME Human Immunodeficiency Virus (HIV) enteropathy This is a controversial entity in which HIV/AIDS patients develop chronic diarrhea without an identifiable pathogen. Some studies have determined this to be the case in up to 20% of patients with chronic diarrhea (321). Although it is postulated that normal flora may become pathogenic in HIV/AIDS, an immune-mediated enterocolitis or direct HIV infection of the gut may be the etiology (304,321-323). Histology typically shows variable and nonspecific features, such as villous abnormality, crypt hyperplasia, and an increase in crypt epithelial apoptosis. Although apoptosis is associated with a variety of injurious agents and medications, it is also the characteristic form of cell death associated with cell-mediated immune cytotoxicity, and the degree of apoptosis in HIV patients mimics that of grade 1

GVHD (324). Interestingly, patients tend to do better once HAART therapy is initiated and there is an increase in CD4 counts; this provides some support to theories that postulate a direct effect of the virus or infection with undetectable pathogens (321). Kaposi Sarcoma Kaposi sarcoma remains the most common HIV-associated GI malignancy, and the GI tract is the second most common organ system involved after the skin (321,325). Patients are usually asymptomatic but may present with nausea and abdominal pain. Gastrointestinal hemorrhage can also be a presenting symptom, although this is typically with more advanced disease. Grossly and endoscopically, Kaposi sarcoma appears as red macules or nodules. Histology usually reveals a mildly atypical spindle cell proliferation with poorly formed, slit-like blood vessels and abundant erythrocytes (Fig. 33.31). Immunostaining for HHV-8 can confirm the diagnosis. Of note, Kaposi sarcoma can be positive for CD117 and DOG-1, so it is important to rule out other spindle cell tumors of the GI tract such as gastrointestinal stromal tumors (GISTs) (321,326).

FIGURE 33.31 Kaposi sarcoma in a duodenal biopsy. The neoplasm is composed of spindle cells and slit-like vascular spaces and expands the lamina propria. The spindle cells also overrun and obliterate the muscularis mucosae, an important clue to the diagnosis.

Epstein-Barr virus–associated smooth muscle tumor Epstein-Barr virus (EBV)–associated smooth muscle tumor is a rare neoplasm that can occur in the setting of HIV/AIDS infection. The GI tract is a common anatomic site, and the lesions present as nodules with central ulceration. Histologically, they appear as classic smooth muscle tumors (leiomyoma or leiomyosarcoma) but with abundant T-cell inflammation (321). EBV infection can be confirmed with in situ hybridization. Treatment relies on reestablishing efficient T-cell immunity such as initiation of HAART in HIV patients or reduction of immunosuppression in patients with organ transplantation. Surgery should be performed whenever tumor masses affect organ function. Chemotherapy and radiotherapy can be used if these other measures do not improve the disease course (327).

EVALUATION OF RESECTION SPECIMENS IN INFLAMMATORY BOWEL DISEASE When other causes of enteritis and colitis are excluded, a group of diseases referred to as idiopathic IBD is left. IBD describes at least the following three entities: CD, UC, and so-called indeterminate colitis. The pathologic features of CD and UC are usually sufficiently distinctive to allow for a specific

diagnosis, especially when the histopathologic features can be correlated with the endoscopic and other clinical findings. CROHN DISEASE AND ULCERATIVE COLITIS The distribution, gross appearance, and histologic characteristics of CD and UC are well described and have been known for some time. Features aiding the distinction of CD and UC are summarized in Tables 33.9 and 33.10. In the colon, and in the absence of prior therapy, which can significantly alter disease distribution and other manifestations, rectal sparing, skip areas of involvement, and preferential right-sided localization favor CD over UC. Discriminating microscopic features of CD include nonnecrotizing granulomas that are unassociated with ruptured crypts, deep fissuring ulcers that run perpendicular to the long axis of the intestine, and transmural inflammation. Granulomas, found in 50% to 70% of cases, are generally poorly formed and few in number and seen more often in the small intestine. Fissuring ulcers are lined by granulation tissue and extend deeply to involve submucosa, muscularis propria, or beyond. Transmural inflammation consists of lymphoid aggregates that tend to concentrate around lymphatic and blood vessels. Intense inflammation may involve nearby blood vessel walls, a feature termed Crohn vasculitis, which, on occasion, may be prominent. Microscopic inflammation at the margins of resection does not correlate with disease recurrence, which usually occurs at the proximal (“neoterminal ileum”) side of anastomoses after surgery (328). The differential diagnosis of CD includes conditions with an ischemic pattern, including those caused by vasculitis and some infections, and other infectious conditions such as tuberculosis. Distinguishing features are summarized in Table 33.11. TABLE 33.9 Distinguishing Gross Features of Crohn Disease and Ulcerative Colitis Feature

Crohn Enteritis

Crohn Colitis

Ulcerative Colitis

Serositis

Yes

Yes

No, except in fulminant colitis

Thick bowel wall

Yes

Yes

No, except when complicated by carcinoma

Stricture

Often

Sometimes

No, except when complicated by carcinoma

Mucosal edema

Yes

Yes

Usually, no

Discrete mucosal ulcers

Yes

Yes

Usually no, except in fulminant colitis

Fat wrapping

Often present

Often present

Usually, no

Fistula

Common

Sometimes

No

Distribution

Focal or patchy

Focal or patchy

Diffuse

Rectal involvement

No

Sometimes

Yes

Inflammatory polyps

Rare

Sometimes

Sometimes

TABLE 33.10 Distinguishing Histologic Features of Crohn Disease and Ulcerative Colitis Feature

Crohn Enteritis

Crohn Colitis

Ulcerative Colitis

Granulomas

Common

Sometimes

No, except when associated with crypt rupture

Fissuring ulcer

Common

Common

No, except in fulminant colitis

Transmural inflammation

Yes

Yes

No, except in fulminant colitis

Submucosal edema

Yes

Yes

Usually, no

Submucosal inflammation

Yes

Yes

Usually, no

Neuronal hyperplasia

Yes

Sometimes

Usually, no

Thickening of muscularis mucosae

Yes, patchy and marked

Yes, patchy

Yes, diffuse (in chronic UC); not typically as thick as in CD

Pseudopyloric (mucous) gland metaplasia

Common

Rare

Extremely rare

Mucosal inflammation and architectural distortion

Focal/patchy

Usually focal/patchy

Diffuse

Paneth cell metaplasia

No

Sometimes

Yes

UC, ulcerative colitis; CD, Crohn disease.

COLITIS—TYPE INDETERMINATE The term colitis—type indeterminate applies to around 5% of resected specimens, classically consisting of cases of acute or severe clinical disease requiring urgent or emergent colectomy (fulminant colitis), in which the pathologic features are ambiguous and do not permit precise separation of CD from UC (329333). A precise definition of the term, however, has not been established. Fulminant cases of UC may have features, including fissuring ulcers and transmural inflammation, which are normally found in CD. Fulminant colitis with toxic megacolon has been associated with UC, but some patients with this complication actually follow a clinical course more like CD (329-333). Using the classification system of UC, CD, or colitis—type indeterminate, a definitive diagnosis of UC requires all of the following features (330,332,334): diffuse disease limited to the large intestine, involvement of the rectum, more proximal colonic disease occurring in continuity with an involved rectum (i.e., no gross or histologic skip lesions), absence of deep, fissure ulcers, no mural sinus tracts, and no transmural lymphoid aggregates or granulomas. Definitive diagnosis of CD requires histologic verification, with the demonstration of transmural lymphoid aggregates in areas that are not deeply ulcerated or the presence of nonnecrotizing granulomas suggest CD (e.g., skip lesions, linear ulcers, mucosal cobblestoning, fat wrapping, terminal ileal inflammation); the specimen should be extensively sampled in an attempt to find these histologic features of CD. Although the term indeterminate colitis has been widely used in a variety of settings in which diagnostic distinction between UC and CD is difficult, its use is probably best reserved for cases of idiopathic colonic IBD that have ambiguous pathologic features even when a resected specimen is available (330,332-340). Other terminology, such as colonic inflammatory bowel disease unclassified or chronic colitis, exact type currently unknown, can be used when the diagnosis is unclear based on clinical, endoscopic, and biopsy findings. Patients who do not have clinical, endoscopic, or radiologic evidence of CD and in whom the pathology of the resected colon remains indeterminate are, in general, suitable candidates for total proctocolectomy and ileal pouch–anal anastomosis (IPAA) (331,334). The use of indeterminate colitis for appropriate patients is of clinical importance, because pouch failure rates in indeterminate colitis (8%-19%) are intermediate between those in CD (34%-55%) and those in UC (6%-8%) (332,336-339,341). ULCERATIVE PROCTITIS Ulcerative proctitis is a localized form of UC with relatively good prognosis (330,342-345). The endoscopic appearance is identical to that of more extensive UC, as are the histopathologic findings. Some cases may also show prominent mucosal lymphoid follicles (follicular proctitis) reminiscent of diversion colitis (Fig. 33.32) (344). Most ulcerative proctitis patients respond dramatically to local corticosteroids or 5-aminosalicylates, but approximately 10% progress to pancolitis (342,346). The subset of patients with follicular proctitis may be less responsive to treatment (344).

FIGURE 33.32 Follicular proctitis in a patient with ulcerative colitis. Prominent lymphoid follicles are associated with cryptitis, superficial erosions, and chronic inflammation in the lamina propria.

INFLAMMATORY BOWEL DISEASE AND DIVERTICULAR DISEASE IBD and diverticular disease are both common and can coexist. In addition, an IBD-like segmental inflammation associated with diverticular disease has been described (347-354). This entity has gone by a variety of names but is now referred to as segmental colitis associated with diverticulosis (SCAD). Both CD and diverticular disease, with or without SCAD, can have focal mucosal inflammation, strictures, and fistulae. All of these changes might be attributed to one disease (usually diverticular disease), but this risks overlooking coexisting IBD (usually CD). Features that suggest CD include fissuring ulcers, involvement outside a diverticular segment, and fistulae other than colovesical or colovaginal (348). Patients with SCAD tend to have fewer complications than those with UC or CD (354). LESIONS ASSOCIATED WITH SURGICAL PROCEDURES Diversion Colitis A segment of colon (most often rectum) that is bypassed from the fecal stream, either surgically or by another means such as a fistula, develops histologic changes associated with defunctioning alone, regardless of the reason for or means of diversion (355-359). The changes are probably a response to stasis and the loss of trophic factors in the feces, thought to predominantly be short-chain fatty acids (360). Patients are usually asymptomatic, but can present with tenesmus and blood discharge, and the mucosa of the diverted segment is erythematous, granular, and friable. Histologically, there is nodular lymphoid hyperplasia with germinal centers, accompanied by chronic lamina propria inflammation, cryptitis, and crypt abscesses (Fig. 33.33), and these changes may be indistinguishable from UC. Over time, if the fecal continuity is not restored, the muscularis mucosae becomes hypertrophic, the submucosa scarred, and the muscularis propria thickened with luminal narrowing (334). Identical histologic changes occur in diverted segments in patients with and without IBD, so a diagnosis of primary IBD, especially CD, should not be made solely on histologic changes seen in diverted segments. In many patients, the rectum is placed out of circuit as part of a resection for IBD. In these cases, the rectum (Hartmann pouch) will have changes of both primary IBD and diversion colitis, which may not correlate directly with the original diagnosis or the clinical outcome (361,362).

FIGURE 33.33 Diversion colitis from a patient with a defunctionalized rectum. Lymphoid follicles, the hallmark of this condition, are accompanied by cryptitis and crypt abscesses, with an appearance very similar to follicular proctitis. The history of diversion is a critical component of the diagnosis.

Ileal Reservoirs (Pouches) and Pouchitis Patients requiring total colectomy can undergo surgery to either create a continent ileostomy (Kock ileostomy) or preserve anal sphincter function and restore the continuity to the intestine (IPAA) (363,364). These operations have in common the creation of a reservoir (pouch), which is formed by connecting loops of the terminal ileum, typically into a “J” shape (hence the alternative name of “Jpouch” for the reservoir created by IPAA). These procedures are usually contraindicated in patients with CD because of increased morbidity (e.g., fistula and abscess), although careful patient selection has led to good outcomes for patients diagnosed with CD prior to IPAA in some cases (128,365). Complications requiring pouch removal can result in the loss of considerable lengths of the small intestine that are sometimes enough to cause short bowel syndrome. Pouch complications include fistula, sinus, obstruction, incontinence, and anastomotic leaks (363). Although many complications result from surgical and mechanical difficulties and others relate to the development of primary pouch inflammation (pouchitis), some of these complicated cases may reflect recurrence of initially undiagnosed CD in the pouch. These cases illustrate the difficultly in differentiating UC from CD in some cases, even after examination of the colectomy specimen (see section “Colitis— Type Indeterminate”). Virtually all of the reports of surgical experiences with IPAA for presumed UC contain approximately 2% to 7% of patients in whom the actual diagnosis proved to be CD (366-370). In addition, there is mounting evidence that a de novo form of CD can develop in the pouch in patients initially (and appropriately) diagnosed with UC (371-373). The most common late complication of IPAA is the development of a primary inflammation of the pouch, termed along with its associated clinical syndrome pouchitis (370,373,374). The reported incidence varies from 8% to 59%, the wide range likely being attributable to the lack of an accepted case definition (370,375,376). Some disease characteristics in patients with UC seem to be associated with the development of pouchitis in patients who undergo IPAA, including severe pancolitis, active appendiceal inflammation, and appendiceal ulceration (377). Patients with pouchitis may experience nausea, vomiting, malaise, fever, and abdominal cramping, along with increased effluent and stool from the ileum that may be watery, foul-smelling, or grossly bloody, which may culminate in incontinence. The ecology of bacterial flora is known to be altered in pouches, and patients with pouchitis usually respond to antibiotics, suggesting a bacterial etiology (378). Some patients, however, become dependent on, or refractory to, antibiotic therapy and may require sulfasalazine, corticosteroids, immunologic therapy with anti–tumor necrosis factor or anti–integrin antibodies, or even pouch excision for the management of pouchitis (363,370,374-376,379-382). There are some reports of success with FMT in this setting, as well (382,383), although this remains under investigation. Endoscopic examination and biopsy of the pouch may be performed to confirm the presence of inflammation or to evaluate the possibility of CD (384). Initial evaluations may include C. difficile and

CMV testing. Nondysfunctional pouch mucosa may have changes reflecting the altered function of the ileum, including mild villous shortening and chronic inflammation with regenerative changes in the crypt epithelium. Intraepithelial lymphocytes may be present (385). Rare neutrophils may also be found in the surface epithelium (379,386,387), but significant acute inflammation is abnormal. Pouches with classic pouchitis have more intense regenerative changes, including decreased mucin production, along with erosions, ulcers, cryptitis, and crypt abscesses (374,379,386-388). There may even be inflammatory polyps (370). “Pouchitis disease activity index” (375) and “pouchitis activity score” (389) criteria have been developed but are not routinely used by pathologists. There is reportedly an inconsistent relationship between patient symptoms and endoscopic and histologic findings in the pouch (367,370,390). Therefore, many clinicians diagnose pouchitis based on clinical features, reserving endoscopy and biopsy for patients with refractory pouchitis or a suspicion of CD (381,384,386). Given the propensity for architectural and inflammatory changes in even nondysfunctional pouches, there are no reliable endoscopic or histologic criteria that differentiate pouchitis from new-onset or recurrent CD in pouches. The clinical syndrome of pouchitis probably represents many different conditions (Table 33.10) (373,386,391,392). Antibiotic-responsive pouchitis may be “classic,” as described earlier, but some cases may be secondary to jejunal bacterial overgrowth (379,386,390,393,394). This could be a result of pouch distention, which causes an “ileal brake” that decreases motility and predisposes to bacterial overgrowth in the proximal small intestine (394,395). Pouchitis syndromes refractory to antibiotic therapy include irritable pouch syndrome, “short-strip” pouchitis, CD, and primary refractory pouchitis (Table 33.) (386,391,392). Patients with irritable pouch syndrome have severe clinical symptoms but lack endoscopic or histologic abnormalities in the pouch. Some of these patients respond to dietary fiber supplements, antidiarrheal and antispasmodic medication, and antidepressant therapy (370,391,392). To obtain a more functional pouch, some surgeons have abandoned the rectal mucosectomy in ileal pouch–anal anastomoses (396), leaving a “rectal cuff” at the distal end of the pouch. What has been termed short-strip pouchitis may be better referred to as “cuffitis,” and likely results from symptoms of residual UC in the retained rectal segment, which can be treated as such (374,381,384,387,390,397). TABLE 33.11 Differential Diagnosis of Crohn Disease Feature

Crohn Disease

Ischemic Bowel Disease

Tuberculosis

Discrete ulcers

Yes

Yes

Yes

Longitudinally running ulcers

Yes

Yes

Usually, no

Stricture

Yes

Yes

Yes

Lymphoid aggregates

Yes

No

Usually, no

Hemorrhage and necrosis

No

Yes (acute)

No

Granulation tissue and fibrosis

No

Yes (late)

Sometimes

Hemosiderin deposition

Usually, no

Often

Usually, no

Preferred location

Ileum and cecum

Watershed zones (e.g., splenic flexure)

Ileum and cecum

Granulomas

Often

No

Yes

Confluence of granulomas

No

Not applicable

Sometimes

Necrotizing granulomas

No

Not applicable

Sometimes

Fibrosis of muscularis propria

Usually, no

Yes

Yes

Some investigators have identified histologic patterns of mucosal adaptation in pouches (388,398400). Approximately 50% to 60% of patients have what has been called type A mucosa with normal histology or only mild mucosal atrophy and no or minimal inflammation. Type B mucosa, which is characterized by transient villous shortening, temporary moderate-to-severe inflammation, and subsequent normalization of the mucosa, is seen in around 40% of patients. Finally, type C mucosa with permanent, persistent atrophy and severe inflammation occurs in approximately 10% of pouches. Colonic-type mucosal features have been reported at least focally in pouches of all types by morphology, mucin histochemistry, IHC, lectin binding, or electron microscopy. This colonic-type metaplasia is most well developed in type C mucosa, but it is never complete. Pouchitis and Dysplasia Epithelial dysplasia can develop in ileal pouches, albeit very rarely (370,378); those with prior colonic dysplasia or cancer and/or those with primary sclerosing cholangitis appear to be at increased risk. Dysplasia seems to be limited to patients (10 year) history of UC (386,400,401). Although colonic mucosectomy may still be performed as part of the IPAA procedure, dysplasia can also develop in the short retained rectal segment, often incorrectly referred to as the anal transitional zone, but better termed the rectal cuff, in patients who have had stapled IPAA (402). Thus, endoscopic surveillance with biopsy is also recommended for these rectal segments on an annual basis. As in the pouch, dysplasia in the rectal cuff is rare (90%

>90%

40%

KRAS mutation

0%

50%

1 cm), contain a villous component comprising at least 25% of the polyp, or display high-grade dysplasia (48). Unfortunately, the morphologic criteria inherent in this definition are somewhat subjective, particularly with respect to assessment of villous architecture and dysplasia. Polyp size remains the most reliable criterion for designating a polyp as an advanced adenoma. Most colorectal adenomas can be completely removed with endoscopic techniques. Small lesions are usually excised using a combination of biopsy forceps and cautery. Pedunculated polyps are removed by surrounding the polyp stalk with a snare through which a current is passed. Electrocautery simultaneously transects the polyp base and coagulates bleeding sites. Sessile lesions can be elevated by injecting saline, epinephrine, or lifting agents into the polyp base, thereby elevating the lesion to facilitate snare excision (Fig. 34.17). Endoscopic mucosal resection (EMR), submucosal dissection, and other advanced methods can be used to remove large polyps (49).

FIGURE 34.17 Endoscopic removal of an adenoma. A sessile tubular adenoma produces an ill-defined thickened area at the crest of a mucosal fold (A), which can be lifted off the muscularis propria by injecting the submucosa with a combination of either saline and epinephrine or a lifting agent and India ink, the latter of which imparts blue discoloration to the mucosa (B). The polyp can then be ensnared with a loop that conducts a current, thereby allowing transection of the polyp base and simultaneous thermal coagulation (C). Epinephrine and/or saline injection causes no sustained submucosal alterations, but gel lifting agents, such as Orise and Eleview, remain in the submucosa. Over time, they appear as eosinophilic ribbon-like or amorphous deposits accompanied by a giant cell reaction (D).

Tubular adenomas tend to be round sessile or pedunculated polyps with a smooth surface that is divided into lobules secondary to intercommunicating clefts in the polyp head, whereas villous adenomas are usually sessile lesions with a shaggy appearance (Fig. 34.18). Polyp morphology is directly related to polyp size: more than 90% of adenomas smaller than 1 cm are tubular adenomas, whereas 50% of adenomas larger than 2 cm contain villous areas. Straight tubular or branched crypts comprise at least 75% of the volume of tubular adenomas, whereas villous adenomas contain slender fronds of neoplastic epithelium that account for at least 75% of the polyp volume (Fig. 34.19). Polyps with a villous component accounting for 25% to 75% of the lesion are classified as tubulovillous adenomas (50).

FIGURE 34.18 Colonic adenomas. A small tubular adenoma expands a fold and imparts reddish brown discoloration to the mucosa (A). A resected tubular adenoma has a multilobular appearance with red-brown discoloration (B). A large villous adenoma appears as a velvety red plaque with a glistening surface owing to adherent mucin (C). A cross section through the same lesion demonstrates long, fingerlike projections that impart a shaggy appearance to the polyp (D).

FIGURE 34.19 Colonic adenomas. Tubular adenomas feature a proliferation of tubular crypts that vary in size and shape and lack orientation to the muscularis mucosae (A). Lesional cells contain decreased cytoplasmic mucin and elongated, hyperchromatic nuclei (B). A villous adenoma displays projections of lamina propria supporting neoplastic epithelial cells (C) with enlarged, pseudostratified nuclei (D).

Tubular, villous, and tubulovillous adenomas show similar patterns of dysplasia that are classified as low or high grade based on a combination of cytologic and architectural features (50). Neoplastic epithelium is supported by normal lamina propria, but crypts are increased in number and show variability in size and shape. Lesional epithelial cells contain enlarged, hyperchromatic nuclei and decreased intracellular mucin; the latter may be present in well-formed goblets or small apical vacuoles. Low-grade dysplasia is characterized by crowded, tubular, or branched crypts that lack orientation to the muscularis mucosae. The cells contain large, cigar-shaped nuclei with coarse chromatin and conspicuous nucleoli that maintain their close relationships to the basement membrane (Fig. 34.20A). Apoptotic epithelial cells are easily detected in the deep crypts, and a neutrophil-rich inflammatory infiltrate is sometimes present. High-grade dysplasia shows complex architectural features with cribriform growth or irregular crypt budding and loss of the relationship between epithelial cells and the basement membrane. High-grade cytologic features include markedly increased nuclear-to-cytoplasmic ratios, loss of cell polarity with full-thickness nuclear stratification, prominent nucleoli, frequent mitotic figures, and foci of cellular necrosis (Fig. 34.20B). Luminal necrotic debris may be present but likely signifies the presence of an invasive adenocarcinoma when extensive, especially if the specimen represents sampling of a larger, incompletely resected mass. Conventional adenomas can display increased numbers of Paneth cells, small nests of endocrine cells, or squamous

metaplasia, but these features do not require specific mention (51-53). Colorectal adenomas show CDX2, MUC2, CK20, and SATB2 staining. They are usually negative for CK7, although patchy positivity can occur, especially in lesions of the proximal colon and distal rectum.

FIGURE 34.20 Colonic adenomas. Adenomas with low-grade dysplasia contain straight tubular crypts lined by polarized cells with basally located, elongated nuclei and scattered apoptotic debris (A). High-grade dysplasia displays crypt architectural abnormalities with cribriform spaces lined by cells with a loss of cell polarity; ovoid nuclei bear no relationship to the basement membrane (B).

Some adenomas contain adenocarcinomas that invade the lamina propria but are confined to the muscularis mucosae. These intramucosal adenocarcinomas usually expand the lamina propria with a proliferation of neoplastic glands that shows back-to-back and/or cribriform growth or small clusters of tumor cells around larger glands (Fig. 34.21). Importantly, infiltrating tumor cells in the lamina propria are unassociated with a desmoplastic tissue reaction in most cases; detection of desmoplasia in biopsy material generally signifies invasion of the submucosa. Intramucosal carcinoma of the colorectum has a negligible risk for metastasis, presumably because there is a paucity of lymphatic channels above the level of the crypt bases (54). For this reason, adenomas containing intramucosal adenocarcinoma are staged as in situ neoplasms (pTis) and adequately treated with complete excision.

FIGURE 34.21 Intramucosal adenocarcinoma. The head of a polyp is expanded by a neoplastic proliferation of architecturally complex glands (A), some of which are cystically dilated and filled with necrotic debris (B). Confluent sheets of glands show cribriform growth (C). Small clusters of cytologically malignant cells bud off larger glands in the lamina propria (D).

Adenomas with Epithelial Misplacement. There are several situations in which benign adenomatous epithelium can be found in the submucosa of an adenoma. Most commonly, adenomatous glands are found in lymphoid aggregates that transgress the muscularis mucosae and extend from the submucosa into the mucosa (Fig. 34.25A). These lymphoglandular complexes are usually found in the right colon where lymphoid tissue is more abundant. Presumably, peristalsis and luminal trauma propel mucosal elements through weak points in the muscularis mucosae, which is discontinuous in lymphoid aggregates. Misplaced epithelium is cytologically similar to that on the surface and is supported by lamina propria. Epithelial misplacement is also relatively common among large pedunculated adenomas of the distal colorectum. Intermittent ischemic injury coupled with increased luminal pressures cause herniation of mucosal elements into the submucosa, a finding which has been termed pseudoinvasion (55-58). Benign misplaced epithelium appears as circumscribed aggregates of neoplastic glands surrounded by a rim of lamina propria (Fig. 34.25B). It is usually accompanied by telltale signs of polyp injury, such as hemorrhage, hemosiderin deposits, and fibrosis. Some misplaced glands are cystically dilated or ruptured or and accompanied by pools of extruded mucin (Fig. 34.25C). Although benign epithelial misplacement may bear a resemblance to invasive carcinoma, the submucosal epithelium of a traumatized adenoma is cytologically similar to that on the polyp surface and it is supported by lamina propria. The lobular arrangement of epithelial elements in combination with inflammatory changes facilitates a diagnosis in most cases. Other helpful diagnostic features are listed in Table 34.2. TABLE 34.2 Features that Distinguish Benign Epithelial Misplacement From Adenocarcinoma in Colorectal Polyps Feature

Adenoma With Misplaced Epithelium

Adenoma With Invasive Adenocarcinoma

Low-grade cytologic atypia

Usually present

Usually absent

High-grade cytologic atypia

Usually absent

Usually present

Architecture

“V” shape: Diameter of proliferation in deep submucosa less than that at surface

Inverted “V” shape: Diameter of proliferation in deep submucosa greater than that at surface

Epithelial arrangement

Lobules of epithelium and lamina propria

Expansile, irregular distribution

Cytologic atypia

Usually low-grade

Usually high-grade

Usually present

Usually absent

OVERLYING SURFACE EPITHELIUM

SUBMUCOSAL EPITHELIUM

Associated elements Lamina propria

Muscularis mucosae

Usually present

Usually absent

Nonneoplastic epithelium

Often present

Usually absent

Mucin pools

Acellular Epithelium and lamina propria in mucin Peripheral strips of bland epithelium

Peripheral strips of atypical epithelium Floating clusters of atypical cells unaccompanied by lamina propria

Granulation tissue

Often present

Usually absent

Thick collagen bands

Often present

Usually absent

Hemorrhage/hemosiderin

Often present

Usually absent

Erosions

Often present

Usually absent

Inflamed ruptured crypts

Often present

Usually absent

OTHER FINDINGS IN SUBMUCOSA

Finally, endoscopic manipulation of adenomas traumatically introduces benign epithelium into the submucosa (59). This type of epithelial misplacement is relatively common, occurring in more than one-third of previously sampled adenomas (60). Biopsy of a polyp disrupts the integrity of the muscularis mucosae. The subsequent ulcer bed extends into the submucosa, containing inflammation, extruded mucin, small groups of dysplastic and nonneoplastic epithelial cells, and abundant granulation tissue. As the injury resolves, disrupted glands and single neoplastic cells become embedded in the submucosa where they are associated with mucin pools and organizing granulation tissue (Fig. 34.22D). Most submucosal epithelium in these lesions displays low-grade cytologic features and is accompanied by lamina propria and muscularis mucosae. Aggregates of nondysplastic epithelium may also be present in the submucosa.

FIGURE 34.22 Benign epithelial misplacement in an adenoma. Adenomas of the right colon may contain lymphoglandular complexes composed of adenomatous glands and lymphoid nodules (A). Pedunculated polyps of the left colon are subjected to luminal forces that cause herniation of mucosal elements into the submucosa. Round pools of acellular mucin and aggregates of benign epithelium (B) are intimately associated with lamina propria, fresh hemorrhage, and hemosiderin deposits (C). Adenomas that have been endoscopically manipulated may contain submucosal epithelium as a result of biopsy. In this situation, the amount of submucosal epithelium decreases at its advancing edge. Atypical glands may communicate with the luminal surface or show acellular mucin pools at the deepest extent (D).

Evaluation of Endoscopic Specimens

Endoscopically removed specimens usually consist of biopsy material, polypectomy samples, EMRs, or endoscopic submucosal dissections (ESDs). Regardless of the nature of the specimen, endoscopically removed material should be entirely submitted for histologic evaluation. Small biopsy samples are generally evaluated with multiple tissue levels prepared on one or several glass slides. Polypectomy specimens are evaluated for the presence of a stalk or base, which represents the resection margin and should be designated with ink. Sectioning of polyps is performed such that the relationships between the lesion and the resection margins are evident in subsequent histologic sections. Sectioning at 2-mm intervals is required for larger polyps in order to ensure complete fixation. EMR and ESD specimens may be pinned on cork or paraffin prior to fixation. In this situation, the base of the excisional specimen is inked, and the entire specimen is serially sectioned while maintaining tissue orientation, if provided. The type of diagnostic information reported by the pathologist varies depending on the nature of the sample. Reports based on evaluation of biopsy material usually indicate the type of adenoma (i.e., tubular, villous, or tubulovillous) and presence or absence of highgrade dysplasia because this type of information is often used to determine the subsequent surveillance interval (2,61). Reports for polypectomy and excisional specimens are more comprehensive, in that pathologists may comment on the abovementioned features as well as the status of the resection margin. Approximately 2% to 5% of endoscopically removed adenomas contain invasive adenocarcinoma in the submucosa (62). Many of these can be treated by complete endoscopic resection alone, provided features associated with disease recurrence and/or regional lymph node metastases are absent. Pathology reports should specifically note the presence or absence of high-grade invasive components, tumor budding, or lymphovascular invasion, as well as the distance from the invasive carcinoma to the resection margin. All of these features are taken into consideration when determining whether an early carcinoma is adequately treated by polypectomy or requires additional surgery (63,64). Distinction between pedunculated and sessile polyps is best left to endoscopists; they often inject the base of a sessile polyp to lift it off of the muscularis propria, thereby creating a wheal that can simulate a stalk in histologic sections. PYLORIC GLAND ADENOMA Pyloric gland adenomas occur throughout the upper gastrointestinal tract. Some lesions in the proximal small intestine and ampullary region have probably been considered to represent Brunner gland adenomas in the past. Pyloric gland adenomas are less common than intestinal-type adenomas. Many are associated with ectopic gastric mucosa, exuberant foveolar hyperplasia, gastric-type dysplasia, and Brunner gland lesions. Pyloric gland adenomas are endoscopically similar to conventional adenomas, producing sessile nodules (Fig. 34.23A). They are composed of tightly packed tubules lined by cuboidal-to-columnar cells that contain tiny vacuoles of pale or faintly eosinophilic mucin dispersed in the cytoplasm (Fig. 34.23B) (38). Small, evenly spaced nuclei maintain their polarity and mitotic figures are infrequently encountered. Pyloric gland adenomas often harbor GNAS mutations and show MUC6 and MUC5AC coexpression by immunohistochemistry (65,66).

FIGURE 34.23 Pyloric gland adenoma of the duodenum. This lesion produced a smooth sessile polyp in the proximal duodenum (A). It is composed of small tubules lined by cuboidal cells with round nuclei, prominent nuclei, and neutral mucin as well as larger glands lined by foveolar-type epithelial cells (B).

INFLAMMATORY BOWEL DISEASE–ASSOCIATED NEOPLASIA Patients with idiopathic inflammatory bowel disease are at increased risk for development of intestinal dysplasia and adenocarcinoma. Risk factors include disease duration in excess of 10 years, extensive mucosal involvement (i.e., pancolitis), primary sclerosing cholangitis, severe mucosal inflammation, and a family history of colorectal dysplasia or carcinoma. Dysplasia risk for patients with ulcerative colitis is highest in the distal colorectum and is estimated to be 5% to 20% after 10 years of disease (67). Colorectal cancer risk among patients with ulcerative colitis is estimated to be 2%, 8.5%, and 17.8% at 10, 20, and 30 years, respectively (68). Patients with Crohn disease have a lower risk, presumably reflecting less frequent pancolitis. Their neoplasms are more evenly distributed throughout the colorectum and distal small bowel. Dysplasia is categorized as visible and invisible based on its endoscopic appearance (69). Visible dysplasia is subclassified as polypoid and nonpolypoid. Polypoid dysplasia resembles a conventional adenoma and may be pedunculated or sessile. Nonpolypoid visible dysplasia is classified as superficial elevated, flat, or depressed depending on whether or not it protrudes into the lumen (Fig. 34.24). Invisible dysplasia is endoscopically undetectable and comprises less than 5% of dysplasias discovered during endoscopic surveillance. Visible dysplasias that can be entirely removed by endoscopy are generally treated with excision and surveillance colonoscopy, whereas those that cannot be removed endoscopically require colectomy. Detection of dysplasia in nontargeted, random biopsies is an indication for colectomy.

FIGURE 34.24 Idiopathic inflammatory bowel disease–associated dysplasia. Visible dysplasia appears as an ill-defined plaque of villous projections in the distal colon (A) that is enhanced with narrow band imaging (B).

Biopsy samples from patients with inflammatory bowel disease are classified as negative for dysplasia, positive for dysplasia, or indefinite for dysplasia. Cases deemed positive for dysplasia are further classified as low-grade or high-grade using criteria similar to those applied to sporadic adenomas. Most colitis-associated dysplasias are of intestinal type and, thus, resemble conventional tubular, villous, or tubulovillous adenomas (Fig. 34.25A and B). Occasional patients may develop serrated neoplasms that, when extensive, simulate serrated polyposis (70). Less common variants include mucinous and mucin-depleted dysplasia. Mucinous dysplasia displays a tubulovillous growth pattern of goblet or non– goblet mucinous epithelial cells with bland nuclear features (Fig. 34.25C). Mucin-depleted dysplasia features crowded crypts lined by columnar cells with enlarged nuclei and eosinophilic cytoplasm (Fig. 34.25D). Some authors recognize a variant of dysplasia they term “crypt dysplasia”. In this situation, dysplastic-appearing cells in the crypts are accompanied by apparent surface maturation. This latter category is widely considered “indefinite for dysplasia” by others, especially when the cytologic features are low-grade. In fact, a provisional diagnosis of indefinite for dysplasia is appropriate when epithelial cells show low-grade cytologic atypia but these abnormalities are associated with neutrophil-rich infiltrates or they are limited to the crypts.

FIGURE 34.25 Idiopathic inflammatory bowel disease–associated dysplasia. Intestinal-type dysplasia features crowded crypts lined by neoplastic cells (A) that show nuclear enlargement with open chromatin and prominent nucleoli (B). Other variants are rich in non–goblet mucinous cells that show disordered growth and mild nuclear abnormalities (C) or mucin-depleted cells with eosinophilic cytoplasm (D).

PATHWAYS OF CARCINOGENESIS THE CHROMOSOMAL INSTABILITY PATHWAY Adenomas of the small bowel and colorectum show similar molecular alterations and most progress to carcinoma through the chromosomal instability pathway. This mechanism accounts for approximately 80% of intestinal adenocarcinomas. Adenomas develop as a result of biallelic APC inactivation on chromosome 5q that leads to constitutive APC/βcatenin/WNT signaling. Sporadic adenomas result from early somatic mutations affecting both copies of APC; approximately 2% of cases develop in patients with germline APC mutations affecting one allele (i.e., familial adenomatous polyposis). Sporadic and heritable adenomas progress to high-grade dysplasia and carcinoma through accumulation of genetic events, such as KRAS mutations, deletions from chromosome 18q, and TP53 inactivation, most of which result from gains and losses of large amounts of genetic material, including entire chromosomes (71). Thus, the chromosomal instability pathway essentially represents the molecular counterpart to the adenoma-carcinoma sequence.

APC encodes a 2843-amino acid protein that regulates cell growth and differentiation through its suppressive effects on WNT signaling. In the absence of WNT signaling, the APC-axin-GSK3b complex induces phosphorylation of β-catenin and targets it for ubiquitination. Activation of WNT signaling occurs when WNT proteins bind to the Frizzled receptor and the LDL receptor–related protein co-receptor on the cell surface (72). Coupling of these molecules induces axin and LDL receptor–related protein interactions that drive phosphorylation of Disheveled, a protein that interferes with the APC-axin-GSK3b complex and prevents degradation of cytoplasmic β-catenin. Biallelic inactivation of APC essentially creates a scenario that simulates constitutive WNT-mediated cell signaling. Dysfunctional APC fails to participate in the complex that normally sequesters β-catenin and facilitates its cytoplasmic degradation. Excess β-catenin translocates to the nucleus where it promotes expression of genes regulating the cell cycle. For this reason, nuclear β-catenin immunostaining can serve as a surrogate marker of aberrant WNT signaling (73). APC also suppresses WNT activation by decreasing transcription of WNT-responsive genes and shuttling β-catenin out of the nucleus (74). Finally, APC alters cytoskeletal interactions and chromosomal dynamics through its effects on microtubules (75-77). The latter role predisposes cells without functional APC to aneuploidy because defective mitotic spindles promote replicative errors (78). Inactivation of APC leads to subsequent genetic alterations. Activating KRAS mutations, which are present in up to 40% of colorectal adenomas and adenocarcinomas, promote mitogen-activated protein (MAP) kinase–mediated signaling. KRAS encodes a GTP-binding protein that loses its GTPase activity when mutated; impaired hydrolysis of active GTP to inactive GDP causes constitutive downstream activation of the MAP kinase pathway. Up to 60% of intestinal carcinomas have deletions in 18q that affect the region containing SMAD2 and SMAD4, both of which participate in the TGF-β signaling pathway (79). Abnormalities of the PI3K pathway result from alterations affecting PTEN or PIK3CA, the net effects of which include decreased apoptosis and prolonged cell survival (80). Loss of TP53 is a late event that usually results from allelic loss of 17p (81). P53 promotes expression of genes that slow the cell cycle, and it regulates proapoptotic genes. These functions normally allow for DNA repair following occasional genetic insults or cause cell death when excessive genetic damage is present (82). Mutations affecting p53 function allow neoplastic cells to survive despite substantial DNA damage because dysfunctional p53 protein cannot trigger apoptosis or induce cell-cycle arrest (83). THE MICROSATELLITE INSTABILITY PATHWAY Approximately 20% of intestinal carcinomas have microsatellite instability (MSI). These tumors are often large and show heterogeneous growth patterns with medullary or solid areas, mucinous differentiation, and signet-ring cells. Most display a host immune response composed of tumor-infiltrating lymphocytes and/or a Crohn-like lymphoid reaction, as described subsequently. Tumors harbor biallelic inactivation of one of the mismatch repair genes: mutL homolog 1 (MLH1), mutS homolog 2 (MSH2), mutS homolog 6 (MSH6), and postmeiotic segregation increased 2 (PMS2). Similar to carcinomas with chromosomal instability, those with MSI can result from autosomal dominant germline mutations in one allele (i.e., Lynch syndrome) or occur sporadically. Sporadic mismatch repair–deficient tumors usually result from methylation of the MLH1 promoter, although they can also develop via double somatic mutations affecting any one of the mismatch repair genes (8487). Sporadic tumors with MLH1 promoter methylation show a predilection for the proximal

colon and tend to affect older patients, particularly women. Approximately 50% contain BRAF V600E mutations (88). Mismatch repair–deficient tumors resulting from somatic mutations have an epidemiology mirroring that of sporadic mismatch repair–proficient tumors, rather than Lynch syndrome–related carcinomas (89). The clinicopathologic features of Lynch syndrome are discussed in subsequent sections. The four major mismatch repair genes encode proteins that bind specific partners to form complexes that patrol the genome to correct replicative errors. The MSH2/MSH6 heterodimer first recognizes and binds to mismatched base-pairs, then it recruits the MLH1/PMS2 heterodimer to repair the mismatch (73). Mismatches tend to be more numerous in microsatellites, which are short mononucleotide or dinucleotide repeats that occur throughout the genome, particularly in its noncoding regions. Errors tend to occur in these areas because DNA polymerase is prone to slippage along repetitive sequences, creating insertion-deletion loops and single base-pair mismatches. Dysfunctional mismatch repair mechanisms fail to correct these errors, leading to their propagation in the second round of replication. Errors of this nature alter the lengths of microsatellite sequences in the tumor DNA compared with DNA from nonneoplastic tissues (86). Single base-pair mismatches result in point mutations, whereas insertion-deletion loops cause frameshift mutations. Mismatch repair deficiency can be assessed with several methods: polymerase chain reaction (PCR) for MSI, immunohistochemistry for mismatch repair protein expression, and next-generation sequencing for both mutation analysis of mismatch repair genes and tumor mutational burden assessment. Molecular testing for MSI is an assay aimed at assessing mismatch repair protein function. The National Cancer Institute formerly recommended a reference panel of five MSI markers, including two mononucleotide (BAT26 and BAT25) and three dinucleotide (D2S123, D5S346, and D17S250) repeats, defining tumors with highfrequency MSI (MSI-H) as those that showed instability in at least two markers, lowfrequency MSI (MSI-L) as those with instability at one locus, and microsatellite stable (MSS) tumors as those without instability at any marker (90). Most laboratories now utilize a panel of five quasimonomorphic mononucleotide repeat markers (BAT25, BAT26, NR-1, NR-24, MONO-27) in a pentaplex PCR because it offers a greater degree of specificity and sensitivity (91,92). The majority of mismatch repair–deficient tumors show MSI-H, and mismatch repair–proficient tumors are MSS. Tumors with instability at one locus are best classified as indeterminate because they may, or may not, be mismatch repair-deficient. Immunohistochemical stains for mismatch repair proteins represent a surrogate marker of protein expression and generally correlate with PCR results. Mismatch repair proteins are normally expressed by proliferating cells, including mismatch repair–proficient carcinomas and lymphocytes; immunohistochemical stains show strong nuclear positivity for mismatch repair proteins in these cell populations. Mismatch repair–deficient tumors show loss of at least one of these markers, and the staining pattern can be used to identify the underlying defective gene according to criteria listed in Table 34.3. Both PMS2 and MSH6 require their binding partners to maintain stable heterodimers. Defective MLH1 is usually characterized by combined loss of MLH1 and PMS2, whereas defective MSH2 generally results in loss of MSH2 and MSH6. However, both MLH1 and MSH2 can form heterodimers with other proteins; loss of either PMS2 or MSH6 function typically does not affect MLH1 or MSH2 staining, respectively. Patchy staining for one or more markers is not uncommon and may be related to tissue hypoxia or variable fixation conditions. However, several staining patterns warrant additional investigation because they may denote mismatch repair

deficiency: punctate or speckled nuclear staining, nuclear membrane staining, nucleolar staining, and weaker staining of the tumor cell nuclei compared with internal controls (93). Neoadjuvant therapy can decrease immunoexpression of mismatch repair proteins despite the presence of mismatch repair proficiency (94). TABLE 34.3 Typical Patterns of Mismatch Repair Protein Immunohistochemical Staining and Underlying Defective Genes Affected Gene

Preserved MLH1

Preserved PMS2

Preserved MSH2

Preserved MSH6

None

Yes

Yes

Yes

Yes

MLH1

No

No

Yes

Yes

PMS2

Usually

No

Yes

Yes

MSH2

Yes

Yes

No

Usually not

MSH6

Yes

Yes

Yes

No

Mismatch repair deficiency promotes neoplasia through several mechanisms. Failure to correct replicative errors leads to multiple base-pair changes that diminish protein function. Although most microsatellites are located in noncoding regions, some mononucleotide repeats occur in coding regions where frameshifts can cause gene inactivation. Transforming growth factor-β type II receptor (TGF-bR2) is an important regulatory gene that contains two microsatellite sequences in its coding region (95). Other regulatory genes affected by mismatch repair deficiency influence cell proliferation (e.g., GRB1, TCF-4, WISP3, activin receptor-2, insulin-like growth factor-2 receptor, axin-2, and CDX), cell cycling and apoptosis (e.g., BAX, caspase-5, RIZ, BCL-10, PTEN, hG4-1, and FAS), and DNA repair (e.g., MBD-4, BLM, CHK1, MLH3, RAD50, MSH3, and MSH6) (86,96). THE CPG ISLAND METHYLATOR PHENOTYPE DNA methyltransferases form methyl cytosines by adding methyl groups to cytosine residues located 5′ to guanine. These cytosine (C) and guanine (G) dinucleotides are often clustered in CG-rich regions (i.e., CpG islands) that commonly occur in the promoters of mammalian genes. Methylation of the promoter regions silences genes from transcription by directly inhibiting transcription factor binding, influencing histone acetylation, and blocking transcriptional machinery from accessing the gene. Epigenetic silencing may account for the first or second or both events that silence important genes such as MLH1. Tumors with DNA hypermethylation at multiple promoters show the CpG island methylator phenotype (CIMP) (97). They tend to occur in the proximal colons of older women and are associated with cigarette smoking (98). Intestinal carcinomas with CIMP are often mismatch repair-deficient with BRAF mutations and show methylation of MINT1, MLH1, RIZ1, and TIMP3 (99,100). Higher levels of CpG island methylation are associated with BRAF mutations compared with KRAS mutations; methylation is uncommon among tumors that are wild-type for both BRAF and KRAS (101). THE SERRATED NEOPLASTIC PATHWAY

The serrated neoplastic pathway accounts for approximately 15% of intestinal carcinomas and shows substantial overlap with previously discussed mechanisms of carcinogenesis, namely, MSI and CpG island methylation. Several potential routes from benign serrated precursors to various types of carcinoma have been proposed, but the best data support the concept that some sessile serrated polyps progress through an intermediate dysplastic step (i.e., sessile serrated polyp with dysplasia) to mismatch repair–deficient adenocarcinomas with BRAF mutations and CIMP. Presumably, sessile serrated polyps with BRAF mutations develop progressive DNA methylation that ultimately inactivates MLH1. Mismatch repair deficiency heralds transformation to a sessile serrated polyp with dysplasia. Additional alterations, such as activation of WNT signaling, occur as late events in the evolution to carcinoma (1). Unlike conventional adenomas, WNT signaling abnormalities in serrated polyps rarely affect APC. Rather, methylation silences WNT antagonists such as soluble frizzled-related protein (SFRP) genes, AXIN2, and MCC, thereby allowing WNT signaling to continue unchecked (102). As previously mentioned, most microvesicular hyperplastic polyps contain BRAF mutations, and one-third to one-half of goblet cell–rich hyperplastic polyps contain KRAS mutations. However, there are no compelling data to suggest either of these polyp types participates in intestinal carcinogenesis. Traditional serrated adenomas with BRAF mutations often contain foci of sessile serrated polyp, but they do not progress via a mechanism that includes DNA methylation and mismatch repair deficiency. KRAS-mutated traditional serrated adenomas show WNT signaling activation and TP53 mutations without mismatch repair deficiency.

MALIGNANT EPITHELIAL NEOPLASMS Pathologists play a critical role in the management of patients with intestinal carcinomas. They provide tumor stage assessment and evaluate carcinomas for a variety of histologic prognostic factors. Some of these are well-established markers of biologic behavior, whereas others have recently gained attention because of their predictive values. NONAMPULLARY CARCINOMAS OF THE SMALL INTESTINE Small intestinal adenocarcinomas arise sporadically, in association with hereditary cancer syndromes, or as a result of chronic inflammatory conditions, such as Crohn disease and celiac disease. They show a striking predilection for the ampulla and are much less common than colorectal carcinomas; nonampullary duodenal adenocarcinomas comprise only 0.3% of gastrointestinal malignancies but account for more than 50% of small intestinal adenocarcinomas. Most tumors are asymptomatic until locally advanced and, thus, the overall prognosis is poor. Presenting symptoms are usually related to luminal obstruction or gastrointestinal bleeding. Adenocarcinomas of the duodenum and jejunum tend to be polypoid, fungating, or annular masses that obstruct the lumen, whereas those associated with Crohn disease often produce strictures. Most are intestinal-type tumors composed of infiltrative glands lined by malignant cells with enlarged, hyperchromatic nuclei; prominent nucleoli; increased mitotic activity; and necrosis (103). Several morphologic variants similar to those that develop in the colorectum have been described and are discussed in detail in subsequent sections (104). Well-differentiated carcinomas have a villoglandular or papillary architecture

and bland cytologic features that may be confused with adenomas when present in superficial biopsy specimens, especially in the setting of Crohn disease. The molecular features of small intestinal adenocarcinomas mirror the spectrum of changes seen in colorectal adenocarcinomas, although the relative numbers of these alterations are different. Nearly 50% of cases show nuclear accumulation of β-catenin with frequent loss of membranous E-cadherin expression. However, APC mutations are less frequently encountered, raising the possibility that WNT signaling pathway abnormalities develop through mechanisms affecting CTNNB1 rather than APC (105). More than twothirds of tumors harbor KRAS mutations in codon 12 (106). Mismatch repair deficiency occurs in 5% to 35% of tumors (104). Alterations affecting SMARC genes underlie some undifferentiated carcinomas with rhabdoid morphology (107). AMPULLARY AND PERIAMPULLARY CARCINOMAS Most (90%) ampullary and periampullary adenocarcinomas arise from intestinal-type adenomas. They typically present at an earlier stage than do nonampullary small intestinal adenocarcinomas because they cause symptoms related to biliary obstruction when they are still relatively small. Affected patients tend to be older adults who present approximately 10 years later than patients with sporadic ampullary adenomas (108). Patients with cancer syndromes, including familial adenomatous polyposis, Lynch syndrome, neurofibromatosis type 1, and MUTYH-associated polyposis, can develop carcinomas at this site (109-116). Ampullary adenocarcinomas arising from the periampullary duodenum are readily evident upon inspection of the duodenal mucosa because they produce ulcerated or fungating masses associated with adenomas (Fig. 34.26A). These lesions should be distinguished from those derived from the intraampullary mucosa, which generally produce more subtle abnormalities in the periampullary mucosa, such as slight elevations, nodularity, or ulcers (Fig. 34.26B). Bisecting the resection specimen along the distal common bile duct reveals either (a) a polypoid nodule that expands the ampullary cavity or (b) a scirrhous tumor that encompass the ampulla without a prominent intraluminal component (Fig. 34.26C). Polypoid intraluminal masses are generally derived from intraampullary papillary-tubular neoplasms, whereas infiltrative tumors are derived from the ampullary ducts (Fig. 34.26D) (117).

FIGURE 34.26 Ampullary adenocarcinoma. A central umbilication surrounded by indurated mucosa (arrow) corresponds to invasive adenocarcinoma arising in a plaque-like intestinal-type adenoma (A). An intraampullary adenocarcinoma produces a nodule that undermines the mucosa (B). Sectioning through the same lesion reveals a gritty stellate tumor that surrounds the distal common bile duct (C). Adenocarcinomas with a prominent intraluminal component are derived from intraampullary papillary-tubular neoplasms (D).

Ampullary carcinomas show intestinal and/or pancreatobiliary differentiation. Carcinomas derived from the periampullary duodenum usually show intestinal-type differentiation with intestinal-type dysplasia in the adjacent mucosa. Pancreatobiliary-type tumors feature small

infiltrating glands composed of cells with cytoplasmic mucin and ovoid, irregular nuclei. Other variants include mucinous adenocarcinoma, poorly cohesive carcinoma, medullary carcinoma, neuroendocrine carcinoma, and adenosquamous carcinoma, as described in subsequent sections (117). Undifferentiated carcinomas with osteoclast-type giant cells and those with a rhabdoid phenotype that show alterations affecting the SWI/SNF chromatin remodeling complex have been described (107,118). The molecular features of ampullary adenocarcinomas overlap with those of nonampullary small-intestinal tumors and carcinomas of the distal common bile duct and pancreas. Intestinal-type ampullary adenocarcinomas frequently express CDX-2 and apomucin MUC2 with variable expression of cytokeratins 7 and 20, similar to other smallintestinal carcinomas. The features of pancreatobiliary-type tumors resemble those of pancreatic ductal adenocarcinomas and bile duct carcinomas with positivity for cytokeratin 7 and a lack of staining for apomucin MUC2 and cytokeratin 20 (119,120). Approximately 65% of ampullary carcinomas harbor TP53 mutations accompanied by abnormalities in p21WAF1/CIP1 (43%), p27Kip1 (79%), p16INK4 (29%), cyclin D1 (29%), and cyclin E (57%). Somatic APC mutations are detected in less than 20% of tumors, and loss of heterozygosity affecting chromosome 5q occurs in less than 50% of cases (121). MSI has been variably reported among ampullary carcinomas and is detected at rates comparable to those seen in colorectal carcinomas (122). COLORECTAL CARCINOMAS Colorectal carcinoma represents a global health issue, with nearly two million new cases each year (123). Rates are highest in industrialized countries and seem to be increasing among young adults (124). Risk factors include sedentary lifestyle, consumption of red meat, alcohol use, and obesity; dietary fiber and prolonged use of nonsteroidal antiinflammatory agents decrease risk (125,126). Smoking is associated with carcinomas of the proximal colon, probably reflecting its role in the development of serrated neoplasms. Risk is also increased among patients with a variety of cancer syndromes, as listed in Table 34.4. Cases are classified based on location as right-sided (i.e., cecum, ascending colon, and transverse colon), left-sided (i.e., splenic flexure to sigmoid colon), and rectal. Most tumors develop in the left colon and/or the rectum, although the overall incidence of tumors in the distal colon is decreasing as a result of endoscopic screening and surveillance. TABLE 34.4 Features of Syndromes Associated With Increased Risk for Intestinal Carcinoma Syndrome

Gene

Inheritance

Li-Fraumeni syndrome

TP53

Autosomal dominant

Lynch syndrome

MLH1, PMS2, MSH2, MSH6

Autosomal dominant

Polyp Types

CANCERS WITH NO OR FEW POLYPS

CANCERS WITH PREDOMINANTLY HAMARTOMAS

Adenomas, if present

Juvenile polyposis syndrome

SMAD4, BMPR1A

Autosomal dominant

Hamartomas

Peutz-Jeghers syndrome

LKB1/STK11

Autosomal dominant

Hamartomas

Cowden syndrome

PTEN, PTCH (SDHB/SDHD)

Autosomal dominant

Hamartomas, adenomas, lipomas, ganglioneuromas

CANCERS WITH PREDOMINANTLY ADENOMAS Familial adenomatous polyposis

APC

Autosomal dominant

Adenomas

Attenuated familial adenomatous polyposis

APC

Autosomal dominant

Adenomas

MUTYH-associated polyposis

MUTYH

Autosomal recessive

Adenomas, serrated polyps

Polymerase proofreadingassociated polyposis syndrome

POLE, POLD1

Autosomal dominant

Adenomas

Hereditary mixed polyposis syndrome

GREM1

Autosomal dominant

Adenomas, serrated polyps, hamartomas

NTHL1-associated tumor syndrome

NTHL1

Autosomal recessive

Adenomas

MSH3-associated polyposis

MSH3

Autosomal recessive

Adenomas

MLH3-associated polyposis

MLH3

Autosomal recessive

Adenomas

Presenting symptoms depend on the anatomic location of the tumor and stage of disease. Early lesions are asymptomatic or present with occult blood loss. Tumors of the proximal colon manifest with iron deficiency anemia or gastrointestinal bleeding; they generally achieve a large size before producing obstructive symptoms. Tumors of the distal colorectum cause abdominal pain, small caliber stools, and hematochezia. Paradoxical diarrhea occurs with obstructing lesions prevent passage of formed stools, allowing only liquid stools to be eliminated. Carcinomas arising from large villous adenomas may produce mucoid diarrhea. Most colorectal carcinomas form exophytic luminal masses with extension into the bowel wall. They may be coated with thick mucus or show central depressions with indurated or rolled borders (Fig. 34.27). They generally grow vertically in the bowel wall rather than laterally, and thus, clear margins are attainable when tumors occur in the abdominal colon. Close margins are most problematic when dealing with low-lying rectal carcinomas. Tumors with abundant mucin often have a gelatinous appearance, and those composed of sheets of tumor cells with little intervening stroma have a fleshy, variegated surface. Carcinomas associated with inflammatory bowel disease may show infiltrative growth in the wall, producing strictures rather than intraluminal growth.

FIGURE 34.27 Colonic adenocarcinoma. Adenocarcinomas of the colorectum grow as exophytic masses with central crater-like ulcers and thick rolled borders (A). Carcinomas tend to grow vertically in the bowel wall, infiltrating the deeper layers and obliterating preexisting tissue planes without extensive lateral spread (B). In this example, the layers of the colonic wall are preserved adjacent to the tumor.

Invasive colorectal carcinoma is defined by the presence of infiltrating tumor cells beyond the level of the muscularis mucosae; tumor cells limited to the mucosa are not considered to represent invasive adenocarcinomas and are assigned a pathologic stage of Tis. Most colorectal carcinomas are adenocarcinomas with intestinal differentiation. They contain large angulated or fused glands lined by cells with round or ovoid nuclei and coarse, heterogeneous chromatin. Numerous mitotic figures and abundant necrotic cellular debris are readily identified. Intestinal-type adenocarcinomas are often associated with a desmoplastic stromal reaction, particularly at the advancing tumor edge (Fig. 34.28A). However, some tumors display an “adenoma-like” growth configuration with striking villous morphology, low-grade cytologic features, and minimal desmoplasia (127).

FIGURE 34.28 Morphologic variants of colorectal adenocarcinoma. Invasive intestinal-type adenocarcinomas feature infiltrative malignant glands intimately associated with desmoplastic stroma; single tumor cells are present at the advancing tumor edge (A). A low-grade mucinous adenocarcinoma is composed of large mucin lakes that contain strips and clusters of tumor cells (B). Signet-ring cell carcinoma consists of poorly cohesive groups of cells with large mucin vacuoles and eccentric nuclei (C). Medullary carcinomas are composed of irregular cords and thick nests of tumor cells embedded in lymphocyte-rich stroma. Numerous tumor-infiltrating lymphocytes are readily apparent (D). Serrated adenocarcinomas contain infiltrating glands with a serrated appearance. Tumor cells with brightly eosinophilic cytoplasm are present in small nests and clusters at the advancing edge (E). A micropapillary adenocarcinoma is composed of rounded aggregates of tumor cells in lacunar spaces (F).

Adenocarcinomas are graded based on the extent of glandular differentiation (128). Carcinomas with more than 95% glandular elements have been historically classified as well differentiated, whereas those with 50% to 95%, 5% to 49%, and less than 5% gland

formation were classified as moderately, poorly, and undifferentiated carcinomas, respectively. Current schemes utilize a two-tier grading system based on the presence of at least 50% glandular differentiation, which improves reproducibility (129). Although some authors suggest that the poorly differentiated areas at the advancing tumor edge should not be considered when assigning tumor grade, these recommendations are not based on any data (123). Recent observations regarding the importance of solid cell nests and tumor budding at the advancing tumor edge indicate that failure to assess the tumor front underestimates biologic risk. Importantly, grading criteria based on the extent of gland formation are applicable only to intestinal-type adenocarcinomas. MORPHOLOGIC VARIANTS OF INTESTINAL CARCINOMA Low-Grade Tubuloglandular Adenocarcinoma This variant is an extremely well-differentiated intestinal-type adenocarcinoma described exclusively in the setting of inflammatory bowel disease (130). Tumors contain scattered tubular glands lined by remarkably bland epithelial cells. Infiltrating glands do not elicit a striking desmoplastic reaction. Low-grade tubuloglandular carcinomas have a higher prevalence of IDH1 mutations than do conventional adenocarcinomas (131). Mucinous Adenocarcinoma Approximately 10% of intestinal carcinomas are mucinous adenocarcinomas. Although they are defined by the presence of extracellular mucin accounting for at least 50% of the tumor volume, this designation is arbitrary and the extent of mucinous differentiation is not predictive of outcome (123,132). Mucinous adenocarcinomas typically display expansile growth with a circumscribed advancing front. They contain strips, clusters, or singly arranged tumor cells floating in mucin pools (Fig. 34.28B). Some tumors display singly dispersed tumor cells and signet-ring cells floating in mucin pools; these lesions are classified as mucinous adenocarcinomas, rather than signet-ring cell carcinomas, and generally behave more aggressively than mucinous tumors that contain cohesive epithelial cells (123,133). Neoplasms composed of distended, mucin-filled glands should be classified as intestinal adenocarcinomas, rather than mucinous adenocarcinomas. Although mucinous adenocarcinomas have been historically classified as high-grade neoplasms, their behavior is determined by underlying molecular abnormalities, particularly mismatch repair status (134). Nearly 50% of mucinous adenocarcinomas are mismatch repair-deficient, in which case they have a better prognosis than stage-matched, mismatch repair–proficient mucinous adenocarcinomas (132,135). However, mismatch repair status is no longer incorporated into grade assessment of mucinous adenocarcinomas (136). Signet-Ring Cell Carcinoma Intestinal carcinomas that show at least 50% signet-ring cell differentiation are classified as signet-ring cell carcinomas. This subtype accounts for less than 1% of intestinal carcinomas and is more prevalent among patients less than 40 years of age, particularly those with inflammatory bowel disease (124,137). Signet-ring cell carcinomas are composed of dyshesive, medium-to-large cells with abundant mucinous cytoplasm and eccentric, hyperchromatic nuclei. They may feature single infiltrating signet-ring cells reminiscent of diffuse-type gastric carcinoma or infiltrative nests of globoid mucin-containing cells unaccompanied by a desmoplastic stromal response (Fig. 34.28C). The latter pattern is

often present in combination with mucinous adenocarcinoma. As previously noted, signetring cells confined to mucin pools are classified as mucinous adenocarcinomas, rather than signet-ring cell carcinomas (123,133). Approximately one-third of signet-ring cell carcinomas are mismatch repair-deficient; most of these are associated with mucinous adenocarcinomas. Signet-ring cell differentiation is a poor prognostic factor among both mismatch repair–proficient and mismatch repair– deficient carcinomas, even when it accounts for a small (e.g., 10%) amount of the tumor volume (133,134,138). For this reason, signet-ring cell differentiation warrants designation as a high-grade carcinoma (136). Medullary Carcinoma Medullary carcinomas are solid tumors with broad-front, pushing invasion and minimal gland formation (139). Tumor cells are arranged in syncytial nests and cords intimately associated with lymphoid stroma and intraepithelial lymphocytes. Lymphoid aggregates are usually present at the advancing tumor edge. Tumor cells contain eosinophilic or amphophilic cytoplasm and large vesicular nuclei with prominent nucleoli (Fig. 34.28D). Criteria distinguishing medullary carcinoma from poorly differentiated intestinal-type adenocarcinoma with solid growth are not well established, and there is a high degree of interobserver variability with respect to the classification of solid colorectal tumors (140,141). Most medullary carcinomas are mismatch repair-deficient. Serrated Adenocarcinoma Serrated adenocarcinomas account for less than 10% of all intestinal carcinomas. They are composed of infiltrative serrated glands lined by epithelial cells with clear or eosinophilic cytoplasm, vesicular nuclei, and prominent nucleoli (Fig. 34.28E). Mucinous differentiation and tumor budding are commonly present at the advancing tumor edge (142). Serrated adenocarcinomas are associated with a poor prognosis when they occur in the left colon; these are usually mismatch repair-proficient and show various combinations of BRAF or KRAS mutations and DNA hypermethylation (143). Only 20% of serrated adenocarcinomas show BRAF mutations, CpG island hypermethylation, and mismatch repair deficiency. These tumors have a better prognosis than neoplasms with KRAS mutations or those that are BRAF mutated with mismatch repair proficiency (1,142). Micropapillary Adenocarcinoma Approximately 10% of intestinal carcinomas contain foci of micropapillary differentiation (144). Micropapillary carcinomas feature clusters or balls of tumor cells within lacunar spaces that are unaccompanied by supportive stroma (Fig. 34.31F). The tumor cells characteristically display reversed polarity with their apical surfaces oriented toward the periphery of the cell cluster. They contain abundant eosinophilic or pale cytoplasm and display high-grade cytologic features. Lacunar spaces reflect a retraction artifact, although most micropapillary carcinomas also show lymphovascular invasion. Micropapillary carcinomas are associated with a worse prognosis than that of stage-matched advanced intestinal adenocarcinomas, even when this component accounts for less than 10% of the tumor volume. Enteroblastoma Biphasic tumors similar to gastroblastoma rarely occur in the small intestine. Clinical data regarding these tumors are extremely limited, but they seem to occur among men and

women, affecting young and older adults (145). Like their gastric counterparts, enteroblastomas arise subjacent to the mucosa, creating a polypoid mass that may cause intermittent abdominal pain or bleeding (Fig. 34.29A). Cases encountered to date show either MALAT1-GLI1 fusions similar to gastroblastomas or ACTB-GLI1 fusions. The former has also been described in plexiform fibromyxomas (146).

FIGURE 34.29 Enteroblastoma of the small intestine. This well-circumscribed, unencapsulated tumor is centered on the submucosa and has a mostly homogeneous cut surface (A). The submucosa is expanded by irregular sheets and aggregates of tumor cells (B) embedded in a densely collagenous stroma rather than the desmoplastic reaction typically seen in adenocarcinomas (C). Nests and ribbons of round tumor cells contain abundant clear cytoplasm and smooth nuclei reminiscent of an endocrine tumor (D). Case courtesy of Dr. Yongmei Diana Yin, Department of Pathology, NewYork Presbyterian Brooklyn Methodist Hospital.

Enteroblastomas show multinodular growth of epithelioid cells arranged in sheets, nests, and tubules intimately associated with plump spindled cells (Fig. 34.29B). Cellular areas are separated by broad bands of collagenous stroma (Fig. 34.29C). The epithelioid cells contain round nuclei with coarse chromatin comparable to nuclei of endocrine cells, but they are negative for endocrine markers (Fig. 34.29D). Tumor necrosis and vascular invasion can be observed. Tumors show an immunophenotype similar to that of gastroblastomas with diffuse vimentin and CD56 staining, as well as patchy CD10 positivity. The epithelioid cells are positive for cytokeratins, claudin-4, and EMA. Spindled cells are negative for myoepithelial markers, CD117, DOG-1, CD34, and S100 protein. Other Variants

Less than 10% of intestinal adenocarcinomas are classified as adenosquamous, neuroendocrine, squamous cell, and undifferentiated carcinomas (123). Undifferentiated carcinomas may contain cells with spindled or rhabdoid morphology and abundant eosinophilic cytoplasm with eosinophilic inclusions accompanied by multinucleated tumor giant cells. A subset of carcinomas composed of rhabdoid cells harbor genetic alterations affecting the SWI/SNF family of proteins, namely, SMARCA4, SMARCA2, and SMARCB1 (107). Rare cases resembling choriocarcinoma can produce human chorionic gonadotropin (Fig. 34.30A). Some intestinal carcinomas contain clear cells with prominent subnuclear vacuoles or nested cells with eosinophilic cytoplasm, resembling hepatoid or yolk sac carcinoma (Fig. 34.30B).

FIGURE 34.30 Variants of colorectal carcinoma. Occasional high-grade colorectal carcinomas resemble choriocarcinoma and contain sheets of large polygonal cells with eosinophilic cytoplasm accompanied by hemorrhage and necrosis (A). Clear cell variants contain subnuclear vacuoles and eosinophilic inclusions reminiscent of the embryonic yolk sac (B).

PROGNOSTICALLY IMPORTANT HISTOLOGIC FEATURES Lymphovascular and Small Vein Invasion Lymphatic channels are normally present in all layers of the intestinal wall, including the full thickness of the small intestinal mucosa and the deepest aspect of the colonic mucosa. Intestinal carcinomas induce lymphangiogenesis in and around the tumor and the density of these vessels increases with depth of invasion. The likelihood of lymphatic vessel invasion and lymph node metastasis increases with more deeply invasive tumors. Both lymphovascular and small vein invasion are poor prognostic factors among intestinal carcinomas. Small vessel invasion appears as single or clustered tumor cells within endothelium-lined spaces (Fig. 34.31A). Lymphovascular invasion can be obscured by the destructive nature of infiltrating malignant glands, so its presence may be better appreciated at the advancing edge of the tumor or in the adjacent mucosa. Ancillary immunostains for D240 or other endothelial markers may facilitate detection of vascular invasion in some cases.

FIGURE 34.31 Prognostically important histologic features of colorectal carcinoma. The presence of tumor cell clusters in lymphatic spaces is an independent poor prognostic factor (A). Venous invasion may be less conspicuous when tumor cells obliterate the vein wall. Clues to the presence of vascular invasion include the presence of unpaired arteries (right upper corner) adjacent to tumor nodules (B). The presence of tumor cells within the perineurium of a peripheral nerve is associated with decreased survival (C). Tumor budding, defined as single cells or small (≤4 cells) clusters of tumor cells, is associated with an increased likelihood of regional lymph node metastases. Larger solid cell clusters frequently accompany tumor budding and are also associated with biologically aggressive behavior (D).

Large Vein Invasion Large vein invasion is an independent poor prognostic feature of intestinal carcinomas, especially when present in soft tissue outside the bowel wall (147). In this situation, venous invasion appears as small nodules that are discontinuous with the primary tumor. Detection can be improved by an understanding of microanatomy. Intestinal arteries are normally accompanied by comparably sized veins as they traverse the bowel wall. Detection of “naked arteries” adjacent to tumor nodules is a clue to the presence of large vein invasion; tumor cells grow out of the adjacent vein and destroy its wall (Fig. 34.31B). Elastin stains can facilitate detection of extramural venous invasion. Perineural Invasion Approximately 10% of intestinal carcinomas show perineural invasion, often in combination with other high-risk features (Fig. 34.31C). Its presence is independently associated with regional lymph node metastases and decreased survival, as well as a more than 2.5-fold increased risk for local recurrence among patients with tumors localized to the intestine (i.e., stage II) at the time of diagnosis (148).

Tumor Budding and Poorly Differentiated Clusters Tumor budding is defined by the presence of single infiltrating cells or groups of up to four neoplastic cells intimately associated with larger invasive glands. This feature is most pronounced at the invasive front of the tumor where the neoplastic cells assume an elongated shape and merge imperceptibly with surrounding stroma, a finding that has been termed “epithelial-mesenchymal transformation.” Tumor budding reflects a loss of intercellular junctions and reversion to a stem cell phenotype with expression of CD133, LGR5, and other stem cell markers. It is more common among mismatch repair–proficient tumors than mismatch repair–deficient neoplasms and predicts aggressive behavior among both types of tumors (149,150). Tumor budding is associated with increased risk for regional lymph node metastases when detected in polypectomy specimens (151). It is classified as low (0-4 buds), intermediate (5-9 buds), and high-grade (≥10 buds) by assessing the number of individual cells and small clusters per 0.785 mm2 in the area of maximal budding, which is usually at the advancing tumor edge. Cytokeratin immunohistochemistry may facilitate evaluation when tumors contain a striking peritumoral inflammatory cell infiltrate or desmoplasia. Similarly, slightly larger nests of five or more neoplastic cells are also associated with a poor prognosis (149). The extent of these poorly differentiated clusters can be graded using a three-tier system similar to that proposed for assessing tumor budding. However, poorly differentiated clusters are usually detected in carcinomas that also contain tumor buds, suggesting that the distinction between tumor budding and larger cell clusters is essentially an academic exercise (Fig. 34.31D). Host Immune Response to Tumor Patients who mount an immune response to their tumors have an improved survival compared with those who lack this finding. The two most common patterns of host immune response include a Crohn-like lymphoid reaction at the advancing tumor edge and tumorinfiltrating lymphocytes within the neoplastic epithelium. Although both features are more common among mismatch repair–deficient tumors, they are independently associated with improved outcome, regardless of mismatch repair status. The Crohn-like lymphoid response to intestinal carcinomas features at least three lymphoid aggregates at the periphery of the advancing tumor edge, often with germinal centers (Fig. 34.32A). This finding is associated with a decreased likelihood of regional lymph node metastases and improved 10-year survival. Tumor-infiltrating lymphocytes represent an immune response to neoantigens produced by cancer cells (Fig. 34.32B). They are considered to be increased when at least two are seen interdigitating between tumor cells in a high-power field (×400 magnification) (152). The extent of tumor-infiltrating lymphocytes is classified as absent, mildly increased (two per ×400 magnification field), or markedly increased (three or more per ×400 magnification field). Most mismatch repair–deficient carcinomas contain high numbers of tumor-infiltrating lymphocytes, although they tend to be less prominent in tumors that show mucinous differentiation.

FIGURE 34.32 Host immune response to colonic carcinoma. A colonic carcinoma with a broad pushing front is rimmed by lymphoid tissue and rounded lymphoid aggregates reminiscent of the tissue reaction seen in patients with Crohn disease (A). Tumor-infiltrating lymphocytes are numerous in a mismatch repair–deficient carcinoma (B).

FIGURE 34.33 Neoadjuvantly treated rectal carcinoma. Treated rectal carcinomas often show regression of viable tumor and replacement by fibrosis or extracellular mucin pools (A). Residual tumor cell nests contain increased numbers of endocrine cells (B).

NEOADJUVANTLY TREATED CARCINOMAS Locally advanced rectal cancers are routinely treated with neoadjuvant therapy to improve resectability and postsurgical quality of life. Most treated tumors show some type of therapeutic response with decreased amounts of viable carcinoma accompanied by mural fibrosis, dystrophic calcifications, or pools of extracellular mucin (Fig. 34.33A). Residual viable tumor cells may show features of endocrine differentiation or marked cytologic atypia (Fig. 34.33B). Tumor grade and carcinoma subtype are not assigned in the setting of neoadjuvant therapy. The extent of tumor regression is predictive of prognosis (153). Tumors with an extensive therapeutic response are associated with improved disease-free survival compared with those that show little or no response to therapy. Extent of regression is evaluated using a four-tier quantitative system: complete response, near-complete response, partial response, or poor response (44). Assessment of tumor regression for staging purposes should be performed on the primary tumor; lymph node deposits are not considered when grading tumor regression. Nonviable tumor and extracellular mucin do not contribute to pathologic stage assignment.

TUMOR STAGING Tumor stage is the most powerful predictor of outcome among patients with intestinal carcinomas. Lymph node–negative (i.e., stages I and II) tumors are generally treated with surgery alone, whereas most patients with metastases to regional lymph nodes or distant sites (i.e., stage III or IV) are considered for adjuvant chemotherapy. The Tumor, Node, Metastasis (TNM) staging system of the American Joint Committee on Cancer (AJCC) and the Union for International Cancer Control (UICC) provide standardized criteria for cancer reporting (43). The T category denotes pathologic tumor stage and describes the deepest point of penetration in the intestinal wall, surrounding fat, or adjacent structures. Tumors confined to the submucosa are staged as pT1, whereas tumors that extend into the muscularis propria or subserosal or adventitial connective tissue are classified as pT2, or pT3, respectively. The pT4 category is subdivided into pT4a and pT4b to denote penetration of the visceral peritoneum and invasion noncontiguous organs or structures, respectively. Distinction between pT4a and pT4b remains a significant diagnostic challenge and relies upon careful gross examination, meticulous sampling, and a clear understanding of microanatomy. Puckered or sclerotic areas on the serosa, creeping fat, plaques, and fibrinous adhesions overlying the tumor are all clues to the presence of serosal penetration by tumor (Fig. 34.34). Serosal penetration is readily recognized when tumor cells on the peritonealized surface are accompanied by mesothelial cell hyperplasia, fibrin deposits, erosion of the mesothelium, or inflammation. However, the biologic risk of cancers within 1 mm of a serosal reaction (i.e., granulation tissue, fibrin deposits, or mesothelial hyperplasia) is greater than that of pT3 tumors more than 1 mm from the serosa and approximates the risk of pT4a tumors (154). For these reasons, carcinomas that communicate with the peritoneal surface through inflammation are classified as pT4a. Many pathologists also assign pT4a to carcinomas less than 1 mm from the serosa when accompanied by a serosal reaction featuring fibrin, granulation tissue, or inflammation (154).

FIGURE 34.34 Serosal penetration by colonic carcinoma. A circumferential carcinoma is associated with creeping fat overlying the antimesenteric aspect of the colon in an area of serosal penetration (A). Cross sections through a large colonic carcinoma demonstrate tumor invading adherent omental fat (arrow) and extending to the serosal surfaces on the antimesenteric aspect (B). Sections through the interface between the tumor and pericolic fat demonstrate broad inflammatory septa containing tumor cells on the peritoneal surfaces (C). The presence of tumor cells on the peritonealized surfaces of the pericolic fat warrants assignment of the pT4a designation (D).

Regional lymph nodes are staged as pN0, pN1, or pN2 depending upon the number involved by carcinoma (44). Nonregional lymph nodes containing metastases are staged as pM1 disease. The estimated 5-year survival rate for patients with at least 18 negative lymph nodes is 76%, compared with 62% among patients with less than or equal to 7 detected lymph nodes. The likelihood of detecting metastatic disease increases as more lymph nodes are identified. For these reasons, at least 12 lymph nodes are required to adequately stage untreated carcinomas. Lymph nodes may be more difficult to detect in resection specimens from neoadjuvantly treated patients, but most of these cases also contain at least 12 lymph nodes. Lymph node metastases measuring less than 0.2 mm are currently classified in the N0 category, although this finding is still associated with decreased disease-free survival intervals (44,155). Tumor deposits are discrete nodules with smooth or irregular contours located within the regional lymph node basin. By definition, they cannot be recognized as lymph node metastases or vascular invasion, although most examples represent entirely effaced lymph nodes or lymphovascular, venous, or perineural invasion with extension into soft tissue and obliteration of the underlying structures. Tumor deposits are associated with a poor prognosis even when numerous lymph node metastases are also present. Given that

cancers associated with tumor deposits have a prognosis comparable to stage III tumors, tumor deposits are reported in the N category when all other lymph nodes are negative in order to upstage stage II patients to stage III disease and consider them for potential adjuvant chemotherapy. The “N1c” category is used in this situation and facilitates long-term follow-up of affected patients in multiinstitutional studies (44). Notably, the N1c category is used only when tumor deposits are detected in patients without metastases in regional lymph nodes, and this designation does not imply a more advanced stage than N1a or N1b. The number of tumor deposits should not be added to the total number of positive lymph nodes. Distinction between completely replaced lymph nodes and tumor deposits is subjective and reproducibility is poor (156). Round shape, peripheral rim of lymphoid tissue, capsule, and subcapsular sinuses are all more typical of effaced lymph nodes (156). However, other explanations should also be considered once the possibility of a lymph node metastasis has been excluded. Tumor nodules close to (5-10 cm

Moderate

>10 cm

High

≤5 cm

High

5-10 cm

High

>10 cm

High

5 MITOTIC FIGURES/5 MM2

5 MITOTIC FIGURES/5 MM2

Reprinted from Miettinen M, Lasota J. Gastrointestinal stromal tumors: pathology and prognosis at different sites. Semin Diagn Pathol. 2006;23(2):70-83. Copyright © 2006 Elsevier. With permission.

Approximately 85% of gastrointestinal stromal tumors have gain-of-function mutations affecting KIT (75%) or PDGFRA (10%), both of which encode type III receptor tyrosine kinases (218,222,223). These mutations result in constitutively activated cell signaling in the absence of ligand binding to the receptor. Two-thirds of KIT mutations affect exon 11, followed in frequency by mutations in exon 9; the remainder occur in exons 13 and 17. Mutations in PDGFRA usually occur in exon 18 (8%), with less frequent mutations in exons 12 and 14. Mutations affecting tyrosine kinase receptors are followed by chromosomal losses affecting tumor-suppressor genes. Tumors that are wild-type for both of these genes typically harbor germline SDHA, SDHB, SDHC, or SDHD mutations or show somatic SDHC promoter methylation (224). Most gastrointestinal stromal tumors have an epicenter in the muscularis propria. They are circumscribed with bulging, heterogeneous cut surfaces; hemorrhage and cystic degeneration are more commonly present in large tumors (Fig. 34.51). Tumors of the intestines are usually composed of short intersecting fascicles of uniform spindled cells. They contain abundant eosinophilic cytoplasm and blunt-ended or ovoid nuclei. Aggregates

of collagen (i.e., skenoid fibers) may be present, especially in clinically benign tumors of the small bowel (Fig. 34.52A). Epithelioid cells arranged in a nested or organoid manner occasionally predominate, especially in SDH-deficient tumors (Fig. 34.52B).

FIGURE 34.51 Gastrointestinal stromal tumor of the jejunum. This high-risk tumor is large with heterogeneous cut surfaces, areas of hemorrhage, and cystic degeneration.

FIGURE 34.52 Gastrointestinal stromal tumors of the small intestine. A spindled cell tumor contains short fascicles of tumor cells with elongated nuclei and eosinophilic cytoplasm. Although the lesion is quite cellular, mitotic activity is negligible. Amorphous skenoid fibers (arrow) are typically seen in small intestinal tumors (A). Another case features sheets of epithelioid cells with indistinct cell borders and uniform round nuclei (B).

Nearly all cases occurring in the small intestine and colorectum show strong diffuse staining for DOG1 and CD117. Other markers that are less commonly expressed include BCL-2 (80%), CD34 (75%), smooth muscle actin (30%), S100 protein (10%), and desmin (5%); rare tumors show weak patchy staining for cytokeratins (225). The extent and pattern of CD117 staining do not correlate with the nature of the underlying mutation or the likelihood of response to imatinib. However, tumors with weak, focal, or negative staining are more likely to harbor PDGFRA mutations. Dedifferentiation can occur de novo in gastrointestinal stromal tumors or following imatinib therapy. Dedifferentiated areas contain anaplastic-appearing cells or other types of

differentiation, including rhabdomyosarcomatous, angiosarcomatous, and leiomyosarcomatous elements (226). Advanced tumors are generally treated with imatinib or other targeted agents. These drugs induce regressive changes characterized by hypocellularity, hyalinization, and necrosis with decreased or absent CD117 staining. SMOOTH MUSCLE NEOPLASMS Leiomyoma Leiomyomas generally arise from the muscularis mucosae of the colon and rectum and tend to be small. Intramural leiomyomas are much rarer and tend to be a bit larger. Leiomyomas are well-circumscribed, unencapsulated nodules that usually span only a few millimeters. They are composed of paucicellular proliferations of smooth muscle cells arranged in short intersecting fascicles (Fig. 34.53). They lack mitotic activity and cytologic atypia. Immunohistochemical stains demonstrate strong, diffuse staining for desmin and actins; the tumor cells are negative for CD117 and DOG1 (227).

FIGURE 34.53 Leiomyoma of the muscularis mucosae. The tumor obscures the muscularis mucosae and contains fascicles of smooth muscle cells without intervening stroma (A). Smooth muscle cells contain bland, uniform nuclei (B).

Leiomyosarcoma Approximately 80% of gastrointestinal leiomyosarcomas occur in the small intestine or colon (227). They occur equally among men and women and show a predilection for older adults. Leiomyosarcomas are biologically aggressive with a high risk for local recurrence and metastasis, especially when they are larger than 5 cm. Leiomyosarcomas produce polyps or bulky, heterogeneous masses. They are quite cellular and consist of long, intersecting fascicles of spindled cells with eosinophilic cytoplasm and blunt-ended, elongated nuclei. Tumor nuclei contain coarse chromatin and prominent nucleoli. Mitotic figures are often numerous, and coagulative necrosis may be present. These tumors are immunopositive for actins and desmin and negative for CD117 and DOG1. FIBROBLASTIC/MYOFIBROBLASTIC LESIONS Desmoid Fibromatosis Intraabdominal fibromatoses arise in the mesentery of the small bowel or colon. They afflict both men and women with near-equal frequency and tend to occur in younger adult

patients. Most cases are sporadic, although 10% to 15% occur in the setting of familial adenomatous polyposis (228). Desmoid tumors are locally aggressive and often recur, but they do not metastasize. Tumors develop through activation of WNT signaling. Those associated with familial adenomatous polyposis have APC mutations and somatic cases generally result from CTNNB1 mutations, although some sporadic tumors harbor APC mutations (229). Rare cases are wild type for both genes and have other alterations that affect WNT signaling. Tumors are often quite large with infiltrative edges and a firm gritty surface (Fig. 34.54A). They consist of long, sweeping fascicles of spindled cells with tapered nuclei and scant eosinophilic cytoplasm intimately associated with thin tendrils of extracellular collagen (Fig. 34.54B). Desmoid fibromatoses often contain scattered thick-walled vessels surrounded by extravasated red blood cells; occasional keloidal collagen or areas of hyalinization may be present (230). Mesenteric fibromatoses show strong nuclear β-catenin staining and are negative for CD117.

FIGURE 34.54 Desmoid fibromatosis. Tumors have tan-white homogeneous cut surfaces with infiltrative edges that encroach on the bowel wall (A). They contain long, sweeping fascicles of spindled cells that intersect obliquely. Tumor cells are intimately associated with fine extracellular collagen and contain faintly eosinophilic cytoplasm with thin tapering nuclei (B).

Inflammatory Fibroid Polyp Inflammatory fibroid polyps are generally sporadic, although they can develop in patients with germline PDGFRA mutations, in which case they may be multiple (231). Most tumors of the small intestine harbor PDGFRA mutations affecting exon 12 and span less than 5 cm (232). Tumors are centered in the submucosa and have a slightly myxoid cut surface (Fig. 34.60A). They contain a disorderly proliferation of spindled and stellate cells embedded in loose stroma (Fig. 34.60B). The tumor cells have bland cytologic features and scant, faintly eosinophilic cytoplasm. They may show condensation around medium-sized blood vessels. The stroma of myxoid tumors is often infiltrated by numerous eosinophils and scattered mononuclear cells, whereas those with a greater degree of hyalinization typically contain less prominent inflammatory cell infiltrates (233). The spindled cells may show immunopositivity for vimentin, actins, and CD34. Scattered CD117-positive mast cells are frequently present, but the lesional cells are negative for this marker (Fig. 34.55).

FIGURE 34.55 Inflammatory fibroid polyp. This small intestinal lesion is located in the submucosa and has a myxoid cut surface (A). Loose fascicles of bland spindled cells are embedded in myxoid stroma rich in inflammatory cells, including eosinophils (B).

Benign Fibroblastic Polyp (Perineurioma) These mucosa-based lesions are usually encountered during colonoscopic examinations performed in older adults and show a slight female predilection. Most are sessile polyps of the rectosigmoid colon (234). Benign fibroblastic polyps feature a uniform population of bland spindled cells confined to the lamina propria. Spindled cells often show condensation around crypts, which may be normal appearing, dilated, or serrated (235). Slightly more than two-thirds of cases contain serrated crypts reminiscent of sessile serrated polyps or microvesicular hyperplastic polyps; these lesions harbor BRAF mutations confined to the epithelial component (Fig. 34.7). Benign fibroblastic polyps containing nonserrated crypts feature similar-appearing spindled cells, but the epithelium lacks BRAF mutations (Fig. 34.56). Their nature is not entirely clear, but they may represent a heterogeneous group of lesions composed of benign fibroblastic neoplasms or inflammatory-type polyps.

FIGURE 34.56 Benign fibroblastic polyp. This lesion contains mostly normal-appearing crypts separated by a spindled cell proliferation that expands the lamina propria and shows pericryptal accentuation. The spindled cells are plump and cytologically bland.

The spindled cells of benign fibroblastic polyps commonly show patchy, often weak immunostaining for EMA, GLUT-1, and/or claudin-1, leading some authors to classify them as perineuriomas (236). However, this practice is discouraged for several reasons. First, the evidence supporting this hypothesis is entirely circumstantial, being largely based on shared immunohistochemical features between the spindled cell components of these polyps and soft-tissue perineuriomas. It is worth noting that the normal colonic mucosa does not contain myelinated nerves or perineural cells and, thus, it would be highly unlikely that the least common soft-tissue nerve sheath tumor is the most common nerve sheath tumor of the colon. Rather, it is more likely that the spindled cell proliferation of benign fibroblastic polyps represents a reactive proliferation of pericryptal fibroblasts resulting from interactions with the epithelium. Most importantly, classification of these lesions based on their mesenchymal components could result in inappropriate management of affected patients. Benign fibroblastic polyps that contain serrated crypts are probably best considered to represent serrated polyps for purposes of surveillance, as they are often associated with other serrated lesions and adenomas, and likely have a biologic risk comparable to that of serrated polyps lacking cellular stroma (237). Calcifying Fibrous Tumor Calcifying fibrous tumors are unusual mural or subserosal nodules that can occur in a variety of locations but show a predilection for the stomach and small intestine. They tend to occur in adults, in whom they are discovered during abdominal imaging, or produce vague abdominal symptoms. Tumors generally span less than 5 cm and are composed of circumscribed, but unencapsulated, aggregates of hyalinized collagen and scattered spindled cells with a vaguely storiform architecture. These paucicellular lesions often display dystrophic calcifications and peripheral lymphoid aggregates (Fig. 34.57). Their pathogenesis is unknown. Some cases contain IgG4-positive plasma cells, raising the possibility that they result from immune-mediated injury (238). Others display genome-wide methylation patterns similar to those of inflammatory myofibroblastic tumors, suggesting a

relationship between these entities (239). Importantly, calcifying fibrous tumors are not associated with any known syndromes and are cured by excision.

FIGURE 34.57 Calcifying fibrous tumor. Aggregates of hyalinized collagen and scattered spindled cells are associated with dystrophic calcifications.

Inflammatory Myofibroblastic Tumor Most inflammatory myofibroblastic tumors of the gastrointestinal tract affect the intestines. They show a predilection for young adults, and variants that feature a prominent epithelioid cell component are more common among men. Similar to other mesenchymal tumors, inflammatory myofibroblastic tumors can produce symptoms related to a mass effect, but they may also cause fever, leukocytosis, anemia, and hypergammaglobulinemia (240). Approximately two-thirds of cases harbor tyrosine kinase receptor gene rearrangements, particularly involving ALK 2p23. Approximately 5% of cases have ROS1 fusions; less common abnormalities affect NTRK3, PDGFRB, and RET (233,241,242). Tumors are composed of variably cellular fascicles of spindled cells with bland nuclear features and pale cytoplasm. The stroma is usually myxoid or sclerotic and contains a mononuclear cell–rich inflammatory infiltrate composed of lymphocytes and plasma cells; granulocytes are not prominent (Fig. 34.58). Epithelioid inflammatory myofibroblastic tumors contain polygonal cells with eosinophilic cytoplasm and round nuclei with prominent nucleoli, often accompanied by numerous stromal neutrophils (243). Immunohistochemical stains are positive for actins with frequent desmin (50%) and/or keratin (30%) staining. Most cases behave in a benign manner; however, death can occur from recurrent tumors (244).

FIGURE 34.58 Inflammatory myofibroblastic tumor. Broad sweeping fascicles of uniform spindled cells are associated with mononuclear cell–rich inflammatory infiltrates. The tumor cells contain ovoid-to-elongated nuclei and eosinophilic cytoplasm.

NEURAL/NERVE SHEATH TUMORS Granular Cell Tumor Granular cell tumors occur throughout the gastrointestinal tract and may be multifocal in the intestinal wall as well as regional lymph nodes. They are usually small, yellow nodules composed of plump spindled and epithelioid cells. The tumor cells contain abundant granular eosinophilic cytoplasm and eosinophilic inclusions (Fig. 34.64A). Nuclei are eccentrically located, somewhat elongated, and cytologically bland. Granular cell tumors show strong S100 protein staining and harbor ATP6AP1 or ATP6AP2 mutations (245). Ganglioneuroma and Ganglioneuromatosis Ganglioneuromatous proliferations of the intestines are most common in the colon and occur in three main settings. Solitary ganglioneuromas appear as polypoid masses unassociated with any heritable syndromes. Ganglioneuromatous polyposis features multiple ganglioneuromatous polyps and may signify the presence of a heritable disorder, namely, type 1 neurofibromatosis and Cowden syndrome. Solitary and multifocal lesions can be sessile or pedunculated, featuring slightly distorted crypts and an expanded lamina propria that contains collections of Schwann cells and clustered ganglion cells (Fig.34.59B). Diffuse ganglioneuromatosis usually indicates the presence of either type 1 neurofibromatosis or MEN2B (246). Diffuse ganglioneuromatosis most commonly affects the colon and appendix (Fig. 34.59C). It features a diffuse transmural proliferation of ganglion cells, satellite cells, unmyelinated axons, and Schwann cells (Fig. 34.59D).

FIGURE 34.59 Neural and nerve sheath tumors of the intestines. Aggregates of plump spindled and epithelioid cells expand the mucosa of this colonic granular cell tumor. The cells contain granular eosinophilic cytoplasm and small dark nuclei (A). Ganglioneuromas feature plump Schwann cells with eosinophilic cytoplasm and scattered or clustered mature ganglion cells (B). Ganglioneuromatosis causes diffuse appendiceal thickening in a patient with type 1 neurofibromatosis (C). Whorled aggregates of spindled cells and ganglion cells produce diffuse mural expansion in the appendix (D). Schwann cell hamartomas feature a dense cellular cuff of Schwann cells in the superficial mucosa (E). A neurofibroma diffusely expands the mucosa and contains bland spindled cells with thin, tapering cytoplasm (F).

Schwann Cell Hamartoma Schwann cell hamartomas are small, asymptomatic polyps that occur throughout the colorectum and affect both men and women. They characteristically show a compact layer of Schwann cells beneath the surface epithelium (Fig. 34.59E). Lesional cells are

cytologically bland with dense eosinophilic cytoplasm and no mitotic figures. Verocay bodies may be present. Schwann cell hamartomas are unassociated with heritable syndromes and have no malignant potential (247). The spindled cells are diffusely positive for S100 protein. Neurofibroma Intestinal neurofibromas are strongly associated with type 1 neurofibromatosis. Those that develop in the gastrointestinal tract are morphologically identical to those occurring in the skin and soft tissues. Neurofibromas feature a diffuse proliferation of spindled cells with faintly eosinophilic tapering cytoplasm and elongated nuclei (Fig. 34.59F). Thin tendrils of collagen are interspersed between lesional cells, many of which express S100 protein. MALIGNANT GASTROINTESTINAL NEUROECTODERMAL TUMOR Malignant gastrointestinal neuroectodermal tumor, also termed “gastrointestinal clear cell sarcoma,” characteristically harbors a fusion translocation involving EWSR1, particularly EWSR1-ATF1 or EWSR1-CRERB1 (248). Most intestinal examples occur in the small bowel. Unlike many sarcomas, these tumors show a predilection for young adults and tend to spread to regional lymph nodes and the liver (249). Malignant gastrointestinal neuroectodermal tumors are bulky masses with a fleshy surface reflecting their high cellularity. Sheets of polygonal cells are arranged in large nests or cords, sometimes in combination with a spindled cell component (Fig. 34.60). The tumor cells contain abundant clear or eosinophilic cytoplasm with round nuclei, vesicular chromatin, and prominent nucleoli. Osteoclast-type giant cells are present in approximately 50% of cases. Immunohistochemical stain results generally show S100 protein and SOX10 positivity, whereas other markers of melanocytic differentiation, such as HMB-45, melan-A, and MITF, are negative or show equivocal staining. Tumor cells are also positive for synaptophysin and CD56. They are negative for CD117 and DOG1.

FIGURE 34.60 Malignant gastrointestinal neuroectodermal tumor. Sheets of tumor cells display spindled or epithelioid cell morphology (A). Lesional cells contain abundant faintly eosinophilic or clear cytoplasm and large nuclei with vesicular chromatin and prominent nucleoli (B).

VASCULAR TUMORS Vascular tumors commonly occur in the small intestine and colon; at least 10% of all small intestinal tumors are vascular lesions. Most vascular tumors, including many hemangiomas and lymphangiomas, are congenital or acquired anomalies, although some likely represent

neoplasms. Regardless of their etiologies, these lesions often present with occult blood loss or acute gastrointestinal hemorrhage. Hemangioma Intestinal hemangiomas are solitary polypoid or sessile masses that protrude into the intestinal lumen. Small hemangiomas contain lobules of capillaries lined by flattened endothelial cells. Mitotic figures are rare. Larger hemangiomas are unencapsulated and mostly circumscribed, although they can infiltrate the mucosa. Dilated vascular channels are lined by cytologically bland cells and supported by sclerotic stroma. Telangiectasia Small intestinal telangiectasias are acquired vascular anomalies. They are most common among patients with scleroderma and dialysis-dependent end-stage renal disease but also occur sporadically and in patients who have hereditary hemorrhagic telangiectasia (250). Telangiectasias appear as flat or stellate areas of vascular enhancement that may or may not blanch upon compression. They develop as the integrity of small veins and venules is lost, resulting in pooling of blood in damaged vessels and engorgement of mucosal capillaries. Angiodysplasia Angiodysplasias develop as an age-related phenomenon. Degenerative changes in the intestinal wall coupled with intermittent occlusion of venous outflow during peristalsis ultimately increase the resting pressure in submucosal veins (251). These veins subsequently dilate, resulting in increased resting pressures within mucosal venules and capillaries. Long-standing elevated venous pressures lead to decreased precapillary sphincter function and development of arteriovenous fistulae. Angiodysplasias appear as clustered dilated mucosal vessels in combination with tortuous aggregates of submucosal veins. Arteriovenous Malformation Arteriovenous malformations are congenital abnormalities that result from aberrant communications between arteries and veins. Patients with multiple or large malformations can develop high-output cardiac failure or portal hypertension. Solitary lesions are usually sporadic, but multiple malformations are seen in patients with Maffucci syndrome, KlippelTrénaunay syndrome, and hereditary hemorrhagic telangiectasia. Treatment consists of selective embolization or surgical resection. Arteriovenous malformations can affect all layers of the bowel wall, producing large purple nodules that protrude into the lumen and a spongy appearance on cross section (Fig. 34.61A). They contain disorganized aggregates of tortuous arteries and veins with thick muscular walls surrounded by ectatic, thin-walled vessels (Fig. 34.61B).

FIGURE 34.61 Arteriovenous malformation. Innumerable engorged vessels underlie a plaque-like area of discoloration in the distal colorectum (A). Abnormal veins and arteries permeate the submucosa and muscularis propria (B).

Hereditary Hemorrhagic Telangiectasia Hereditary hemorrhagic telangiectasia (i.e., Osler-Weber-Rendu syndrome) is an autosomal dominant disorder affecting approximately 1 in 10,000 individuals (252). Patients develop mucosal telangiectasias, arteriovenous malformations, and angiodysplasias in multiple organ systems. Most affected patients present with epistaxis, hemoptysis, and gastrointestinal bleeding in childhood or early adulthood. Affected patients usually have germline HHT1 (ENG) or HHT2 (ALK1, ACVRL1) mutations. These genes encode endoglin and activin receptor-like kinase (ALK1), respectively (253). Interestingly, some patients with juvenile polyposis syndrome due to SMAD4 mutations also have hereditary hemorrhagic telangiectasia (254). All three genes encode proteins involved in the TGF-β signaling pathway, which regulates vascular remodeling and plays a role in maintaining vascular wall integrity (255,256). Blue Rubber Bleb Nevus Syndrome Patients with blue rubber bleb nevus syndrome develop multiple venous malformations of the skin and gastrointestinal tract, particularly the small intestine (257). Venous malformations are congenital. Cutaneous lesions are most numerous on the upper trunk and arms, appearing as small, circumscribed blue or purple nodules that blanch upon compression and slowly refill. Intestinal lesions expand the submucosa, producing smooth blue nodules composed of dilated veins (258). Lymphangioma Lymphangiomas are benign malformations composed of variably dilated lymphatic spaces. They are most common in the small intestine where they form soft, yellow polyps (Fig. 34.62A). They contain variably sized cystic spaces that expand the mucosa and distort villi, some of which are filled with faintly eosinophilic secretions and macrophages (Fig. 34.62B). Larger lesions have a plaque-like appearance on the mucosal aspect and often involve the full thickness of the bowel wall or even extend into the mesentery (Fig. 34.62C). They have a spongy appearance and contain thick, milky fluid. Large lesions feature dissecting vascular channels in the muscularis propria and subserosa. Lymphoid tissue may be present in vascular walls (Fig. 34.62D).

FIGURE 34.62 Lymphangioma. Most intestinal lymphangiomas are small, smooth polyps with slight yellow discoloration (A). They are composed of ectatic lymphatic channels in the mucosa and submucosa, some of which contain foamy macrophages (B). A large lymphangioma has a spongy white appearance on the mucosal surface (C). Large, irregularly shaped vascular channels dissect through the muscularis propria and mesentery. Some of them are invested with interrupted bundles of smooth muscle, and others contain organized lymphoid tissue (D).

Kaposi Sarcoma Gastrointestinal Kaposi sarcoma usually occurs in patients who are immunosuppressed following organ transplantation or in association with acquired human immunodeficiency syndrome (AIDS); up to 50% of patients in the latter group develop gastrointestinal Kaposi sarcoma (259). Gastrointestinal involvement may precede development of cutaneous lesions and is often asymptomatic. However, some patients develop diarrhea, protein-losing enteropathy, or abdominal pain. Tumors appear as multiple red-brown or purple nodules. They are composed of irregularly shaped vascular channels dissecting through the lamina propria, often accompanied by spindle cells with large, elongated nuclei and extravasated red blood cells. Intracellular or extracellular hyaline globules are characteristic (Fig. 34.63). Lesional cells express CD34, CD31, and human herpesvirus-8 (HHV8). They are negative for S100 protein, desmin, and muscle-specific actin.

FIGURE 34.63 Kaposi sarcoma. A large duodenal plaque is composed of an ulcerated spindled cell proliferation that effaces the mucosal architecture (A). The spindled cells accompany angulated vascular channels and hemorrhage. A few cytoplasmic globules (arrows) are present (B).

ADIPOCYTIC TUMORS Lipomas The ileocecal valve region is prone to submucosal fat accumulation, resulting in a thickened ileocecal valve that protrudes into the lumen. Lipohyperplasia of the ileocecal valve is essentially a normal variant and does not cause symptoms, but it may be clinically confused with an adenoma or carcinoma on radiologic or endoscopic examination. Lipomas are commonly recognized throughout the gastrointestinal tract, and most are not sampled because gastroenterologists make the diagnosis at the time of endoscopy. Lipomas are soft, yellow polyps that are ballotable when prodded with the endoscope. They usually arise from the submucosa and protrude into the lumen; ulcerated lipomas occasionally present with signs and symptoms related to bleeding (Fig. 34.64). Lipomas are well-circumscribed, unencapsulated masses composed of mature adipocytes supported by delicate fibrous septa. Intramucosal lipomas are much less common than submucosal lesions; approximately one-third are related to the Cowden syndrome as previously discussed (169). Intramucosal lipomas appear as poorly circumscribed aggregates of mature adipocytes surrounded by lamina propria and, unlike pseudolipomatosis secondary to insufflation, are S100 protein positive (Fig. 34.40E and F).

FIGURE 34.64 Colonic lipoma. The submucosa is expanded by a circumscribed but unencapsulated nodule of mature adipose tissue.

METASTASES AND SECONDARY NEOPLASMS Virtually, any type of neoplasm can secondarily involve the small bowel or colon, forming polyps or masses that simulate primary tumors. In fact, small intestinal metastases are more common than primary carcinomas (260). Tumors can secondarily involve the wall as a result of hematogenous or lymphovascular dissemination, direct tumor extension from another organ, or serosal seeding in the setting of peritoneal disease. Clinical symptoms are variable and depend on the size and location of the lesion within the bowel wall. Metastases that ulcerate cause gastrointestinal bleeding or iron deficiency anemia (261,262). Small, superficial lesions are mostly asymptomatic, whereas large tumor deposits cause obstruction and abdominal pain. The diagnosis of a secondary malignancy is usually straightforward because the clinical history of another neoplasm is known. Tumor multifocality, high-grade cytologic features, and extensive mural disease with minimal mucosal involvement are features that favor a metastasis (Fig. 34.65A). Lymphovascular invasion is usually difficult to recognize in biopsy samples of primary carcinomas; extensive permeation of vascular spaces by tumor cells in a biopsy sample is a clue to the possibility of a metastasis (Fig. 34.65B).

FIGURE 34.65 Metastatic carcinomas to the intestines. Pulmonary carcinomas frequently metastasize to the small intestine. They often display sheet-like growth of large cells with necrosis that can be a helpful diagnostic clue (A). Extensive lymphovascular invasion in a biopsy sample raises the possibility of secondary involvement by an extraintestinal carcinoma. This patient proved to have an occult pancreatic ductal adenocarcinoma (B). Metastases to the bowel mucosa show maturation when they colonize the basement membrane. This duodenal bulb tumor represents a metastasis from a colonic primary; note the abrupt transition between neoplastic and nonneoplastic epithelium in a single villus (C). Metastatic breast cancer in the colon often shows lobular morphology, regardless of the nature of the primary tumor. Tumor cells contain large cytoplasmic vacuoles and inspissated secretions (D). Prostatic adenocarcinoma shows a nested growth pattern in the rectal mucosa that simulates an endocrine neoplasm (E). Metastatic endometrioid carcinoma in the colon simulates an adenoma. However, the neoplastic cells lack cytoplasmic mucin (F).

CARCINOMAS Most intestinal metastases originate elsewhere in the gastrointestinal tract (263). Other common sites of origin include gynecologic and breast tumors that have spread to the peritoneum. Pulmonary and renal cell carcinomas show hematogenous spread, producing solitary lesions that simulate primary intestinal carcinomas (264-266). Metastases do not elicit a desmoplastic tissue response when they invade the intestinal lamina propria and may show well-differentiated areas that mimic an in situ lesion when they colonize the basement membrane (Fig. 34.65C). Clues suggesting a metastasis include the lack of an adjacent “shoulder” of adenoma overlying the invasive component, signet-ring cell areas or other uncommon high-grade growth patterns, and an absence of intestinal differentiation. Mammary carcinomas produce multiple small polyps or masses in the bowel wall, mesentery, and retroperitoneum. They typically show a lobular phenotype in the tubular gut, infiltrating the lamina propria and expanding the mucosa without destroying the crypt epithelium (Fig. 34.65D) (267). Prostatic adenocarcinoma is common among older men and can mimic primary rectal neoplasms. Nearly 5% of male patients undergoing resection for rectal adenocarcinoma have metastatic deposits of prostatic adenocarcinoma in mesorectal lymph nodes that can mimic spread of the rectal primary (268). Prostatic cancers can also directly invade the rectal wall and display a nested growth pattern that simulates the appearance of an endocrine tumor (Fig. 34.65E). This differential diagnosis is compounded by the frequent presence of prostate-specific acid phosphatase (PSAP) immunostaining among well-differentiated rectal endocrine neoplasms.

Müllerian carcinomas frequently involve the gastrointestinal tract. Serous carcinomas generally show high-grade cytologic features and may be accompanied by psammomatous calcifications. Endometrioid carcinomas can simulate the morphologic features of intestinal carcinomas, although they lack cytoplasmic mucin and generally show a lesser degree of cytologic atypia (Fig. 34.65F). Endometriosis of the gastrointestinal tract rarely gives rise to a spectrum of Müllerian neoplasms, including carcinomas, endometrioid stromal sarcomas, and adenosarcomas, especially under the influence of exogenous hormonal therapy (56). Neoplastic changes are accompanied by benign endometriosis, thereby facilitating a correct diagnosis upon evaluation of resection specimens. Challenging cases can be resolved with a combination of immunohistochemical stains for CK7, CK20, CDX-2, and estrogen and progesterone receptors. MALIGNANT MESOTHELIOMA Malignant mesotheliomas occasionally infiltrate the full thickness of the intestinal wall and extend into the mucosa, thereby simulating a primary malignancy. The epidemiology of these tumors is essentially the same as that of peritoneal mesothelioma. Malignant mesotheliomas of the intestines are generally bulky, heterogeneous tumors with hemorrhage (Fig. 34.66A). They tend to grow with an epicenter outside the bowel wall and display cystic degeneration, hemorrhage, and/or necrosis (Fig. 34.66B). Most tumors are composed of plump or cuboidal epithelioid cells with abundant eosinophilic cytoplasm and prominent nucleoli that may be arranged in nests and tubules, simulating a carcinoma in biopsy samples (Fig. 34.66C). Occasional cases contain poorly formed fascicles of plump spindled cells with rounded nuclei and prominent nucleoli (Fig. 34.66D). Confirmatory immunohistochemical stains include calretinin, WT-1, D2-40, and cytokeratin 5/6 (269). Loss of nuclear BAP1 staining is associated with aggressive behavior.

FIGURE 34.66 Malignant mesothelioma. The tumor is a large heterogeneous mass with areas of hemorrhage and necrosis (A). Its epicenter is outside the muscularis propria, forming a solid and cystic plaque on the outer aspect of the colon (B). Mesotheliomas that infiltrate the mucosa contain small glands that simulate an adenocarcinoma. However, the tumor cell nuclei are small and round with prominent nucleoli (C). Some mesotheliomas are composed of plump spindled cells in a haphazard arrangement (D).

MALIGNANT MELANOMA Malignant melanoma shows a propensity for metastasis to the gastrointestinal tract, particularly the small intestine. Gastrointestinal metastases are frequently amelanotic, and nearly 50% of patients have no known cutaneous melanoma or history of melanoma (270). Typical features include dyshesive epithelioid cells with a plasmacytoid appearance and abundant eosinophilic cytoplasm (Fig. 34.67A). Spindled cells may be arranged in sweeping fascicles that simulate a sarcoma (Fig. 34.67B). Enlarged nuclei with open chromatin, eosinophilic macronucleoli, and intranuclear pseudoinclusions are helpful morphologic features that should raise the possibility of metastatic melanoma. Most cases show strong, diffuse staining for SOX-10, S100 protein, HMB-45, A103 (MART-1), and microphthalmia transcription factor. Of note, malignant melanoma may express CD117 in more than 50% of cases.

FIGURE 34.67 Malignant melanoma. Nests of epithelioid, somewhat plasmacytoid, cells contain abundant eosinophilic cytoplasm and large nuclei with macronucleoli (A). Spindle cell melanomas are highly cellular lesions composed of intersecting fascicles of plump spindled cells with large, elongated nuclei and prominent nucleoli (B).

MIMICS OF INTESTINAL NEOPLASIA INFLAMMATORY-TYPE POLYPS Inflammatory-type polyps are the most common nonneoplastic polyps of the intestines. They usually develop as a result of chronic mucosal injury due to idiopathic inflammatory bowel disease. Less common causes include persistent infection, long-standing medication-related injury, and chronic mural diseases, such as endometriosis and diverticulosis, that evoke mucosal inflammation (Fig. 34.68A). Inflammatory-type polyps occurring in the setting of idiopathic inflammatory bowel disease typically show features of chronic enteritis or colitis with plasma cell–rich inflammation, crypt architectural distortion, and a variable amount of neutrophilic inflammation, as described in Chapter 33. They may contain bizarre stromal cells that simulate sarcoma (Fig. 34.68B).

FIGURE 34.68 Inflammatory-type polyps. Chronic mucosal injury due to ipilimumab features heaped up pseudopolyps of intact mucosa flanked by extensive ulcers (A). Polypoid granulation tissue can contain bizarre fibroblasts with markedly enlarged nuclei and macronucleoli (B). Segmental polyposis can develop following placement of expandable metal stents placed over obstructing colonic adenocarcinomas (arrow); inflammatory-type polyps represent mucosal elements protruding through the stent (C). Mounds of residual mucosa and submucosa are surrounded by basophilic necrosis at points of contact between the stent and the mucosa (D).

Inflammatory polyps can also develop as a result of instrumentation or at prior surgical sites, such as stomas and anastomoses. Self-expanding metal stents are commonly deployed across segmental stenoses to prevent impending bowel obstruction due to carcinoma, diverticulitis, and other causes. The mesh of the stent compresses the mucosa at points of contact, causing pressure necrosis, whereas the intervening mucosa protrudes through the stent (Fig. 34.68C). The resultant pseudopolyps are composed of near-normal mucosa flanked by ulcers with basophilic necrosis, fibrin deposition, and pseudomembranes (Fig. 34.68D) (271). MUCOSAL PROLAPSE POLYPS Prolapse-related changes can occur anywhere in the intestine where the mucosa is subjected to pulsion and intermittent ischemia, such as diverticulosis, ostomy sites, and adjacent to masses (272,273). Mucosal prolapse polyps and solitary rectal ulcer syndrome show a predilection for older women but can occur in younger individuals and males. Patients present with rectal bleeding, straining with defecation, mucoid diarrhea, constipation, and a sensation of incomplete rectal evacuation (274). Rectal prolapse results from downward displacement of the rectal wall during defecation (275). Straining

compresses the mucosa, resulting in intermittent ischemia and episodes of repair that ultimately leads to chronic ulcers and/or inflammatory-type polyps. Affected patients develop patchy inflammation, ulcers, polyps, or stenosis mimicking malignancy (274). Lesions are most commonly found on the anterolateral rectal wall (276). Mucosal erosions and fibrin deposits are accompanied by hyperplastic and often serrated crypts with regenerative epithelial cell changes (277). Smooth muscle cells emanate from the muscularis mucosae into the mucosa and surround the crypts (Fig. 34.69) (278).

FIGURE 34.69 Mucosal prolapse polyp. Mucosal prolapse polyps contain serrated, regenerative-appearing crypts lined by cells with cytoplasmic depletion (A). Smooth muscle cells emanate from the muscularis mucosae into the mucosa, which shows superficial granulation tissue (B).

ANGIOGENIC POLYPS Serotonin-producing endocrine tumors of the intestines can cause polypoid angiomatoid vascular proliferations in the overlying or distant mucosa. These lesions consist of aggregates of dilated capillaries accompanied by fibromuscularization of the lamina propria and regenerative epithelial changes. They were initially thought to result from elaboration of vascular growth factors by the endocrine neoplasm, but are now believed to reflect mucosal prolapse (279,280). ENDOMETRIOSIS Endometriosis shows a predilection for the rectosigmoid colon and ileocecal region. Extensive mural disease can cause gastrointestinal bleeding or bowel obstruction secondary to intermittent intussusception or luminal narrowing, whereas appendiceal endometriosis may mimic acute appendicitis (281). Endometriosis can sometimes harbor mutations affecting ARID1A, PIK3CA, KRAS, and PPP2R1A (282). Endometriosis forms gray or black patches on the serosal surface. When present in the muscularis propria, it induces smooth muscle cell hypertrophy and hyperplasia that distort and expand the muscularis propria, causing strictures that simulate malignancy (Fig. 34.70A). Endometriosis can also produce clustered polyps or ulcerated tumors when it extends into the mucosa (283). Foci of endometriosis contain endometrioid glands cuffed by variable amounts of cellular stroma with hemorrhage or hemosiderin deposits (Fig. 34.70B). Glands are often dilated and lined by cuboidal to short columnar epithelial cells with tubal metaplasia. Stromal cells are usually spindle shaped, although decidualized cells are polygonal with abundant eosinophilic cytoplasm. Neoplastic transformation can occur as previously described (284).

FIGURE 34.70 Colonic endometriosis. Endometriosis induces hypertrophy of the muscularis propria, producing an annular mass that simulated malignancy (A). Round aggregates of endometrioid glands are intimately associated with stromal cells. The surrounding muscularis propria is hypertrophic and disorganized (B).

HEMATOPOIETIC LESIONS LYMPHOID HYPERPLASIA Gut-associated lymphoid tissue (GALT) consisting of mature lymphocytes and plasma cells is normally present throughout the intestinal mucosa, although its distribution varies somewhat by site. Lymphoid aggregates and follicles are less frequently identified in the proximal small intestines of adults. In fact, the presence of numerous benign lymphoid aggregates in the duodenum raises the possibilities of selective IgA deficiency, common variable immunodeficiency, giardiasis, human immunodeficiency virus (HIV) infection, or a precursor to lymphoma (285,286). On the other hand, nodular lymphoid aggregates are normally present in the mucosa and superficial submucosa of the ileum and colon. These nodules may enlarge in a variety of inflammatory disorders, particularly idiopathic inflammatory bowel disease and viral infections, producing smooth mucosal excrescences or sessile polyps. Large nodules may be pedunculated when they occur in the rectum (287). Lymphoid nodules often show central umbilication or a rim of erythema that simulates aphthous ulcers (Fig. 34.71A). Aggregates of lymphoid tissue are centered in the submucosa but can transgress the muscularis mucosae and expand the mucosa, especially in children and young adults (Fig. 34.71B). Adjacent crypts and the surface epithelium are commonly infiltrated by mononuclear cells, neutrophils, and eosinophils. Lymphoid nodules consist of B-cell–rich follicles surrounded by T-cells. Primary follicles express BCL-2 and are negative for BCL-6, whereas germinal centers express BCL-6 and are negative for BCL-2. Follicular cells are polyclonal; the ratio of cells expressing κ and those expressing λ is approximately 2:1.

FIGURE 34.71 Prominent lymphoid aggregates. The Peyer patches of the ileum can be endoscopically visualized, especially in children and young adults. Pale sessile polyps are scattered throughout the ileum, some of which show central depressions (A). Abundant lymphoid tissue can also form polyps in the rectum. Mucosa-associated lymphoid tissue contains numerous follicles and germinal centers (B).

EPSTEIN-BARR VIRUS–POSITIVE MUCOCUTANEOUS ULCER Epstein-Barr virus (EBV)-positive mucocutaneous ulcer is a B-cell lymphoproliferative disorder that usually develops in immunosuppressed patients, solid-organ transplant recipients, and older adults who may not be immunocompromised (288). This indolent condition is characterized by ulcers of the skin, oral mucosa, and gastrointestinal tract. Intestinal lesions appear as solitary, circumscribed nodules composed of a polymorphous lymphocyte-predominant infiltrate that features numerous eosinophils (Fig. 34.72A). The infiltrate also contains scattered large neoplastic cells with clear cytoplasm, some of which resemble Reed-Sternberg cells with large nuclei, vesicular chromatin, and prominent nucleoli (Fig. 34.72B). These large cells contain EBV-encoded RNAs (Fig. 34.72C). They also stain for CD30, PAX5, and MUM1, but show variable, often decreased, staining for CD20 and CD45 and are negative for CD15 (Fig. 34.72D). Nearly 50% of cases spontaneously resolve, often with a reduction of immunosuppression (289).

FIGURE 34.72 Epstein-Barr virus (EBV)-associated mucocutaneous ulcer. This lymphoproliferative lesion of the duodenum features a polymorphous infiltrate of B-cells and T-cells, as well as plasma cells, eosinophils, and macrophages (A). Scattered large cells with abundant clear cytoplasm and round nuclei with open chromatin (arrow) resemble Reed-Sternberg cells (B). Numerous small and large cells contain EBV-encoded RNAs by in situ hybridization (C). The large cells are also immunopositive for CD30 (D). Case courtesy of Dr. Rebecca Waters, Department of Pathology and Laboratory Medicine, University of Texas MD Anderson Cancer Center.

LYMPHOMA The gastrointestinal tract is the most common site of extranodal lymphoma, accounting for 30% to 40% of all primary extranodal lymphomas. It is also a common site of secondary involvement by lymphomas that develop in other organs. Intestinal lymphomas make up nearly 50% of gastrointestinal lymphomas: 30% to 40% develop in the small bowel, and 10% develop in the colon (290). In fact, lymphomas are the most common malignant tumors of the small intestine. Specific lymphoma subtypes are prone to affect different regions of the bowel, as described subsequently. Symptoms are similar, regardless of subtype. Large masses can cause gastrointestinal bleeding or B symptoms as well as abdominal pain due to intermittent bowel obstruction or perforation. Low-grade B- and T-cell lymphomas can manifest with lymphomatous polyposis (291,292). Lymphomas are staged according to the Lugano classification, which has been incorporated into the eighth edition of the AJCC Cancer Staging Manual (293). The classification of hematologic malignancies requires integration of morphologic, immunophenotypic, molecular, and genotypic features. Comprehensive discussions of myeloid disorders and lymphomas are provided in Chapters 16 and 17, respectively.

Extranodal Marginal Zone Lymphoma of Mucosa-Associated Lymphoid Tissue Extranodal marginal zone lymphomas account for nearly 25% of intestinal lymphomas and occur in older adults. Most tumors show translocations that result in either a chimeric BIRC3-MALT1 protein [t(11;18)(q21;q21)] or dysregulation of BCL10 [t(1;14)(p22;q32)], MALT1 [t(14;18)(q32;q21), or FOXP1 [t(3;14)(p14;q32)], leading to aberrant NF-κB mediated cell signaling (294-296). These tumors are low-grade B-cell lymphomas composed of small neoplastic lymphocytes accompanied by variable numbers of plasma cells and nonneoplastic mononuclear cells. Tumor cells may simulate centrocytes or appear as monocytoid B-cells with pale cytoplasm and small dark nuclei. Plasmacytoid differentiation is variably present. Lesional cells can surround and infiltrate preexisting lymphoid follicles (i.e., follicular colonization), which imparts a nodular appearance to the infiltrate and simulates follicular lymphoma. Tumor cells can also infiltrate the epithelium and form lymphoepithelial lesions, although this finding is more common among gastric tumors than those of the intestines. Scattered large cells resembling centroblasts and immunoblasts are commonly present in low-grade lymphomas. Extranodal marginal zone lymphomas can transform to diffuse large B-cell lymphomas composed of sheets or aggregates of large cells. Immunohistochemical features are similar to those of nonneoplastic marginal zone B-cells and include positivity for CD20, CD79a, and BCL2, as well as variable staining for CD5 and CD23 (297). Immunoproliferative Small Intestinal Disease ( –Heavy-Chain Disease) Immunoproliferative small intestinal disease (IPSID) is a subtype of extranodal marginal zone lymphoma that shows unique clinical and pathologic features. It commonly presents in young adulthood with malabsorptive diarrhea, weight loss, abdominal pain, and clubbing of the digits (298). The disorder mostly occurs in developing countries of the Mediterranean region where poverty and poor hygiene lead to a high incidence of infectious enteritis, particularly Campylobacter jejuni infection (299). Most affected patients have α-chain disease characterized by abnormal serum IgA devoid of light chains. The disorder is usually limited to the jejunum and duodenum where it produces diffuse mural thickening. Early disease manifests with mucosal and submucosal expansion by dense lymphoplasmacytic infiltrates. Over time, the infiltrate progressively extends into the deeper bowel layers and regional lymph nodes (Fig. 34.73A). Lymphoepithelial lesions and follicular colonization by centrocyte-like cells may occur. Small lymphocytes are accompanied by sheets of plasma cells that express cytoplasmic and surface α-chain without light chains (Fig. 34.79B). This feature results from deletions that eliminate most of the immunoglobulin heavy-chain variable region and all of the CH1 domain of the constant region, leaving only the C-terminal region intact. The truncated heavy chain cannot bind to the variable and constant regions of light chains. Tumor cells express CD19 and CD20, but are generally negative for CD5, CD10, and CD23 (297). Transformation to diffuse large Bcell lymphoma can occur as disease progresses.

FIGURE 34.73 Immunoproliferative small intestinal disease. The mucosa is expanded by a destructive lymphoid infiltrate with germinal centers (A). The interfollicular areas contain sheets of neoplastic cells with plasma cell differentiation (B).

Follicular Lymphoma Follicular lymphoma shows a predilection for the small bowel and colon and tends to occur among older adults. Unlike tumors of the duodenum, follicular lymphomas arising elsewhere in the intestines often show extension into the muscularis propria. Patients may also develop multiple polyps throughout the colon and small intestine (i.e., lymphomatous polyposis). The morphologic features of the tumor cells are indistinguishable from those of duodenal-type follicular lymphomas, albeit tumors of the distal small bowel and colon often contain more numerous centroblast-type cells. Nodular proliferations of neoplastic “follicles” contain follicular dendritic cells as well as small B-cells and larger centroblast-like cells. The small B-cells resemble centrocytes, showing a slight degree of nuclear irregularity. Tumors are graded based on the extent of centroblast-like differentiation. Neoplastic follicles contain CD21-positive dendritic cells and may show sclerosis. Tumor cells express B-cell markers in addition to CD10, BCL6, and BLC2. They harbor t(14;18) translocations that result in BCL2IGH fusions (300). Duodenal-Type Follicular Lymphoma. Duodenal-type follicular lymphomas account for 4% of gastrointestinal lymphomas and show a predilection for the second part of the duodenum, although they may be accompanied by disease in the distal small bowel as well. Tumors are generally discovered incidentally during endoscopic examinations performed for other reasons. Most patients are older adults with an equal sex distribution, but approximately 10% of patients are young adults (301). These tumors harbor t(14;18) (q32;a21) alterations similar to primary nodal follicular lymphomas but also express α4β7integrin, which is a mucosal homing receptor expressed by GALT (302). Duodenal-type follicular lymphomas are almost always low-grade and pursue an indolent course (303). Duodenal-type follicular lymphomas are morphologically similar to nodal tumors and follicular lymphomas that develop elsewhere in the gastrointestinal tract. They are composed of neoplastic follicles that expand the mucosa and submucosa, imparting a polypoid appearance. Lymphoid nodules lack tingible-body macrophages and mantle zones (Fig. 34.74A). Most of the tumor cells resemble centrocytes with small round or cleaved nuclei (Fig. 34.74B). They may be encountered in lymphoid nodules and in the intervening lamina propria. Neoplastic cells express CD20, CD10, BCL2, and BCL6 (301,304). Some

authors have suggested that these follicles lack a well-developed network of CD21-positive dendritic cells, which may help distinguish this variant from other follicular lymphomas.

FIGURE 34.74 Follicular lymphoma of the duodenum. Neoplastic follicles expand the mucosa and lack germinal centers (A). They contain small B-cells with cleaved, irregular nuclei (B).

Mantle Cell Lymphoma Mantle cell lymphoma is a mature B-cell neoplasm that typically presents at advanced stage with bone marrow and lymph node involvement. Most patients are older adults, and males are affected more frequently than females. More than 95% of cases harbor t(11;14) (q13;q32) translocations between CCND1 and IGH (305). Translocations involving CCND2 or CCND3 and IGK or IGL account for cases lacking CCND1-IGH. Clinical findings include ulcers, masses, and lymphomatous polyposis. The latter features a myriad of sessile polyps and confluent plaques, or a cobblestone-like appearance of widely spaced nodules separated by intervening normal mucosa (Fig. 34.75). Approximately 50% of cases present with an epicenter in the right colon.

FIGURE 34.75 Lymphomatous polyposis. This patient with mantle cell lymphoma has innumerable sessile nodules and plaques throughout the large and small intestines.

Tumor cells resemble antigen-naïve pre–germinal center B-cells that normally reside in the mantle zone surrounding germinal centers in secondary follicles. Small tumor nodules straddle the muscularis mucosae, involving the deep mucosa and submucosa with sparing of the superficial mucosa. Epithelial invasion and ulceration develop as nodules enlarge. Tumor cells are uniform and display nodular or sheet-like growth. Small-to-medium cells contain nuclei with irregular contours, indistinct nucleoli, and scant cytoplasm. Large transformed cells are rarely identified, although the blastoid variant features intermediatesized cells with round nuclei, fine chromatin, and a high-proliferative rate. The lymphoma cells express both CD19 and CD20, often with weaker coexpression of CD5 and CD43 (305). They show strong, diffuse staining for cyclin D1 and light-chain restriction. Virtually, all cases are negative for CD10 and CD11c. Diffuse Large B-Cell Lymphoma Diffuse large B-cell lymphoma is the most common intestinal lymphoma and shows a predilection for the ileocecal region of middle aged to older adults. These tumors are more common among patients with underlying immunodeficiency. They may develop de novo or result from transformation of a low-grade lymphoma. Approximately 25% of cases harbor BCL6 rearrangements, reflecting germinal center origins (306). Other common changes include alterations affecting BCL2 and MYC; tumors with alterations affecting both genes are associated with a worse prognosis (307). Diffuse large B-cell lymphomas are bulky masses with homogeneous, fleshy cut surfaces, often accompanied by regional lymph adenopathy. Most tumors feature sheets of large cells with abundant cytoplasm and large, vesicular nuclei, peripheral condensed chromatin, and one or more nucleoli. Centroblast-like cells contain round nuclei with multiple small nucleoli, whereas immunoblast-like cells contain prominent, centrally located nucleoli. Other cells are anaplastic with pleomorphic nuclei and abundant cytoplasm that may simulate ReedSternberg cells. Mitotic activity is usually brisk, and cellular necrosis may be identified. Nonneoplastic T-cells, B-cells, multinucleated cells, and granulocytes may be present in abundance, obscuring the nature of the lesion. Diffuse large B-cell lymphomas typically express one or more B-cell markers. Germinal center–type diffuse large B-cell lymphomas frequently show strong BCL6 positivity, whereas the activated B-cell type shows strong MUM1 staining (308). Burkitt Lymphoma Burkitt lymphoma occurs in endemic, sporadic, and immunodeficiency-associated forms. Endemic disease rarely manifests with gastrointestinal involvement, whereas sporadic and immunodeficiency-associated tumors produce bulky abdominal disease associated with pain, constipation, weight loss, and bleeding. Sporadic tumors show a predilection for children and young adults, accounting for 30% to 50% of pediatric lymphomas (309). Males are affected more often than females. Immunodeficiency-associated tumors more commonly occur in the setting of HIV than other types of immunocompromise. EBV plays a pathogenic role in the evolution of approximately 30% of sporadic and immunodeficiency-associated tumors, particularly when they develop in adults (310). Burkitt lymphoma features uniform, medium-sized tumor cells with round nuclei, multiple nucleoli, and basophilic cytoplasm, often with a cohesive appearance. Cytoplasmic lipid vacuoles may be apparent in cytology preparations. Tumors show brisk mitotic activity and abundant necrotic cellular debris in tingible-body macrophages that impart a “starry-sky”

appearance (Fig. 34.76A). Tumor cells show membranous IgM staining with light-chain restriction as well as immunopositivity for CD19, CD20, CD79a, PAX5, BCL6, and CD10. They are negative for CD5, CD23, and BCL2 (311). Immunostains for Ki-67 label the vast majority of tumor cells (Fig. 34.76B). Most cases have t(8;14)(q24;q32) translocations involving MYC on chromosome 8 and the immunoglobulin heavy-chain region on chromosome 14; less frequent alterations include t(2;8)(p12;q32) and t(8;22)(q32;q11), affecting IGK and IGL, respectively (311). Approximately 70% of sporadic cases harbor mutations in the TCF3 transcription factor or its negative regulator ID3, resulting in prolonged cell survival (312).

FIGURE 34.76 Burkitt lymphoma. Scattered tingible-body macrophages are associated with sheets of medium-sized tumor cells (A) that show a high-proliferative rate with Ki-67 immunohistochemical stains (B).

Plasmablastic Lymphoma Plasmablastic lymphomas are aggressive B-cell lymphomas that show plasmablastic or immunoblastic morphology and express plasma cell–associated antigens (305). These tumors usually occur in immunodeficient patients, particularly those with HIV infection or transplant recipients, and show a predilection for males. Tumors feature sheet-like growth of atypical immunoblasts and plasmablasts. They often show with brisk mitotic activity, cellular necrosis, and granulomatous infiltrates. Tumor cells often contain MYC translocations, especially among HIV-infected patients (313). Importantly, lesional cells are negative for conventional B-cell markers such as CD20, and show decreased staining for PAX5 and CD45. Plasma cell markers, including CD79a, MUM1, and CD138, are positive, and staining for MYC is often present (314). Enteropathy-Associated T-Cell Lymphoma Enteropathy-associated T-cell lymphomas account for less than 5% of all gastrointestinal lymphomas (315). Affected patients are older adults who develop symptoms 15 to 20 years after the diagnosis of celiac disease. Patients present with malabsorptive diarrhea despite gluten withdrawal, abdominal pain, gastrointestinal bleeding, or small intestinal perforation. Some patients do not have a history of celiac disease but have features of celiac sprue in the background mucosa at the time of lymphoma resection. Approximately one-third of cases are preceded by type II refractory sprue (316). Manifestations include benign-appearing ulcers (i.e., ulcerative jejunitis), solitary or multiple plaques, ulcerated masses, and diffuse mural thickening (317). Imaging studies may document intestinal perforation or mesenteric lymphadenopathy. Tumors are composed

of sheets of cells that permeate the wall, destroying mucosal elements and the muscularis propria (Fig. 34.77A). They are medium to large in size and contain ovoid-to-round nuclei (Fig. 34.77B). Some nuclei may be pleomorphic. Mitotic figures are numerous, and cellular necrosis is readily apparent. Tumors often contain a striking inflammatory infiltrate composed of eosinophils, plasma cells, and macrophages (318). Enteropathy-associated Tcell lymphomas express CD3, CD7, and CD103, but are negative for CD4, CD5, CD8, and CD56 (319).

FIGURE 34.77 Enteropathy-associated T-cell lymphoma. Tumor cells diffusely infiltrate all layers of the bowel wall, destroying the muscularis propria and mucosal elements (A). Medium-sized tumor cells have a uniform appearance and contain dark nuclei with a thin rim of cytoplasm (B).

Monomorphic Epitheliotropic Intestinal T-cell Lymphoma Monomorphic epitheliotropic intestinal T-cell lymphoma, formerly termed type II enteropathyassociated T-cell lymphoma, is derived from intraepithelial T-cells but is unassociated with celiac disease. Affected patients are older adults and usually males who present with abdominal pain, weight loss, perforation, or gastrointestinal bleeding (320). Tumors are bulky masses composed of small- to medium-sized lymphocytes with slightly irregular nuclei and a rim of pale or clear cytoplasm (318). Tumor cells often show infiltration of surface and crypt epithelium. They are positive for CD2, CD3, CD7, CD8, CD56, and TIA-1 and are negative for CD4 and CD5 (321). They show frequent amplifications of 8q24 affecting MYC (322). Extranodal NK/T-Cell Lymphoma Extranodal NK/T-cell lymphoma is a high-grade lymphoma associated with EBV and genetic variants at HLA-DPB1 (323). It occurs in adults, particularly Asians and indigenous populations from Mexico, Central America, and South America (324). Patients present with fever, malaise, weight loss, lymphadenopathy, and hemophagocytic lymphohistiocytosis, as well as bowel obstruction, perforation, or gastrointestinal bleeding (325). Most tumors are large, fungating masses with ulcers and mucosal friability. They are composed of a sheetlike proliferation of atypical cells that permeates the bowel wall, often with an angiocentric growth pattern and destructive vascular invasion (326). Tumor cells contain abundant clear or eosinophilic cytoplasm with either small angulated nuclei or larger nuclei with open chromatin and prominent nucleoli. Most tumors show extensive necrosis and apoptotic debris with easily identifiable mitotic figures (327). Extranodal NK/T-cell lymphomas are positive for CD2, CD56, and CD3ε, but do not express CD3 or other T-cell markers (328).

They are characteristically EBV-positive by in situ hybridization and show cytotoxic T-cell differentiation with granzyme B and TIA-1 expression (329). Other T-Cell Neoplasms Nonepitheliotropic T-cell lymphomas can occur in the intestines. They are usually unifocal plaques or polypoid masses unassociated with celiac disease. Lesional cells are large with pleomorphic nuclei, necrosis, and readily identifiable mitotic figures. Infiltration of the epithelium by tumor cells is lacking. Expression of TIA-1 is common (330). Approximately 10% of tumors are related to EBV. These neoplasms are associated with an aggressive clinical course. Indolent T-cell lymphomas can affect the small intestine and colon. Affected patients complain of abdominal pain, diarrhea, or weight loss. Most are adults, and women are affected more commonly than men. Endoscopic findings include mucosal thickening and nodularity that may be accompanied by erythema. The lamina propria is expanded by a dense lymphoid infiltrate, unaccompanied by crypt destruction or epitheliotropism that may simulate the malabsorptive pattern of chronic enteritis (331). Tumor cells are small, matureappearing lymphocytes with scant cytoplasm and round nuclei (332). They express CD3 and often stain for CD8 as well as TIA-1. A minority express CD4 (333). Most patients have a protracted clinical course, although a minority develop progressive disease. Posttransplant Lymphoproliferative Disorder Immunosuppressed individuals who undergo bone marrow or solid-organ transplantation are at increased risk for EBV-driven lymphoproliferative disorders. Chronic immunosuppression leads to impaired T-cell function and facilitates proliferation of EBVpositive B-cell populations. Risk increases with higher levels of immunosuppression and cyclosporine usage (334,335). Symptoms include a mononucleosis-like syndrome, diarrhea, abdominal pain, and declining graft function. Small lesions produce ulcers or polyps, whereas larger tumors appear as fungating masses (336,337). Treatment consists of decreased immunosuppression in conjunction with chemotherapy, surgery, and/or radiation. Polymorphous variants respond well to reduced immunosuppression, whereas monomorphic disease is generally aggressive (338). The World Health Organization classifies posttransplant lymphoproliferative disorders according to morphologic and molecular features (339). Early polyclonal B-cell proliferations consist of slightly atypical lymphoid infiltrates accompanied by plasma cells and scattered immunoblasts that do not efface the mucosal architecture (340). The infiltrate may simulate that of infectious mononucleosis or display follicular hyperplasia and contains oligoclonal populations. The polymorphic variant may be polyclonal or monoclonal, featuring a destructive infiltrate of lymphocytes, plasma cells, centrocytes, and immunoblasts. Large cells resembling Reed-Sternberg cells can be present. They express CD30 and CD20, but are negative for CD15. The monomorphic variant is a destructive, high-grade tumor that is histologically and immunohistochemically similar to other lymphomas unrelated to immunosuppression (338,341). Most (85%) cases are B-cell neoplasms with morphologic and immunophenotypic features of diffuse large B-cell lymphoma or Burkitt lymphoma; a subset of tumors show plasmacytic differentiation (342,343). Monomorphic neoplasms usually express CD30 and harbor clonal immunoglobulin gene rearrangements as well as alterations affecting TP53, MYC, and BCL6 (340). Approximately 15% of tumors are T-cell neoplasms that resemble peripheral T-cell lymphoma, not otherwise specified. Rare cases

in the small bowel and colon resemble classic Hodgkin disease. In situ hybridization for EBV-encoded small RNAs (EBER) is often positive, particularly in B-cell neoplasms. MYELOID SARCOMA Myeloid sarcomas are solid, extramedullary tumors composed of neoplastic myeloid cells that efface the underlying tissue architecture. They occur across a wide age distribution, but most frequently affect older adults, particularly men (344). Tumors usually develop in patients known leukemia, but they can occur in patients with myelodysplastic syndrome or without a prior history of neoplasia (345,346). Approximately 80% of de novo myeloid sarcomas progress to leukemia within 1 year (347). Intestinal tumors cause symptoms related to intermittent bowel obstruction or bleeding (347). Myeloid sarcomas produce endoscopically apparent polyps or bulky tumors that may be multifocal. Their cut surfaces are homogeneous, pink, and fleshy. Tumors feature a sheetlike proliferation of medium-to-large dyshesive cells that efface the normal architecture of the bowel wall. Neoplastic cells contain large round or multilobate nuclei with prominent nucleoli and granular eosinophilic cytoplasm (346). Tumor cells are often accompanied by mature-appearing granulocytes. Mitotic figures are readily apparent, although confluent necrosis is uncommon (348). Immunohistochemical stains for CD68, myeloperoxidase, lysozyme, CD13, CD33, and CD117 are often positive (347). Most myeloid sarcomas stain for CD43, but are negative for other T-cell markers. They also harbor molecular alterations similar to leukemic infiltrates (344,349,350). SYSTEMIC MASTOCYTOSIS Enterocolic aggregates of atypical mast cells with aberrant CD2 and CD25 expression occur in two settings: secondary involvement of the gastrointestinal tract by aggressive systemic mastocytosis and incidental lesions detected in asymptomatic patients who undergo endoscopic examination for other reasons. Patients with aggressive systemic mastocytosis present with anorexia, fever, weight loss, and histamine-induced flushing or hypotension (351). Nearly 80% have intestinal involvement, in which case they develop abdominal pain and diarrhea. Most patients with aggressive systemic mastocytosis have D816V KIT mutations that render their tumors resistant to tyrosine kinase inhibitor therapy (352). Aggressive systemic mastocytosis produces mucosal abnormalities, such as loss of normal folds, nodularity, erosions, and ulcers. Biopsy samples contain atypical mast cell infiltrates in the lamina propria that efface the normal architecture. Neoplastic mast cells may show condensation around epithelial elements, or an even distribution throughout the lamina propria (353). These ovoid or spindled cells contain pale eosinophilic cytoplasm and reniform nuclei with dense chromatin and inconspicuous nucleoli. Eosinophils are present in high numbers, obscuring the mast cell infiltrate in some cases. Mucosal mast cells show strong diffuse staining for CD117 and mast cell tryptase, as well as abnormal expression of CD25 and CD2 (354). Incidentally discovered lesions are currently classified as indolent systemic mastocytosis, although there is no compelling evidence that affected patients have a systemic disorder. Patients are typically adults without symptoms referable to histamine elaboration or malabsorption. Incidental lesions may produce small polypoid excrescences, although they are also detected in flat mucosa or other types of polyps, such as sessile serrated polyps and adenomas. Incidental lesions are histologically indistinguishable from those of

aggressive systemic mastocytosis, but patients do not have any other evidence of systemic disease (Fig. 34.78). For this reason, they are probably best considered to represent atypical enterocolic mast cell aggregates than a generalized neoplastic condition (355,356).

FIGURE 34.78 Enterocolic mast cell aggregates. Collections of atypical mast cells are occasionally detected in the intestinal mucosae of asymptomatic adult patients with no evidence of a systemic mast cell disorder. These aggregates are morphologically indistinguishable from those associated with aggressive systemic mastocytosis. Abnormal mast cells and irregular sheets of eosinophils expand the lamina propria and efface the crypt architecture (A). Spindled and ovoid mast cells contain elongated nuclei with dense, homogeneous chromatin and pale cytoplasm (B).

LANGERHANS CELL HISTIOCYTOSIS Langerhans cell histiocytosis affects the small intestine or colon in two situations. Pediatric patients with multisystem involvement by Langerhans cell histiocytosis (e.g., Letterer-Siwe disease) can develop gastrointestinal involvement, in which case they typically present with failure to thrive and protein-losing enteropathy (357). These patients have multiple ulcerated lesions or thickened and irregular folds throughout the small intestine or colon. Nearly 50% of such cases harbor BRAF V600E mutations, and thus, patients may be offered BRAF inhibitor therapy (358). On the other hand, adult patients usually have a solitary polyp or ulcerated nodule in the colon. These patients are generally asymptomatic and have an excellent prognosis (359). Solitary and multiple lesions are histologically similar. Infiltrates are centered on the mucosa, which is expanded by ovoid cells with large round or bean-shaped nuclei that show longitudinal grooves (Fig. 34.79A). Larger cells contain multilobate nuclei and abundant faintly eosinophilic cytoplasm (Fig. 34.79B). They are intimately associated with a mixed inflammatory cell infiltrate rich in eosinophils. Lesional cells express CD1a, S100 protein, and CD207 (langerin).

FIGURE 34.79 Langerhans cell histiocytosis. These mucosal aggregates of Langerhans cells were detected in a polyp of an average-risk patient undergoing screening colonoscopy. Sheets of large cells expand the mucosa (A). They contain abundant faintly eosinophilic cytoplasm and irregularly shaped, convoluted nuclei (B).

FOLLICULAR DENDRITIC CELL SARCOMA Follicular dendritic cell sarcomas contain cells that simulate features of antigen-presenting cells found in lymphoid follicles. They show no age or gender predilection and rarely cause systemic manifestations (360). Abdominal symptoms are related to a mass lesion, including pain, diarrhea, and gastrointestinal bleeding. Tumors are multinodular masses that can show cystic degeneration or frank necrosis when large (361). Most follicular dendritic cell sarcomas contain plump spindled or ovoid cells with faintly eosinophilic cytoplasm arranged in sweeping fascicles and storiform patterns, often with a whorled appearance. Elongated nuclei contain vesicular chromatin and small, conspicuous nucleoli. These tumors are quite cellular, and those with a greater degree of nuclear pleomorphism may show brisk mitotic activity. Follicular dendritic cell sarcomas characteristically contain small B-cells and T-cells dispersed throughout the tumor (361). They show strong immunopositivity for CD21, CD23, and CD35 and variable positivity for S100 protein and CD68 (362). Nearly 20% contain BRAF mutations (363). Some follicular dendritic cell sarcomas are EBV positive and display prominent lymphoplasmacytic infiltrates, often accompanied by granulomatous inflammation and eosinophils (364). These tumors tend to occur in young or middle-aged adults and show a predilection for women, particularly those of Asian descent. HISTIOCYTIC SARCOMA Histiocytic sarcomas can involve the small intestine and colon, producing symptoms related to obstruction or bleeding. These large, solitary masses are composed of sheet-like proliferations of large round or ovoid cells with abundant eosinophilic cytoplasm. The nuclei tend to be ovoid with vesicular chromatin and prominent nucleoli. Hemophagocytosis is sometimes present. Tumor cells are immunopositive for CD163, CD68, and lysozyme, with variable S100 protein staining (365).

THE VERMIFORM APPENDIX SERRATED APPENDICEAL LESIONS

Diffuse Mucosal Hyperplasia Diffuse mucosal hyperplasia is a common finding in appendectomy specimens, particularly when patients undergo delayed appendectomy following appendicitis. In this situation, the appendix features a circumferential proliferation of expanded crypts enriched in matureappearing goblet cells with slightly serrated architecture, often in combination with ruptured diverticula and organizing appendicitis. Mucosal hyperplasia can closely simulate the features of a low-grade appendiceal mucinous neoplasm, especially when accompanied by diverticula and extruded mucin (366,367). Clues to a diagnosis of hyperplasia include preserved crypt architecture, abundant lamina propria, relatively normal muscularis mucosae, and localized mural or subserosal mucin (Fig. 34.80A). Importantly, mucosal hyperplasia features numerous mature goblet cells rather than non–goblet mucinous epithelial cells (Fig. 34.80B). Similar to other hyperplasias elsewhere in the gastrointestinal tract, they can show KRAS mutations in approximately one-third of cases (367). This finding may account for the overrepresentation of reported KRAS mutations among appendiceal serrated lesions (368).

FIGURE 34.80 Postinflammatory hyperplasia of the appendiceal mucosa. An interval appendectomy specimen contains a ruptured diverticulum with organizing periappendiceal mucin and a circumferential proliferation of hyperplastic crypts (A). Unlike a mucinous neoplasm, the lesion features mature goblet cells and evenly spaced crypts supported by abundant lamina propria and a normal-appearing muscularis mucosae (B).

Hyperplastic Polyp Hyperplastic polyps protrude into the lumen but do not involve the entire appendiceal circumference. They are small, spanning no more than a few millimeters, and resemble microvesicular hyperplastic polyps of the colorectum. These polyps contain both goblet cells and non–goblet mucinous epithelial cells. The latter contain tiny vacuoles of cytoplasmic mucin. Unlike microvesicular hyperplastic polyps of the colorectum, however, appendiceal lesions infrequently harbor BRAF mutations, but may contain KRAS mutations (369,370). Sessile Serrated Lesion Appendiceal sessile serrated lesions occur in adults. Most are detected in appendectomy specimens that show appendicitis or right colectomy specimens obtained for other indications. Thus, their reported prevalence and epidemiology are influenced by factors that cause appendicitis or result in colectomy. For example, sessile serrated lesions of the appendix have been reported in association with carcinomas of the right colon or serrated polyposis, but these data are based on findings in right colectomy specimens obtained due

to neoplasia in the proximal colon (369,371). Appendiceal sessile serrated lesions share morphologic features with their colonic counterparts (Fig. 34.81A). They show plaque-like, circumferential expansion of the mucosa by elongated, often dilated crypts with lateral branching or budding above the muscularis mucosae (Fig. 34.81B). Mutually exclusive BRAF and KRAS mutations are detected in near-equal numbers of appendiceal sessile serrated lesions, suggesting that they do not progress via the classic serrated neoplastic pathway of the colorectum (369).

FIGURE 34.81 Sessile serrated neoplasms of the appendix. Nondysplastic sessile serrated lesions involve the luminal circumference, expanding the mucosa with a proliferation of elongated, serrated crypts (A). Crypts show persistent dilatation in the deep mucosa with budding or branching above the muscularis mucosae (B). Dysplastic serrated lesions also involve the circumference of the mucosa, with preservation of the muscularis mucosae (C). Serrated crypts are lined by dysplastic mucin-containing epithelial cells with enlarged, hyperchromatic nuclei (D).

Dysplastic Serrated Polyps Dysplastic serrated polyps of the appendix share overlapping features with colonic traditional serrated adenomas and sessile serrated polyps with dysplasia, although their morphologic features do not precisely align with those of colorectal polyps and multiple growth patterns may be seen in the same lesion. For these reasons, there is no clear advantage to subclassifying dysplastic serrated lesions as sessile serrated polyps with dysplasia and traditional serrated adenomas (282). Dysplastic serrated polyps are circumferential neoplasms supported by lamina propria and relatively normal muscularis mucosae, submucosa, and muscularis propria, which aids distinction from mucinous neoplasms (Fig. 34.81C). Lesional crypts may contain cells resembling those of a traditional

serrated adenoma or conventional colorectal adenoma, or contain dysplastic serrated crypts with lateral branching and budding in the deep mucosa (Fig. 34.81D). CONVENTIONAL (COLONIC-TYPE) ADENOMA Appendiceal adenomas resembling conventional adenomas of the colon are uncommon. Most examples are detected in appendices that accompany colectomy specimens from patients with familial adenomatous polyposis. Many conventional adenomas involving the proximal appendix represent extension of cecal adenomas into the appendiceal orifice. Unlike sessile serrated lesions, conventional adenomas of the appendix do not involve the full circumference of the lumen. They contain crowded crypts lined by dysplastic epithelial cells similar to those of tubular and villous adenomas of the colon (Fig. 34.82). These lesions can precede adenocarcinomas of the appendix.

FIGURE 34.82 Conventional adenoma of the appendix. A polyp projects into the lumen (A). It is composed of irregularly distributed crypts lined by cells with elongated, hyperchromatic nuclei (B).

MUCINOUS NEOPLASMS Mucinous neoplasms occur in both men and women and tend to affect older adults. Most show low-grade cytologic features and lack infiltrative invasion, even when they spread to other sites. The malignant behavior of these tumors despite their low-grade cytologic features and absence of destructive invasion have led to several misconceptions regarding their biologic potential and the widespread belief that these well-differentiated adenocarcinomas represented peritoneal spread of benign (i.e., adenomatous) epithelium. For this reason, they have been classified in the past as tumors of uncertain malignant potential or disseminated peritoneal adenomucinosis (372,373). The World Health Organization and others advocate use of the term “low-grade appendiceal mucinous neoplasm” for virtually all mucinous neoplasms with low-grade cytologic features (373). Although this approach simplifies the task of pathologists, application of “low-grade appendiceal mucinous neoplasm” to such a broad spectrum of lesions limits its clinical utility and increases the likelihood of misclassification of mucosal hyperplasia as a neoplasm. Available data overwhelmingly support the view that mucinous neoplasms confined to the appendix proper are benign, whereas those that spread to the serosa or peritoneum are at risk for progressive disease and essentially behave similar to adenocarcinomas (374). Thus, the argument can be made to classify benign lesions as mucinous adenomas, those with spread to the peritoneum as well-differentiated mucinous adenocarcinomas and reserve the

term “low-grade appendiceal mucinous neoplasm” to denote uncertainty regarding biologic risk. Mucinous Adenoma We use mucinous adenoma to denote a neoplasm confined to the appendiceal mucosa. These lesions may not produce any gross abnormalities or cause localized or fusiform appendiceal dilatation. They generally lack complex architectural features, displaying villous projections or flat, undulating epithelium (Fig. 34.83A). The crypts have mostly straight luminal edges, with slight serration near the lumen (Fig. 34.83B). The lamina propria is decreased compared to that of the nonlesional appendix. Most adenomas show low-grade cytologic atypia with enlarged, hyperchromatic nuclei, pseudostratification, and rare mitotic figures. High-grade dysplasia with complex architectural abnormalities, nuclear pleomorphism, and loss of cell polarity is uncommon. Appendiceal mucinous adenomas are cured by appendectomy, regardless of the degree of dysplasia.

FIGURE 34.83 Mucinous adenoma of the appendix. The appendix is diffusely dilated owing to accumulation of luminal mucin and epithelial proliferation (A). The lesion contains a circumferential proliferation of mucinous epithelial cells with a villiform architecture; the muscularis propria is essentially normal (B). Neoplastic cells are supported by thin fibrovascular cores and decreased lamina propria (C). These barrel-shaped mucinous cells contain abundant cytoplasm and slightly enlarged, basally located nuclei (D).

Low-Grade Appendiceal Mucinous Neoplasm We use “low-grade appendiceal mucinous neoplasm” to imply uncertain biologic risk, such as when the relationship between the neoplastic epithelium and muscularis mucosae is obscured and when mucin or epithelium extends through the wall. Such lesions usually produce localized or fusiform appendiceal dilatation with tenacious mucin that may be visible on the serosal surface (Fig. 34.84A). Tumors with acellular mucin on the serosa are at extremely low risk for progression to pseudomyxoma peritonei, whereas patients with any amount of neoplastic epithelium outside the appendix can develop peritoneal dissemination (375). Thus, extraappendiceal mucin should be entirely submitted for histologic evaluation. Appendices encased in mucin or replaced by mucinous neoplasia are almost always associated with peritoneal tumor deposits and should be classified as well-differentiated adenocarcinomas as described subsequently.

FIGURE 34.84 Low-grade appendiceal mucinous neoplasm. The dilated appendix contains thick, tenacious mucin (A). Neoplastic epithelium extends through the appendiceal wall with a diverticulum-like growth pattern; the distinctions between the lamina propria, muscularis mucosae, submucosa, and muscularis propria are obscured, and the muscularis propria is completely replaced by fibrosis at the point of deepest penetration by the tumor (B). Papillary projections lined by nongoblet, tall mucinous epithelial cells rest on lamina propria– depleted stroma with elastosis and fibrosis of the wall layers (C). Flat, slightly undulated epithelium contains barrel-shaped mucinous cells that show nuclear pseudostratification (D).

Low-grade appendiceal mucinous neoplasms have a villous, undulating, or flat lining, often with diverticulum-like growth of neoplastic epithelium in the wall (Fig. 34.84B). The mucosa contains markedly decreased lamina propria with atrophy of lymphoid tissue, mucosal fibrosis, and obliteration of the muscularis mucosae; fibrosis, hyalinization, or calcification of the appendiceal wall is generally present (Fig. 34.86C). Tall columnar cells are filled with slightly basophilic mucin (372). Tumor cells contain slightly enlarged, hyperchromatic nuclei that occupy less than one-third of the cell volume. Mitotic figures are infrequently identified (Fig. 34.84D). Paneth cells, endocrine cells, and absorptive cells are inconspicuous or entirely absent; their presence in abundance should raise the possibility of mucosal hyperplasia, especially when patients have a prior history of appendicitis. High-Grade Appendiceal Mucinous Neoplasm Rare appendiceal mucinous tumors show architectural features of low-grade mucinous neoplasms but display high-grade cytologic atypia. These tumors show mural fibrosis and broad, pushing invasion by overtly malignant mucinous epithelium. Micropapillary, cribriform, or flat proliferations of tumor cells display enlarged, pleomorphic nuclei with brisk mitotic activity and single-cell necrosis (372). They are assigned pathologic tumor stage similar to

appendiceal adenocarcinomas (374). High-grade appendiceal mucinous neoplasms harbor KRAS mutations and frequent TP53 alterations (376). Tumors that have spread beyond the appendix are commonly associated with peritoneal dissemination of high-grade disease. Mucinous Adenocarcinoma and Pseudomyxoma Peritonei Pseudomyxoma peritonei is a clinical term that describes massive mucin accumulation in the peritoneal cavity, accompanied by omental caking and gelatinous serosal deposits (Fig. 34.85A). Female patients can also develop mucin accumulation in the ovaries that simulates a primary neoplasm (Fig. 34.85B). Most cases represent metastases derived from mucinous appendiceal neoplasms. Patients are usually older adults, and men are affected at rates similar to women. Peritoneal tumor deposits consist of mucin pools and scant epithelium surrounded by hyalinized fibrosis. Low-grade mucinous adenocarcinomas contain strips of epithelium located at the peripheries of mucin pools without infiltrative invasion (Fig. 34.85C). Neoplastic epithelium shows mild cytologic atypia with a paucity of mitotic activity (Fig. 34.85D). High-grade tumors contain more cellular mucin pools with overtly malignant epithelium (Fig. 34.85E). Mucin pools may be small and infiltrative, or accompanied by invasive glands with desmoplasia. High-grade tumors that contain signetring cells have a worse prognosis and are distinguished from other high-grade tumors (Fig. 34.85F). Progression from low-grade to high-grade peritoneal disease can occur when patients with low-grade mucinous adenocarcinoma develop multiple recurrences. However, extensive high-grade features, including single infiltrative signet-ring cells, should lead one to suspect a nonappendiceal site of origin or metastasis from a non–mucinous appendiceal carcinoma (377).

FIGURE 34.85 Disseminated mucinous adenocarcinoma of the appendix. Mucinous adenocarcinomas derived from low-grade appendiceal lesions typically produce gelatinous ascites with omental caking (A), often accompanied by large ovarian metastases in female patients (B). Low-grade disease features large pools of mucin with scant peripheral strips of mucinous epithelium (C). Tumor cells contain abundant, slightly basophilic mucin and mildly atypical nuclei reminiscent of an adenoma (D). High-grade mucinous carcinoma is much more cellular. Floating cords of tumor cells and glands show a greater degree of nuclear atypia (E). Some high-grade tumors contain signet-ring cells, similar to high-grade mucinous adenocarcinomas of the colorectum (F).

The natural history of disease is dictated by tumor grade. Patients with low-grade mucinous adenocarcinoma in the peritoneum have 5- and 10-year survival rates of 75% and 68% compared with approximately 55% and 13%, respectively, for high-grade carcinomas (378). Treatment of low-grade disease consists of peritonectomy and resection of the omentum, uterus, ovaries, and fallopian tubes, as well as involved organs of the

gastrointestinal tract. Patients who can be surgically debulked with complete tumor cytoreduction are generally offered hyperthermic intraperitoneal chemotherapy (HIPEC) supplemented by additional cycles of early postoperative intraperitoneal chemotherapy (EPIC). Complete cytoreduction improves survival to approximately 86% at 5 years. Unfortunately, complete debulking may not be possible for patients with high-grade peritoneal disease, and thus, these individuals may be eligible for systemic therapy only. GOBLET CELL ADENOCARCINOMA (CRYPT CELL CARCINOMA) Goblet cell adenocarcinomas are mostly unique to the vermiform appendix; similar tumors may rarely occur in the colon. These tumors are more common among middle aged to older adults and affect both men and women equally. Most goblet cell adenocarcinomas confined to the appendix are incidentally discovered in patients with symptoms of acute appendicitis, whereas those that have spread beyond the appendix present with abdominal pain or a mass. Goblet cell adenocarcinomas tend to occur in the mid-to-distal region of the appendix, producing an ill-defined mural thickening that is difficult to identify at the time of gross examination (Fig. 34.86A). These tumors are composed of nests and tubules that resemble abortive colonic crypts. They permeate the deep mucosa and infiltrate the appendiceal wall in a circumferential manner, intimately associated with a densely collagenous stroma (Fig. 34.86B). Tumor cells have a globoid appearance because of distention by large cytoplasmic mucin vacuoles (Fig. 34.86C). At least 50% of goblet cell adenocarcinomas contain high-grade areas that display signet-ring cell, mucinous, intestinal, or poorly differentiated areas (Fig. 34.86D). Tumors are graded using a three-tier system based on the extent of low-grade areas (379). Perineural invasion is frequently present and has no bearing on prognosis. Goblet cell adenocarcinomas show alterations affecting WNT signaling, but alterations in KRAS, DPC4, and TP53 typical of colorectal adenocarcinomas are lacking (380).

FIGURE 34.86 Goblet cell adenocarcinoma of the appendix. Goblet cell neoplasms produce ill-defined circumferential thickening of the appendiceal wall (A). The contain nests of tumor cells that resemble crypts embedded in dense collagenous stroma (B). Large vacuoles of basophilic mucin compress peripherally arranged nuclei in low-grade areas (C). High-grade areas show infiltrating single cells and cords of non–goblet cells (D).

Goblet cell adenocarcinomas show a predilection for metastasis to regional lymph nodes, peritoneum, and ovaries. Their management is not standardized, although many patients with incidental tumors in appendectomy specimens undergo subsequent right colectomy with lymph node staging. Extraappendiceal tumor deposits often contain high-grade carcinomatous elements, particularly signet-ring cells. Prognosis is related to tumor stage and grade. OTHER ADENOCARCINOMAS Appendiceal adenocarcinomas show a predilection for older adults. Mucinous and signetring cell carcinomas are slightly more common among women, whereas other types of carcinoma are more frequent among men. Patients may present with appendicitis symptoms, but most have a palpable or radiographically apparent mass in the right lower quadrant. Any epithelial neoplasm of the appendix can give rise to adenocarcinoma, which frequently shows some degree of mucinous differentiation regardless of the nature of the precursor lesion. Appendiceal adenocarcinomas resemble their colonic counterparts and show intestinal-type, mucinous, or signet-ring cell differentiation. Carcinomas associated with serrated precursors feature infiltrative glands with a tubular or serrated appearance and high-grade cytologic features accompanied by extracellular mucin (Fig. 34.87).

FIGURE 34.87 Invasive appendiceal adenocarcinoma. A high-grade adenocarcinoma is associated with a serrated neoplasm involving the circumference of the appendix (A). Irregularly shaped infiltrative glands have serrated contours. Pools of mucin surround ruptured and dilated malignant glands (B).

WELL-DIFFERENTIATED ENDOCRINE NEOPLASMS Endocrine tumors are identified in less than 1% of appendectomy specimens and are more common among adults. They occur in a slightly younger age group than other gastrointestinal endocrine tumors, probably reflecting the frequency of appendectomy procedures among young patients. Appendiceal endocrine neoplasms are graded and staged in accordance with the eighth edition of the AJCC TNM Staging Manual (381). Most tumors behave in a benign manner. Less than 1% of tumors spanning less than 2 cm metastasize, and those that do metastasize spread first to regional lymph nodes followed by the liver. Thus, lesions ranging up to 1 cm are generally treated with appendectomy alone, whereas right colectomy with lymph node dissection is considered when tumors are larger than 2 cm. Tumor grading is assigned based on a combination of mitotic activity and extent of Ki-67 staining. Enterochromaffin Cell Endocrine Neoplasm (Classic Carcinoid Tumors) Enterochromaffin cell endocrine tumors are histologically similar to endocrine tumors of the ileum and cecum. They show a predilection for the distal appendiceal tip and appear as firm yellow nodules (Fig. 34.88A). Tumors are composed of sheets of large, tightly packed nests and acini that feature polygonal cells with abundant cytoplasm. They may contain eosinophilic granules. Tumor cell nuclei are round with coarse chromatin and small nucleoli. Enterochromaffin cells show strong immunopositivity for chromogranin A, synaptophysin, and serotonin. Most (70%) tumors are smaller than 1 cm and show a low (135 mg/dL) in many patients, but it may be normal in up to 40% of patients with biopsy-proven AIP type 1 (166). To date, there have been studies of AIP and antibodies to lactoferrin, carbonic anhydrase isoforms II and IV, pancreatic secretory trypsin inhibitor (PSTI; product of the SPINK1 gene), as well as to less sensitive or specific markers of autoimmunity, such as antinuclear antibody and rheumatoid factor association. Although there are some strengths of association with PSTI antibodies, none of these biomarkers appears to be sensitive or specific enough to serve as distinctive evidence of AIP (167-169). There is a localized to diffuse swelling of the pancreas, centered in the head, with irregular narrowing of the pancreatic ductal system (162-165,170-175). The characteristic appearance on CT imaging for diffuse pancreatic involvement is a sausage-shaped enlargement with homogeneous attenuation, moderate enhancement, and a peripheral halo at the rim of hypoattenuation. Although these findings may mimic a pancreatic head carcinoma (in focal disease), pancreatic ductal narrowing in the pancreas is highly suggestive of AIP. ERCP shows focal, diffuse, or segmental

attenuation of the main pancreatic duct with loss of right angle branches (165,171). EUS is also used, especially to guide FNA or biopsy of the hypoechoic parenchyma (171,172,175-177). The three major histopathologic features associated with AIP type 1 (and IgG4-related disease in general) are the following: 1. Dense lymphoplasmacytic infiltrate with transmigration into the ductal epithelium (ductitis) (Fig. 35.17) 2. Venulitis that may progress to obliterative phlebitis (Fig. 35.18) (150,152154,157,158,160,165,170,174,178-180) 3. Fibrosis of the pancreatic parenchyma that can vary in distribution and extent

FIGURE 35.17 Type 1 autoimmune pancreatitis. One of the major histopathologic features is a dense rim of (bandlike) periductal inflammation composed of numerous lymphocytes, plasma cells, as well as eosinophils.

FIGURE 35.18 Type 1 autoimmune pancreatitis. Vascular involvement is typical. In the most typical lesions, the inflammatory cells undermine the endothelium and produce fibrous obliteration of the veins. The remainder of the lumen appear like a defect in the middle of an inflammatory focus (obliterative phlebitis).

The inflammatory infiltrate is composed of lymphocytes that are predominantly T cells, scattered aggregates of B cells, plasma cells, and eosinophils, which may be prominent enough to raise the

possibility of “eosinophilic pancreatitis.” Although the inflammatory cells tend to aggregate around and within the ducts (ductitis) (162-164,171,174,181), this infiltrate invariably extends into the lobules, the peripancreatic adipose tissue, veins (venulitis), as well as the intrapancreatic portion of the bile duct. Pancreatic parenchymal fibrosis can vary and may be mild to marked fibroblasts/myofibroblasts buried within the inflammatory infiltrate. Among long-standing cases of AIP type 1, a storiform pattern of fibrosis can be observed (158). In rare cases, the amount of fibroblastic activity resembles inflammatory myofibroblastic tumor (162,182). The presence of venulitis may progress to a dense lymphoplasmacytic infiltrate that obliterates peripheral veins (obliterative phlebitis) (183). Fully obliterated veins may require elastin stains for identification. Obliterative thrombophlebitis, especially involving larger venules, appears to be significantly more common in AIP type 1 than in other inflammatory injury of the pancreas, including tumors (183). Calcification, fat necrosis, and cyst formation are not seen. It is important to note that the histologic features of AIP type 1 can closely mimic histologic findings seen with pancreatic involvement by Rosai-Dorfman disease, which is a rare histiocytic disorder (184). Based on immunohistochemical studies, the majority of plasma cells are positive for IgG4 (Fig. 35.19). The finding of more than 50 IgG4+ plasma cells/high-power field (hpf) is considered highly specific for AIP type 1 (150,158,165,185-187). On biopsy specimens, the presence of more than 10 IgG4+ plasma cells/hpf has been proposed as one component of a comprehensive diagnostic panel (171). However, an elevated IgG4+-to-IgG+ plasma cell ratio of more than 40% is more meaningful than IgG4+ plasma cell counts alone in establishing the diagnosis (158,160). Of note, there is no gold-standard approach for counting IgG4+ plasma cells. Because the IgG4+ cell distribution may be patchy, counting only areas of intense IgG4 focus (“hot spots”) might be more representative. It should be kept in mind, though, that neither an increase in serum IgG4 nor the finding of elevated numbers of IgG4+ plasma cells in tissue is specific for AIP type 1 (or IgG4related disease in general). Thus, the diagnosis of AIP type 1 requires both characteristic histologic features (described earlier) and increased numbers of IgG4+ plasma cells (or an elevated IgG4+-toIgG+ ratio) in tissue (158).

FIGURE 35.19 Type 1 autoimmune pancreatitis. The majority of plasma cells are positive for immunoglobulin G4.

To identify the full spectrum of changes occurring in AIP, one must recognize its five cardinal features (the Mayo Clinic’s HISORt criteria): suggestive Histology showing lymphoplasmacytic infiltrate with storiform fibrosis, Imaging showing a diffusely enlarged pancreas, Serology showing elevated IgG4 levels, or evidence of Other organ involvement and Response to steroid therapy

(171,188). AIP should be suspected in patients with obstructive jaundice, pancreatic mass/enlargement, or pancreatitis who have one or more HISORt criteria (171,188). Type 2 Clinical data from histologically confirmed AIP type 2 (previously known as idiopathic duct-centric pancreatitis) cases show that they have distinctly different profile compared with AIP type 1 cases. AIP type 2 seems to be a pancreas-specific disorder. It is not associated with either other organ involvement or with serum IgG4 elevation typically seen in AIP type 1. However, lack of other organ involvement or the absence of serologic abnormalities in patients with AIP does not necessarily imply the diagnosis of type 2, as type 1 also can be without other organ involvement and seronegative. Although inflammatory bowel disease seems to be associated with both forms, these are more common in type 2. Approximately 30% of reported cases of AIP type 2 have associated inflammatory bowel disease, such as ulcerative colitis. Patients with AIP type 2 are, on average, a decade younger than AIP type 1 patients and do not show a sex predilection. Currently, AIP type 2 lacks a serologic biomarker (152,154). The most distinctive feature of the AIP type 2 is a dense periductal lymphoplasmacytic inflammation accompanied by neutrophilic microabscesses within the lumen (Fig. 35.20), the socalled granulocytic epithelial lesion (GEL), involving medium- and small-sized ducts as well as in acini (150,152,153-156,165,189). The inflammation is generally not as dense as that seen in type 1, although it often leads to the destruction and obliteration of the duct lumen. The interlobular fibrosis lacks fibroblastic/myofibroblastic cell infiltrate, and storiform-type fibrosis is rarely prominent. Although some veins are focally involved by the lymphoplasmacytic infiltrate, overt obliterative phlebitis is uncommon. AIP type 2 cases has none or very few (90%) (249,288-296,457). The tumor-suppressor gene SMAD4 is found on chromosome 18q21.1 and encodes for a nuclear transcription factor involved in the transforming growth factor (TGF)-β signaling pathway. SMAD4 alterations are reported to be inactivating and present in approximately 50% of PDACs. Moreover, loss of SMAD4 by immunohistochemistry in PDAC is associated with worse patient clinical outcome (290,297-300). Considering SMAD4 loss is late finding in the pathogenesis of PDAC, it can be used for diagnostic purposes in scenarios where the differential diagnosis is between PDAC and reactive (nonneoplastic) ducts, such as those associated with chronic pancreatitis. Analysis of gene expression has revealed a number of other overexpressed molecules within PDAC (e.g., fascin, mesothelin, claudin-4, S100AP, S100A6, and S100P), some of which have been proposed as potential diagnostic immunohistochemical markers (301-305). A list of prognostic factors for exocrine neoplasms of the pancreas is illustrated in Table 35.4, although it is by no means exhaustive. TABLE 35.4 Prognostic Factors Associated With Prognosis in Exocrine Neoplasms of the Pancreas Invasion Tumor type Tumor grade Tumor size and local extension (duodenum, soft tissue, stomach, spleen, etc.) Location Multicentricity Angiolymphatic invasion Perineural invasion Resection margin status Lymph node metastases Distant metastases Unresectability Total pancreatectomy CA19-9 levels >400 IU/mL Up to 10% of patients with pancreatic cancer have a family history of this disease (283,306,307). Also, some pancreatic cancers arise in patients with recognized genetic syndromes, including hereditary pancreatitis, familial atypical multiple mole melanoma, BRCA2 kindred, Peutz-Jeghers syndrome, and in Lynch syndrome families (247,304). CDKN2A appears to be the molecular link in the patients with familial atypical multiple mole melanoma (FAMMM) syndrome (308,309).

Morphologic Variants of Pancreatic Ductal Adenocarcinoma In addition to the fundamental histologic pattern described earlier, PDACs can exhibit a variety of morphologic variants that may be helpful in their accurate diagnosis. Foamy Gland Variant. The foamy gland variant is characterized by abundant very pale foamy/microvesicular cytoplasm, in which the vesicles are very fine and even. The glands are well formed, and the nuclei are polarized to the periphery of the neoplastic cells; thus, the overall pattern is deceptively benign in appearance. In addition to its distinctive cytoplasmic characteristics, this variant can be distinguished from benign noninvasive ducts by the common presence of an apical chromophilic condensation in the apical membrane, forming a brush border–like zone (Fig. 35.33). The cytoplasmic borders are also distinct. Moreover, the nuclei, if pushed at the periphery, are often hyperchromatic and raisinoid, with frequent irregularities caused by the vesicles indenting the nucleus. If the nuclei are more central and preserved, then the contour irregularities are less

prominent, but nucleoli may be visible. Occasionally, foamy cells form stromal clusters, mimicking collections of macrophages. Foamy cell change is also fairly common in cases after neoadjuvant treatment (310).

FIGURE 35.33 Ductal adenocarcinoma, foamy gland variant. Apical chromophilic condensation in the apical membrane forming a brush border–like zone (highlighted by mucicarmine stain) is characteristic.

Large Duct Variant. Although most PDACs are characterized by small tubular pattern, in some cases, the infiltrating glands may be fairly large and well defined and thus mimic preinvasive neoplasia (311,312), such as PanINs or intraductal neoplasms. In fact, the tubular elements can be so large and cystic that these have been also mistaken as another precursor to PDAC, IPMNs. However, the large duct variant of PDAC often has very angulated contours and wide, open lumen formation (Fig. 35.34) in contrast with preinvasive neoplasia, in which duct contours are typically smooth and undulating, and the lumen is compressed or filled with epithelial elements. In addition, in large duct variant of PDAC, although some ducts may have well-organized papillary elements within, in most units, the epithelium will be flat or irregular. Often, the cytologic features are foamy variant or show other subtle features described for adenocarcinomas earlier. Partial duct rupture is not uncommon, and often, the lumen has neutrophils or necrotic, granular debris (Fig. 35.35).

FIGURE 35.34 Ductal adenocarcinoma. Microscopically, large duct variant is characterized by irregularly distributed cystically dilated ducts. Diameter of the majority of these ducts range from 0.5 mm to 1 cm.

FIGURE 35.35 Ductal adenocarcinoma. Contour irregularities are especially pronounced in the large duct variant, in which intraluminal debris and neutrophils are also prominent. Some glands may even be partially ruptured. Note foamy cells with distinct cytoplasmic borders and hyperchromatic and raisinoid nuclei on the left upper corner.

Although most PDACs are solid scirrhous lesions, some cases can present as a cystic lesion. There are several mechanisms for this to develop. Some examples appear to be a markedly cystic version of the large duct variant, whereas others appears to be due to secondary duct ectasia of the upstream pancreatic ductal system that leads to the cystic mass. Often, the cyst lining shows colonization (cancerization) by invasive glands. In some cases, the cystic component is attributable to a residual IPMN component that is now replaced by the PDAC. Poorly Cohesive (With/Without Signet-Ring Cells) Variant. Focally, a PDAC can exhibit cordlike and even individual cell infiltration pattern, but this is invariably in the context of and closely admixed with a conventional tubular pattern of PDAC. In fact, abortive glandular formation is often evident. Furthermore, these areas typically have significant cytologic atypia and pleomorphism that is unusual for poorly cohesive–type carcinomas arising from other sites within the GI tract. The monotony and insidious pattern that characterizes gastric poorly cohesive carcinoma, mammary lobular carcinoma, or plasmacytoid urothelial carcinoma is typically nonexistent in PDACs. In fact, if a diffuse infiltrative carcinoma with the usual pattern is identified in the pancreas, this typically proves to be of ampullary or duodenal origin rather than pancreatic or a metastasis from another site. Papillary Adenocarcinoma Variant. Papilla formation is a common and defining feature of ductal differentiation in the pancreas. As such, any ductal neoplasm in the pancreas may show some papilla formation. For tumors like IPMN and ITPN, the papilla formation is an integral part of the tumor that it has been incorporated into their name. Florid papillary nodules, detectable grossly, are also seen in approximately 15% of MCNs. Moreover, papilla formation can be observed in PanINs and PDACs, and especially in the large duct variant of PDAC. The cases reported in the literature under the heading of “papillary adenocarcinoma” are likely cases that would be classified today as either a pancreatobiliary-type IPMN or a large duct variant of PDAC. In addition, ampullary and extrahepatic cholangiocarcinomas can also exhibit papilla formation and ought to be considered in the differential diagnosis. A true micropapillary pattern, with solid clusters of cells suspended in lacunar spaces, usually with intraepithelial and stromal tumor-infiltrating neutrophils, is very rare in the pancreas. In fact, if there is a poorly differentiated carcinoma with extensive micropapillary pattern in the pancreas, it typically proves to be of ampullary origin (313). Cytologic Diagnosis

Aspirates from PDAC yield specimens of variable cellularity (Fig. 35.36), and EUS-guided FNA is the most common modality used in current practice. Consistent cytologic features include a paucity of acinar cells and increased numbers of ductal-type epithelium that are arranged in sheets, threedimensional clusters, or single cells. These findings contrast that of normal pancreatic ductal epithelium, which form large, flat, and cohesive sheets of cells that are evenly dispersed and have well-defined borders. Contaminants from the GI tract may also be problematic; however, these can be distinguished from adenocarcinoma by their honeycomb arrangement, the presence of goblet cells, and a distinct brush border. The latter two features are often seen in duodenal epithelial contaminants, but not in gastric epithelium. The neoplastic cells of PDAC vary in size, shape, and degree of cohesiveness. Cell borders are usually easily identified, and tumor cells have scant-toabundant cytoplasm. When tumor cells form disorganized “drunken” honeycomb sheets, their nuclei are often only minimally enlarged, and cells may show palisading, slight crowding, or overlapping. Nuclear enlargement is a very helpful feature in the diagnosis of PDAC, especially when well differentiated and if benign clusters are available for comparison (Fig. 35.37). The degree of nuclear atypia and cellular cohesion varies according to the degree of differentiation of the carcinoma (Figs. 35.36, 35.37, 35.38, 35.39). For less differentiated tumors, more bizarre nuclei and single cells are seen. A greater than 4:1 variability in nuclear size (the so-called fourfold anisonucleosis) is considered a very helpful diagnostic clue, although this refers only to a given cell cluster and not the entire specimen (314,315). When present, foamy gland features (which include foamy/microvesicular cytoplasm; distinct cytoplasmic borders; mild cellular disorganization; and hyperchromatic, irregular, raisinoid nuclei) are also helpful clues to the diagnosis (314). In addition, the presence of large intracytoplasmic vacuoles containing targetoid mucin or debris is also diagnostic of PDAC. Marked nuclear contour irregularity is seldom a prominent feature in benign conditions (like chronic pancreatitis) as it is in adenocarcinoma. In some examples of welldifferentiated adenocarcinoma, the nuclei appear more hypochromatic than hyperchromatic and may be easily overlooked as benign ductal cells if one is not careful (315).

FIGURE 35.36 Fine-needle aspiration: ductal adenocarcinoma. Many atypical epithelial cells are present in loosely cohesive groups and as scattered single cells.

FIGURE 35.37 Fine-needle aspiration: ductal adenocarcinoma, moderately differentiated. Neoplastic cells have larger and more irregular nuclei than the benign ductal cells.

FIGURE 35.38 Fine-needle aspiration: ductal adenocarcinoma, well differentiated. Note slight variation in nuclear size and shape and a suggestion of a glandular arrangement.

FIGURE 35.39 Fine-needle aspiration: ductal adenocarcinoma, moderately differentiated. Cells show moderate variation in nuclear size and shape.

Differential Diagnosis in Surgical Specimens For the pathologist, the principal problems relate to distinguishing PDAC from chronic pancreatitis and other pancreatic neoplasms. Chronic inflammation and fibrosis accompany PDAC (247,287), and any pancreatic neoplasm that obstructs the main pancreatic duct or its major branches may cause ductal dilatation, parenchymal atrophy, and fibrosis, thereby confusing the gross anatomic findings and, possibly, the microscopic features (Table 35.5) (247). TABLE 35.5 Comparison between Chronic Pancreatitis and Ductal Adenocarcinoma Chronic Pancreatitis

Ductal Adenocarcinoma

Distribution of lesions

Focal, segmental, or diffuse

Most common in head

Gross appearance

Irregular scarring; homogeneous fibrosis, ductal cysts; paraduodenal abnormalities stones

Hard, poorly demarcated solitary mass

Light microscopic features

Atrophy; fibrosis; chronic inflammation of variable degree with preservation of basic lobular architecture Ductal dilatation; protein plugs and intraductal calcifications in some cases Atrophy, hyperplasia, and metaplasia of ductal epithelium with minimal atypia

Abnormal distribution and ectopic localizations of tubules (intravascular, perineural, next-to-medium-sized thickwalled vessels, or in the form of isolated solitary ducts) Contour irregularities Large, irregular nuclei; conspicuous nucleoli; frequent mitoses; foamy cytology Necrotic cells in some glands or tubules

Islets of Langerhans

No alterations (early stages) Abnormal (late stages)

Minor abnormalities

Associated lesions

Pseudocysts

Obstructive changes

Fundamentally, the most important differential is with benign noninvasive ducts (140,316). Because there are no myoepithelial or basal layers, unlike in the breast or the prostate, there are no immunohistochemical markers to determine noninvasive ducts. Therefore, cytoarchitectural changes, associations, and the distribution are to be relied upon for this distinction. However, PDAC has a very peculiar ability to form very well-differentiated units not only in the stroma but also when they infiltrate into other structures including the nerves and vessels. In fact, even in poorly differentiated cases, the cells that infiltrate into the nerves and vessels may form well-defined ductal units that can mimic native ducts (140). In low-power examination, the lobularity of the process, its organoid nature, and formation of welldemarcated nodules are helpful to distinguish adenosis-type lesions from invasive carcinomas. Invasive carcinomas often show haphazard distribution and contour irregularities (140). Invasive ducts are often localized in places they do not belong. For example, the presence of small- to medium-sized ducts in the interlobar region, the presence of ducts next to medium-sized/thickwalled blood vessels, perineural invasion, ducts surrounded by a thin cuff of smooth muscle cells (which is indicative of vascular invasion), isolated solitary ducts in adipose tissue (without any accompanying acini or islets), and the presence of ducts in duodenal musculature (away from ampulla major or ampulla minor region) are all in keeping with adenocarcinoma (140,204,247,278,317,318). In addition to angulated contours, invasive adenocarcinoma glands often show open, round lumina formation, in contrast with the compressed lumen and undulating contours of noninvasive

glands (Fig. 35.40). Intraluminal debris with necrotic material or neutrophils and vacuolations within the ductal structure are also features of adenocarcinoma (319). In contrast, the presence of enzymatic (corpora amylacea–like) secretions is more in keeping with a benign process (140,204).

FIGURE 35.40 Comparison of well-differentiated adenocarcinoma (A) and chronic pancreatitis (B).

In terms of cellular and nuclear findings of PDAC, many of the features described in the section “Cytologic Diagnosis” are also applicable to histologic preparations. Typically, the nuclei are enlarged, hyperchromatic, and pleomorphic, and there is loss of polarity in adenocarcinomas. In contrast with atrophic acini with ductulization, which typically show a basophilic appearance because of the attenuation of acinar cytoplasm and paradoxical high nuclear-to-cytoplasmic ratio, carcinoma cells typically acquire fair amount of acidophilic cytoplasm. If the cytoplasm is abundant, the presence of foamy gland features including pale microvesicular cytoplasm, raisinoid nuclei, and apical chromophilic condensation at the apex are characteristic (310). Among stromal changes, there is significant overlap between benign conditions and PDAC. However, the presence of myxoid basophilic stroma, especially with vacuolation, is significantly more common in PDAC. Individual hyperchromatic boxcar-shaped nuclei in the background of a paucicellular stroma without granulation tissue–type changes are also often indicative of an adenocarcinoma (140,204). Differential Diagnosis from Similar Tubular Adenocarcinomas PDACs are morphologically very similar, if not identical, to cholangiocarcinomas. In fact, for a biopsy from the pancreatic head, it is preferable to designate the carcinoma as “pancreatobiliary” because its precise origin cannot be determined definitively without consideration of advanced imaging. Pancreatobiliary adenocarcinomas also have substantial overlapping features with other foregut carcinomas, in particular, esophageal and gastric tumors, not only at the morphologic level but immunohistochemically as well (261). Subtle findings may have to be relied upon. Whereas intestinal-like features, poorly cohesive cell pattern, and clear cell pattern (with the nuclei located suprabasally) are much more common in gastric carcinomas, in contrast, foamy gland, vacuolated, and large duct patterns are more common in pancreatobiliary adenocarcinomas. Ovarian carcinomas can also have overlapping features with pancreatobiliary adenocarcinomas. This extends to the clinical findings, in particular, abdominal carcinomatosis and increased serum CA19-9 or CA125 levels. The findings that favor ovarian origin are overall basophilia, formation of

large interconnecting and anastomosing glandular elements with projection and papillary configuration, and, if present, the monotony of nuclei with prominent nucleoli. Although their specificity has not been tested extensively, a panel composed of PAX8, ER, PR, SMAD4, CK7, and CK20 may be helpful in distinguishing ovarian serous carcinomas from pancreatobiliary adenocarcinomas in omental or peritoneal biopsies, which often proves to be a challenging differential diagnosis. ER−/PR−/PAX8−/SMAD4− phenotype would point toward pancreatobiliary adenocarcinomas, whereas ER+/PR+/PAX8+/SMAD4+ would favor an ovarian primary (261,320). If present, coexpression of CK20 and, less reliably, CK7 would also be more compatible with a diagnosis of a pancreatobiliary adenocarcinoma (321). In the liver, metastatic small intestinal and colonic adenocarcinomas may mimic metastatic PDAC and cholangiocarcinoma. The findings that favor colonic origin are overall basophilia, a columnar appearance of nuclei with pseudostratification, larger glandular units with branching, and intraluminal necrotic material (dirty necrosis). If present, the vacuolated pattern of pancreatobiliary adenocarcinomas, which is quite different than the cribriform pattern of intestinal carcinomas, can be helpful in this distinction. Immunohistochemistry may also aid in favoring one origin over another, with a CK7+/CK20−/CDX2− profile being in favor of pancreatobiliary adenocarcinomas, whereas intestinal tumors showing the opposite pattern (261). Of note, in the appropriate context, demonstration of albumin by RNA in situ hybridization would be supportive of an intrahepatic cholangiocarcinoma. A peculiar aspect of metastatic pancreatobiliary adenocarcinoma is its predilection to undergo a multilocular cystic transformation when it metastasizes to the ovary and may be misdiagnosed as a primary ovarian mucinous tumor. This is particularly challenging owing to the ability of metastatic pancreatobiliary adenocarcinoma to exhibit borderline-like and cystadenomatous growth patterns when involving the ovary. Fortunately, metastatic pancreatobiliary adenocarcinomas to the ovaries are commonly bilateral, only moderately enlarged with a mean/median size of less than 12 cm, and frequently nodular. These features have been established as useful for confirming that mucinous tumors in the ovary are likely to be metastatic (322,323). In addition, immunohistochemical staining for SMAD4 can be a useful as loss of SMAD4 expression is seen in 60% of ovarian metastases; however, preserved expression of this protein neither confirms nor refutes a diagnosis of metastatic pancreatobiliary adenocarcinoma (324). Similarly, pancreatobiliary metastases in the lungs also often mimic primary mucinous bronchioloalveolar carcinomas due to their striking lepidic growth and mucinous cytology. If present, foamy gland cytology and the degree of cytologic atypia especially in the glands between the alveolar spaces are helpful to the diagnosis. Similar to ovarian metastases, SMAD4 immunohistochemistry can also be a helpful adjunct; however, SMAD4 loss is only seen in 40% of metastatic pancreatobiliary adenocarcinomas to the lung. Furthermore, a small subset of non–small cell lung cancers can exhibit loss of SMAD4 (325). Interestingly, KRAS p.G12C mutations are preferentially found in primary lung adenocarcinomas and rarely encountered in metastatic pancreatobiliary adenocarcinomas to the lung (326). Surgical Pathology Reporting and Prognostic Evaluation Although conventional tubular formation is identifiable in virtually all PDACs, a significant proportion of the cases have nonglandular patterns in a mixture. There are various grading systems that have been proposed. The grading scheme advocated by the American Joint Committee on Cancer (AJCC) staging system utilizes the amount of tubule formation, with 95% for well, 50% to 95% moderate, and less than 50% for poor differentiation. Recently, a grading scheme similar to Gleason for prostate, also incorporating the presence of ill-defined glandular patterns (similar to Gleason pattern 4), was proposed (273). In daily practice, however, most pathologists appear to apply a gestalt approach rather than any of these specific grading schemes. For staging, surgeons and oncologists fundamentally classify PDAC into three categories: resectable, locally advanced, and metastatic. The College of American Pathologists (CAP) synoptic

reporting of resectable PDAC utilizes the AJCC/TNM staging system (276). Fundamentally, Tstaging is based on the size of the tumor. Accurate measurement of the size of PDAC requires close correlation of subtle gross findings with microscopic confirmation, which can be difficult if the sampling is not done systematically. As discussed later, T-staging of neoadjuvant-treated PDAC based on tumor size also presents its own challenges. In contrast, N-staging of resectable PDAC is less contentious but requires complete inspection of all regional lymph nodes from the specimen. This often requires submission of the entire peripancreatic soft tissue. As for margins, there are three specific structures on which all authors agree to be reported for pancreaticoduodenectomy specimens: the pancreatic neck, common bile duct, and uncinate process (superior mesenteric artery/retroperitoneal margin). There are other surfaces of the pancreatoduodenectomy specimen of which authors express different views. The vascular bed or groove, where the portal and mesenteric veins lie originally, is regarded as a part of the uncinate margin by some (327), but a specific surgical margin by others. Many investigators report this as a separate surface (vascular bed) (328), considering it may provide an important landmark for postsurgical radiotherapy if involved. The posterolateral surface of the pancreas covered by serosa where it joins to the duodenum is also regarded as part of the “retroperitoneal margin” by some authors (328). However, the anterior free surface is not considered by most, although we report it (329-331). Other findings to be examined and documented microscopically include vascular and perineural invasion. Vascular invasion can manifest in different formats: carcinoma cells in thrombotic nodules, detached micropapillary clusters, and the most common and most challenging tubule formation with the carcinoma cells lining the endothelium. The latter often forms a very well-defined ductal structure and can be mistaken for normal ducts or PanIN (278,280). Evaluation of Neoadjuvant-Treated Cases Neoadjuvant treatment is increasingly employed in the management of PDAC, presumably selecting patients who would benefit from surgery. The tumors that have received prior treatment often show a distinctive sclerosis and cytologic changes, including vacuolization of cells, foamy cell changes, and individual hyperchromatic cells lying in the stroma. Blood vessels also often show reactive changes. Extensive sampling may be necessary to identify residual carcinoma in some cases. Complete remission with neoadjuvant therapy is extremely uncommon in PDAC, requiring examination of the entire specimen microscopically and reevaluation of the original biopsy material before making such a determination. In assessment of the response, a few schemes for the histologic grading of the extent of residual carcinoma in posttreatment pancreatectomy specimens have been proposed: Ishikawa et al proposed to group the tumor response into three categories based on the percentage of severely degenerative cancer cells, specifically into third one (332). Evans et al (333,334) proposed a fourtier grading system based on the percentage of tumor cell destruction (residual viable tumor cells): grade I, less than 10% tumor cell destruction; grade II, 10% to 90% tumor cell destruction; grade III, less than 10% viable-appearing tumor cells; and grade IV, no viable tumor cells. The CAP suggests using a four-tier grading system for the extent of residual carcinoma: grade 0, no viable residual tumor (pathologic complete response); grade 1, marked response (minimal residual cancer with single cells or small groups of cancer cells); grade 2, moderate response (residual cancer outgrown by fibrosis); and grade 3, poor or no response (extensive residual cancer) (329). Alternatively, Chatterjee et al (336) described a two-tier approach in classification of response: response group 1 includes the cases with pathologic complete response (CAP grade 0) and those with minimal residual tumor ( 5 mm) intraductal epithelial neoplasm of mucin-producing cells. Within the literature, these cystic neoplasms have been referred to as ductectatic MCNs, mucinous duct ectasia, mucin-producing tumor, adenoma(tosis) of the ducts, and intraductal papillary neoplasms. Clinically, they are subdivided in three groups based on location within the pancreatic ductal system (42,419,421,453): 1. Branch duct type, which typically present as cystic tumors in the head of the pancreas, mostly in the uncinate (454,455), and, by definition, showing minimal, if any, dilatation of the main pancreatic duct 2. Main duct type is characterized by dilatation of the main duct, usually at least 7 mm, accompanied by nodularity (456-460). Main duct IPMNs may be extensive and may also involve the entire pancreas in some cases (461,462). 3. Mixed duct type is a combination of the other two types. Multifocality is well established in IPMNs; thus, pancreatectomy specimens ought to be carefully evaluated for the presence of multiple mucinous cysts (463-466). However, genetic studies have found multifocal IPMNs to be polyclonal, and these findings suggest that patients harboring multiple IPMNs may have an underlying field defect in IPMN tumorigenesis (467). Branch duct–type IPMNs have become one of the most common “incidentalomas” because of recent improvements and advancements in radiographic imaging modalities (468). If fact, it estimated that at least 1% of the adult population harbors an IPMN. Often, a small cystic lesion is detected in the head of the pancreas during a workup for other conditions (469). Consequently, IPMNs are found at a broad age range; however, they are significantly more common in the elderly. Median age at diagnosis is approximately 66 years, and there seems to be a slight male predominance (460,470-472). Those that are symptomatic often present with vague abdominal complaints related to ductal obstruction and low-grade pancreatitis (460,472). Main duct cases are more commonly associated with acute and/or chronic pancreatitis (473). Whether this is due to ductal obstruction leading to pancreatitis, or pancreatitis being a risk for the development of main duct–type IPMN, is unknown. But it is well documented that some patients present with episodes of acute pancreatitis but lack cholelithiasis or a history of alcohol abuse (42,236,240,277,455,458460,471,474-482). Bulging of the papilla into the duodenal lumen, seen radiographically, or a “fishmouth” appearance to the ampulla on esophagogastroduodenoscopy (EGD) is considered to be pathognomonic of a main duct–type IPMN. In addition to their association with PDAC, patients with IPMNs also have an increased incidence of other malignancies, synchronous or metachronous, suggesting careful preoperative evaluation (e.g., upper endoscopy and colonoscopy) is clinically advised (483-487). It is not clear whether this reflects a specific propensity in these patients to develop neoplasia or coincidence due to older age

or both. However, IPMNs have been linked to certain cancer-associated syndromes, such as PeutzJeghers syndrome and Lynch syndrome. A previous history of diabetes, especially with insulin use, chronic pancreatitis, and other tumors especially of the GI tract, and family history of PDAC have all been documented with higher frequency in patients harboring IPMNs (258,488,489). Whether these are true biologic associations, epiphenomena of old age or organ injury, or representation of a common propensity to develop these disorders has yet to be determined. Different management protocols have been devised for branch duct–type versus main duct–type IPMNs (419,458,490). Most incidental cysts of the pancreas prove to be branch duct IPMNs and are associated with a low incidence of malignant transformation. For this reason, routine surveillance is commonly employed for those that are not associated with patient symptoms, small in size (97% female) and, rather than of ductal origin, may arise from embryologic remnants of primordial germ cells that transmigrate into the pancreas during development (1,243,419,425,458,577,578). However, there are a few bona fide MCNs in males with definitive ovarian-type stroma (577,579-584).

MACROSCOPIC FEATURES MCNs typically form a thick-walled multilocular cyst in the body or the tail of pancreas (298,394,577,578,585). The neoplasms may be irregular in shape and have a well-developed capsule with a smooth, glistening (sometimes translucent) external surface (Fig. 35.67). They range up to 36 cm in diameter, with a large size predictive of a more aggressive biologic behavior (296,394,586). The tumor can be adherent to adjacent organs and may be surrounded by dense fibrosis (similar to a pseudocyst). Part of the surface may be irregular and dense because of this fibrosis or because malignant epithelium has extended through the capsule. Sectioning demonstrates the dense collagenous capsule of variable thickness, cavities of different sizes (the largest often several centimeters in diameter), thick mucoid contents, and (if present) papillary excrescences on the interior surfaces. Hemorrhage, degenerative changes, and loss of epithelium occasionally are extensive and, therefore, suggest a pseudocyst (Fig. 35.68). In most, there is no obvious communication with the ductal system, which distinguishes MCNs from IPMNs (236,240,577,585,587). The adjacent pancreatic parenchyma may exhibit compression atrophy.

FIGURE 35.67 Mucinous cystic neoplasm. A unilocular cyst that involves the pancreatic body/tail and does not communicate with the main pancreatic duct (yellow arrow).

FIGURE 35.68 Mucinous cystic neoplasm. The epithelium is lost, with a replacement by degenerated material. Note the heavy stromal fibrosis. These features alone are insufficient to diagnose a mucinous cystic neoplasm.

MICROSCOPIC FEATURES The neoplasms have columnar epithelium similar to that of the large pancreatic ducts (and colon) and have the requisite and characteristic ovarian-type stroma composed of plump spindle cells (Figs. 35.69, 35.70, 35.71, 35.72, 35.73, 35.74, 35.75) (296,421,474,578,588-591). Occasionally, the stroma contains foci of dense, hyalinized tissue, resembling the corpora albicantia of the ovary (Fig. 35.75) (296,394). The lining cells include nonciliated sialomucin-producing columnar cells, goblet cells, absorptive-type cells, cuboidal cells, and (rarely) Paneth cells. Epithelial loss and poor preservation may make examination more difficult. Abrupt transitions to papillary formations are common (Fig. 35.70) and range from microscopic to large complex structures visible to the naked eye. Similar to IPMNs, careful search of multiple sections frequently reveals a spectrum of architectural and cytologic atypia (Fig. 35.71) that has been traditionally graded as adenoma (lowgrade), borderline (intermediate-grade), or CIS (high-grade) and currently classified using a two-tier system consisting of low-grade and high-grade dysplasia (384,425,592). Typically, small (5 cm) and more complex examples with florid papillary nodules (393,586,595). In a recent study, 90% of invasive carcinomas arising in association with MCNs were found to be of the tubular type (694), with most of the remainder being an undifferentiated carcinoma with or without osteoclastic giant cells (581,596-598). Interestingly, colloid carcinomas, which can be found in the setting of an IPMN, are seldom, if ever, encountered in association with an MCN (594). There are also mesenchymal neoplasms that presumably derive from the ovarian stroma of these tumors, including pleomorphic sarcoma (Fig. 35.76) (599,600). These can be difficult to distinguish from an undifferentiated carcinoma (396,596,598).

FIGURE 35.69 Mucinous cystic neoplasm. The epithelium is generally regular with multiple papillary projections and cyst formation. There is a capsule of heavy fibrous connective tissue between the tumor and the pancreatic parenchyma (lower).

FIGURE 35.70 Mucinous cystic neoplasm. Note the higher grade dysplastic epithelium on the left.

FIGURE 35.71 Mucinous cystic neoplasm. There is bland cyst lining composed of columnar, mucin-producing cells (upper left) immediately adjacent to infiltrating adenocarcinoma within the ovarian-type stroma.

FIGURE 35.72 Mucinous cystic neoplasm. Part of the lining is ulcerated. Atypical epithelial cells, presumably carcinoma, lie in tiny vessels at the base of the ulcer.

FIGURE 35.73 Mucinous cystic neoplasm. Although the lining epithelium is bland, carcinoma has infiltrated the stroma.

FIGURE 35.74 Mucinous cystic neoplasm. The ovarian-type stroma can vary from cellular (left) to slightly more hyalinized (right).

FIGURE 35.75 Mucinous cystic neoplasm. In some tumors, “corpora albicantia”–type sclerosis is seen.

FIGURE 35.76 Mucinous cystic neoplasm. The edge of a sarcoma is visible in the stroma.

CYTOLOGIC DIAGNOSIS Cytology can identify the thick copious mucin and mucin-producing epithelial cells, but the ovariantype stroma is not usually identified in the samples. Therefore, MCNs cannot be reliably distinguished from IPMNs. However, with the advent of micro forceps biopsies, tissue sampling from the pancreatic cyst wall is possible and, in some cases, is able to sample the underlying wall with the identification of ovarian-type stroma. Diagnostic criteria and reporting of MCNs are identical to those discussed with IPMNs (1,526). IMMUNOHISTOCHEMICAL AND MOLECULAR FEATURES The epithelial cells express immunoreactivity with keratins, B72.4, CA19-9 (Fig. 35.77), CEA, MUC5AC, MUC1, and EMA (296,394,596,601-604). The ovarian-type stroma is vimentin, smooth muscle actin, h-caldesmon, calponin, progesterone receptor, and, to a lesser degree, estrogen receptor and inhibin immunoreactive (296,394,589,591,601,605-609). Scattered neuroendocrine cells (chromogranin positive) are frequently noted among the columnar lining cells (Fig. 35.78) (296,394,596,604,610). Such neuroendocrine cells are often argyrophilic and contain amines or regulatory peptides, including serotonin, somatostatin, gastrin, and PP.

FIGURE 35.77 Mucinous cystic neoplasm. CA19-9 immunohistochemical reactivity is accentuated in the higher grade dysplastic epithelial cells.

FIGURE 35.78 Mucinous cystic neoplasm. Chromogranin immunoreactive neuroendocrine cells lie among the mucinous cells.

Molecular studies have found recurrent mutations in KRAS, but typically seen in cases of highgrade dysplasia rather than low-grade dysplasia (263). Further, TP53 and SMAD4 alterations and chromosomal aneuploidy are often seen in association with high-grade dysplasia and invasive carcinoma (263,296,394,474,528,589,591,604,606,608,611-616). The ovarian-type stroma is reported to be devoid of any genomic alterations; however, the Wnt signaling pathway has been reported to be activated and may contribute to development of MCNs (617). CLINICAL OUTCOME In terms of clinical behavior, recent studies have shown that if the possibility of high-grade dysplasia and invasive carcinoma can be ruled out definitively with thorough sampling of the lesion, then the prognosis of low-grade cases is excellent. Those classified as high grade also have a very good prognosis but may experience recurrences and metastasis, presumably owing to missed foci or invasion or progression (394,577,584,587,618,619). In a recent study of thoroughly examined cases, even microinvasive cases appeared to have a good prognosis (580); however, this issue is controversial. It appears that if there is established invasive carcinoma of tubular type, the prognosis is often very poor (394,618,620).

SEROUS NEOPLASMS SEROUS CYSTADENOMA Also called microcystic or glycogen-rich cystadenomas, serous cystadenomas can occur at any age, but they are more common in elderly patients, with a predilection for women (240,621-633). They are often asymptomatic (621,634,635) and discovered incidentally during physical examination, intraoperatively (for another reason), at autopsy, sporadically, or as part of von HippelLindau (vHL) disease (628,630,636-638). If the mass is located in the pancreatic head, it can obstruct the biliary tract or the GI tract (639,640). Occasionally, extensive bleeding originates from one of these tumors. Rarely, the lesions are multiple, specifically when associated with vHL. In fact, those arising in vHL are often more a patchy transformation in the pancreas, although some may form a well-defined localized mass (628,630,641-644). A “honeycomb” appearance on CT or MRI, associated with a central scar, is characteristic (625,626,634,645-647). However, the diagnosis is often not accomplished preoperatively by imaging studies. EUS-guided FNA typically reveals minimal or no tumor cells; thus, it is difficult to establish a diagnosis and rule out other tumors

(648,649). The tumor cells are bland, cuboidal, and arranged in loose clusters or monolayers. The cytoplasm is usually cleared or vacuolated. However, the cells are frequently stripped of cytoplasm, showing only small, round nuclei with fine but dense, homogeneous nuclear chromatin (Fig. 35.79). Fibrovascular stroma can be present (647,650,651). Considering the difficulty in establishing a diagnosis of a serous cystadenoma in the preoperative setting, molecular testing has been extremely useful with the detection of VHL alterations.

FIGURE 35.79 Serous cystadenoma. A fine-needle aspiration smear shows hypocellularity and cohesive, flat sheets of cells with bland, round nuclei. Stripped nuclei and proteinaceous debris are seen in the background.

The mean diameter of the benign tumors is approximately 5 cm (range: 0.5-27 cm), but now, smaller tumors are found using improved imaging techniques (639). They occur anywhere in the organ and appear as partly encapsulated, lobulated masses typically composed of innumerable tiny cysts (occasionally with a few larger ones), which impart the highly distinctive and entity-defining microcystic (sponge-like) appearance on sectioning (Fig. 35.80). The smaller tumors are likely to be soft and spongy. Irregular central scars, frequently calcified, occur with moderate frequency in the larger tumors. The fluid in the cysts is clear and watery, appearing colorless, yellow, or blood stained. Foci of hemorrhage can occur (652). Conspicuous blood vessels may be noted on the outer surfaces of the tumors. Rarely, a lymphangioma may present with similar cysts, but they tend to have lymphocytes in the wall (653).

FIGURE 35.80 Serous cystadenoma. Many cysts of various sizes are visible on the cut surface with a central stellate scar.

Microscopic examination of most serous tumors reveals two principal patterns: (a) groups of minute, regular cysts (a honeycomb pattern resembling immature pulmonary tissue) formed by cuboidal epithelium; and (b) larger, more irregular cysts (up to several centimeters in diameter) lined by low cuboidal to flat cells. The two patterns are usually mixed (Fig. 35.81) (624,626,628,629,631,632,654). Rare tiny papillae, even with branching, are formed by the regular cuboidal cells (Fig. 35.82). Nuclei are small, round, compact, and uniform, with inconspicuous nucleoli. On rare occasions, groups of cells have larger and/or irregular nuclei. Some cases have more oncocytoid cells with granular cytoplasm. The presence of a capillary meshwork immediately adjacent to (almost within) the epithelium is highly characteristic (655,656) and has been noted as a striking analogy with other clear cell tumors arising in association with vHL, including RCCs and hemangioblastomas (655).

FIGURE 35.81 Serous cystadenoma. There are numerous cysts, ranging from small to large. The pancreatic parenchyma is at the bottom.

FIGURE 35.82 Serous cystadenoma. Tiny papillae are present.

Dense fibrous trabeculae run through the serous cystic tumors, and in addition to the central scars commonly present, small irregular nodules of fibrous tissue may be encountered. These scars can be calcified. Dense stroma can occur in the lesions with large cysts. Some of the stroma may be hyalinized or myxoid. Rarely, lymphocytes are present in part of the stroma. Trapped islets, ducts, and segments of acinar tissue may lie near the periphery. Both histologically and immunophenotypically, these neoplasms appear to recapitulate centroacinar cells (627,656-658). The cytoplasm is typically clear (Fig. 35.83) because of glycogen (PAS positive, diastase sensitive) (Fig. 35.84) and reacts with broad-spectrum and low-molecularweight keratins, EMA, and inhibin (624,659). Ductal mucin markers (B72.3, CA19-9, CEA, and MUC1) are either negative or only focally positive, although MUC6 is usually positive (659). Molecules implicated in clear cell tumorigenesis (glucose uptake and transporter-1 [GLUT-1], hypoxia-inducible factor-1α [HIF-1α], and carbonic anhydrase IX) are also consistently expressed (655).

FIGURE 35.83 Serous cystadenoma. The tiny cysts are lined by regular, cuboidal cells with uniform nuclei and clear cytoplasm, although sometimes, the cytoplasm may appear slightly eosinophilic (right).

FIGURE 35.84 Serous cystadenoma. The periodic acid-Schiff stain shows a variable amount of glycogen in different parts of the same tumor.

VHL gene allelic deletions (chromosome 3p) are detected in microcystic adenomas from patients with vHL, providing molecular evidence of their neoplastic nature and integral association with vHL disease (628,630,644). However, VHL gene alterations may also be detected in sporadic cases (554). Genetic abnormalities typical of DA (such as KRAS and TP53 mutations) have not been identified in serous neoplasms (614,630,660) nor have mutations in GNAS or RNF43, which are found in other cystic or intraductal pancreatic neoplasms (551,552).

Electron microscopy demonstrates simple cells attached to one another by desmosomes and containing generous collections of glycogen, with scattered mitochondria and profiles of endoplasmic reticulum. Sparse, short microvilli are present on the apical surfaces. Bundles of filaments lie in the apical and basal parts of the cells (631). Myoepithelial cells have been reported to lie beneath some of the epithelial cells, with myofibroblasts and endothelial cells embedded in thick collagen bundles (624,656). Serous cystadenomas are very slow-growing tumors (661), with an estimated doubling time of 12 years (662). Therefore, watchful waiting is a distinct option for smaller tumors if a definitive diagnosis can be accomplished (621,623,639,654,662,663). For symptomatic cases and/or larger ones, surgical removal is still the treatment of choice (654,664). Serous cystic tumors can be seen in association with a variety of other pancreatic neoplasm, including PanNETs (643,665,666), IPMNs (667), and PDACs (668-671), among others. In most cases, the two processes are independent. In some, there may be an underlying association, such as vHL, leading to simultaneous PanNET and serous adenoma. There are isolated cases reported in which a cytologically obvious carcinoma arose within a microcystic serous cystadenoma (“carcinoma ex microcystic adenoma”) (672). The biologic behavior of these rare cases is yet to be defined. SEROUS OLIGOCYSTIC ADENOMA Some serous oligocystic adenoma (SCA) are macrocystic or oligocystic, largely or completely composed of much larger cysts with fewer loculi, and devoid of central fibrosis or calcification (240,629,673,674). Obviously, such a tumor lacks the internal vascularity of the microcystic examples. Only a single cyst may be evident, thus simulating IPMNs, MCNs, and pseudocysts, especially radiographically (625,629,646,675-677). Such a cyst may be missing much of its lining, thus requiring multiple sections to find the epithelium. The border of the tumor may be irregular and rather poorly defined. The lining of the cysts displays the characteristic serous cytology. SOLID SEROUS ADENOMA A solid serous tumor has been described, composed of nests, sheets, and trabeculae separated by thick fibrous bands. Tiny acini are formed by the typical glycogen-laden cells of serous neoplasms (678-682). Separation from solid neoplasms or other primary clear cell neoplasms may be difficult; metastatic RCC can also be a challenge, even more so in the setting of vHL, where both lesions may be concurrently present. Special studies and immunohistochemical analysis should help (411). MALIGNANT SEROUS CYSTIC NEOPLASMS (SEROUS CYSTADENOCARCINOMA) There are only very rare case reports of serous cystic neoplasms with recurrence and metastasis, either showing concurrent or later development of invasion into the spleen, stomach, small intestines, and adrenal gland and metastasis to the liver (683-686). In general, these cases were otherwise similar to adenomas, although perhaps with more pleomorphism. Some of these cases are associated with hemorrhage, raising the question of whether the growth of the tumor in the secondary sites is due to adhesions. For those associated with liver lesions, the possibility of synchronous (multifocal) tumor development has been raised. There does not seem to be any clear documentation of a fatality due to a bona fide example of a conventional serous cystic neoplasm.

NEUROENDOCRINE NEOPLASMS Tumors with neuroendocrine differentiation include WDNETs, their precursor lesions (incipient, dysplastic-type lesions), high-grade (poorly differentiated) neuroendocrine carcinomas (NECs), mixed neuroendocrine-other carcinomas, and primitive neuroectodermal tumors (PNETs) that may

show neuroendocrine features. Although any of these may arise primarily in the pancreas and thus technically qualify as a pancreatic neuroendocrine neoplasm, the term pancreatic neuroendocrine tumor is mostly reserved for the WDNETs (islet cell tumors/islet cell carcinomas), and in the following discussions, PanNET is used to refer only to these WDNETs of the pancreas. Poorly differentiated NECs are discussed separately and should not be regarded as under the group of grade 3 NETs as previously classified (688). WELL-DIFFERENTIATED NEUROENDOCRINE TUMORS Previously called islet cell tumors/islet cell carcinomas and later pancreatic endocrine neoplasms, the WDNETs of the pancreas are now referred as pancreatic neuroendocrine tumors (687,688). Fundamentally, these are pancreatic counterparts of APUDomas (Amine Precursor Uptake and Decarboxylation tumors) or carcinoids. PanNETs are now regarded as low-grade malignant neoplasia, perhaps with the exception of small precursor-type proliferations (tumorlets) detected incidentally or in the setting of syndromes such as MEN, which are designated “microadenoma” if they are less than 0.5 cm (1,688,689). Demographics and Clinical Features PanNETs are said to represent only approximately 1% to 2% of all pancreatic neoplasms, although it should be noted that approximately 1% of pancreata examined at autopsy contain these rare neoplasms, usually small ones that have caused no signs or symptoms (46,688). There has been a relative increase in the number of cases, but this is probably accounted for by the increasingly sensitive radiographic and laboratory evaluations performed (690). Specifically, octreotide scans (somatostatin receptor scintigraphy using somatostatin analogs that attach to receptors overexpressed by PanNETs) have resulted in the detection of much smaller tumors than could be discovered with other imaging modalities (691,692). PanNETs show no significant gender predilection (possibly a slight female predominance) and can occur at any age, with the majority reported between 30 and 60 years, significantly younger than PDACs (688). The age may be younger when syndrome associated. The presentation symptoms vary significantly by location of the tumor and functionality status (see the following discussion); hypoglycemia, gastric or duodenal ulcers, and substantial fluid loss through watery diarrhea and other symptoms may result from inappropriate hormone secretion. Those located in the head and close to the ampulla and the common bile duct may present with obstructive symptoms or bleeding due to primary ulceration. PanNETs occur anywhere in the pancreas, although preferentially in the body and tail. Most tumors are solitary, but multifocal tumors are common in MEN1 (46). PanNETs may arise in the background of familial hereditary syndromes, including MEN1, vHL, tuberous sclerosis, neurofibromatosis type 1, and other rare syndromes. PanNETs within these syndromes may be multifocal and seen within a background of precursor-type small lesions and abnormalities. Functionality and Cell Types PanNETs have traditionally been separated into functional and nonfunctional categories based on their serologic activity and corresponding symptoms and signs. Functional tumors produce syndromes related to excessive production of a normal pancreatic hormone (insulin, glucagon, somatostatin, or PP) or an ectopic GI hormone (gastrin or vasoactive intestinal peptide [VIP]) and, occasionally, inappropriately for adrenocorticotropic hormone (ACTH), parathyroid-like hormone, calcitonin, and growth hormone–releasing hormone (688,693). Serotonin normally is present in extremely limited amounts in the pancreas, but rarely, a PanNET is the source of a carcinoid syndrome. Immunohistochemistry corroborates the hormone produced in some, but not all, cases. It appears that in many cases, the hormone produced is released readily to the serum, rendering it not or minimally detectable by immunohistochemistry. Expression of multiple hormones in a given tumor is also not uncommon and may not correlate with the serologic activity. In addition, the

intensity of immunohistochemical staining varies from cell to cell, and the patterns of immunohistochemical staining are variable within the same tumor (Fig. 35.85). Moreover, some tumors change their hormonal activity during the progression of the disease, with recurrent or metastatic foci releasing a different hormone type. For these reasons, the value of immunohistochemical hormone analysis within a tumor is fairly limited.

FIGURE 35.85 Pancreatic neuroendocrine tumor. Note the difference in immunoreactivity of chromogranin between tumors (top). Insulin immunoreactivity is concentrated at the capillary pole (left lower), whereas insulin is strongly, but only focally, immunoreactive in this tumor (right lower).

The insulin-producing neoplasms constitute the majority of those tumors that develop clinical manifestations as a result of their hormonal production (694-699). Most occur in adults, show a slight female predominance, are solitary, show a head and tail region predilection, and are small. Whipple triad (symptoms of hypoglycemia, plasma glucose levels 30/10 hpf), and necrosis is often extensive. The small cell variant (Fig. 35.100) shows small to intermediate cells with coarse salt-and-pepper chromatin, high nuclear-to-cytoplasmic ratio, inconspicuous nucleoli, prominent nuclear molding, and crush artifact. Mitotic figures are easily identifiable (>50/10 hpf), and there is extensive tumor necrosis (727,759,760). In cases with the typical cytologic features of small cell carcinoma, it is not necessary to document neuroendocrine differentiation by immunohistochemistry. However, for large cell PanNECs, positive immunohistochemical staining for chromogranin and/or synaptophysin should be obtained to confirm the diagnosis. It should be noted here that PanNECs are very uncommon tumors, and most cases that are diagnosed as such prove to be MiNENs with a significant acinar component once studied more carefully (759).

FIGURE 35.99 Poorly differentiated neuroendocrine carcinoma, large cell type. These reveal various growth patterns (trabecular pattern is depicted here). The tumor cells are often round to polygonal, and the nuclei have either vesicular chromatin or prominent nucleoli. Apoptotic cells and mitotic figures are abundant.

FIGURE 35.100 Poorly differentiated neuroendocrine carcinoma, small cell type. The tumor cells are relatively small with a high nuclear-to-cytoplasmic ratio, hyperchromatic nuclei, and nuclear molding.

As discussed earlier, better delineation of PanNECs from G3 PanNETs has been elucidated in some recent studies (761). Well-differentiated but highly proliferative PanNETs typically have a Ki67 index below 40% to 50% (761), whereas poorly differentiated PanNECs typically show Ki-67 index higher than 55%. More importantly, small and large cell PanNECs were shown to be genetically related but distinct from ordinary PanNETs (759): approximately 26% to 45% of sporadic PanNETs show loss of expression for DAXX and ATRX, which correlates with mutations in the DAXX and ATRX genes (746,759). In contrast, PanNECs retain DAXX and ATRX immunohistochemical expression. Also, PanNECs show abnormal immunolabeling with p53 and Rb in 95% and 74% of cases, respectively, which correspond to mutated TP53 and RB1 genes (759), whereas p53 and Rb immunolabeling is intact in PanNETs. Of note, for PanNECs that retain Rb, there is usually loss of p16 staining, indicating that the two may be mutually exclusive in PanNECs (759).

The clinical course is usually rapidly fatal with widespread metastases and a median survival less than a year (759). Platinum-based therapeutic agents have shown some promise in controlling their growth; however, their overall prognosis remains grim (762). In contrast to PanNETs, PanNECs are AJCC/TNM staged analogous to PDACs, considering their molecular similarities and aggressive clinical course. Differential Diagnosis WDNETs can show significant nuclear pleomorphism, or small cell change (higher nuclear-tocytoplasmic ratio resembling small cell carcinomas) (730) and features such as a diffuse or markedly infiltrative growth pattern and necrosis can also be identified (1). On casual examination, these falsely suggest a PanNEC. If careful mitotic count and immunolabeling for Ki-67 are not performed, misclassification can occur, which can have profound therapeutic consequences (763). The small cell variant of PanNEC may resemble metastatic small cell carcinoma of lung. Thyroid transcription factor-1 (TTF-1) stain is not helpful in distinction as small cell carcinoma is TTF-1 positive in a variety of extrapulmonary sites. Clinical information and a history of previous carcinoma are especially important in the accurate diagnosis of such cases. The crushed and molded tumor cells of small cell poorly differentiated NEC may resemble a highgrade lymphoma. Morphologic distinction is often impossible, and immunohistochemistry is required for diagnosis. Other small, round, blue cell tumors, such as PNET, may also involve the pancreas primarily or secondarily and need to be distinguished from the small cell variant of PanNEC, especially in younger patients (760). PNETs generally have small, round, monotonous nuclei with inconspicuous nucleoli and scant cytoplasm, although pancreatic examples can be more epithelioid and can express keratin strongly (760). The proliferative rate is variable but can overlap with that of PanNEC. Immunolabeling for CD99 can be helpful because most PNETs show strong, diffuse membranous staining. In questionable cases, molecular studies [t(11;22)] can be performed to further explore this diagnosis (760,764). The distinction of ACC and mixed acinar carcinomas from PanNECs is more problematic (759). Both entities usually have a high proliferative rate; ACCs can have a diffuse growth pattern, and large cell–type PanNECs can have prominent nucleoli. The correct diagnosis may not be established without a thorough immunohistochemical evaluation for acinar and neuroendocrine differentiation markers. Given the rarity of primary pancreatic PanNECs, relative to acinar neoplasms, it is thus recommended that a diagnosis of poorly differentiated NEC should not be rendered unless acinar differentiation has been excluded immunohistochemically.

ACINAR NEOPLASMS ACINAR CELL CARCINOMA Despite the fact that the majority of the normal pancreas is composed of acinar cells, ACCs are uncommon and represent 1% to 2% of all pancreatic neoplasms in adults and 15% of those in children. Males are affected more frequently (1,742,765-771). Clinical Findings Most patients have nonspecific symptoms, including abdominal pain, bloating, nausea, vomiting, diarrhea, and weight loss (767,772,773). A palpable abdominal mass and elevated liver enzymes are common findings. Serum AFP level is also elevated in some patients (774). Ten percent to 15% of the patients, usually those with liver metastases, develop the lipase hypersecretion syndrome, a manifestation of excessive lipase secreted by the tumor into the serum (769-771,775). The syndrome is characterized by polyarthropathy, nonbacterial thrombotic endocarditis, and multifocal fat necrosis in the subcutaneous tissues, bone marrow, and abdomen with or without peripheral

blood eosinophilia. Radiographic findings generally identify a solid, well-demarcated, hypovascular mass but are otherwise nonspecific (776,777). Macroscopic Features ACCs may arise in any portion of the pancreas. Most are large, with a median size of 7 cm (767); generally fairly well circumscribed; and even partly encapsulated. They are usually tan to red, soft, and fleshy. The external surface is bosselated; the cut surfaces are pink to tan, homogeneous, and fleshy, with thin fibrous bands separating the tumor into lobules (Fig. 35.101). Areas of hemorrhage, necrosis, and cystic degeneration can be present. Rare ACCs involve the ductal system and reveal intraductal polypoid projections as well as cystic dilatation of the ducts (778). Invasion of the spleen, duodenum, or other adjacent organs can occur.

FIGURE 35.101 Acinar cell carcinoma. The cut surface is pale, fleshy, and lobulated.

Microscopic Features Microscopically, at low magnification, ACCs are markedly cellular and are devoid of the intervening desmoplastic stroma characteristic of DAs (Fig. 35.102). The periphery of the carcinoma may appear circumscribed, although extension into adjacent parenchyma is common. Of the several different architectural patterns described, the most common histologic patterns are acinar and solid (Figs. 35.102, 35.103, 35.104). The acinar pattern consists of small lumina surrounded by cells of various sizes with eosinophilic granular cytoplasm and basal nuclei (1,768). A number of the socalled microglandular adenocarcinomas were recently shown to be ACCs (779-782). In the solid regions, most cells have central nuclei and little cytoplasm. The tendency to have basal nuclei is often more evident in cells adjacent to stroma or to the capsule. The absence of abundant cytoplasm, especially in solid regions, may suggest a poorly differentiated tumor, but additional sections and special studies will help to bring out the more typical acinar cell features (1,742,768,771). Less common patterns include the glandular pattern (large and often irregular lumina—the result of dilated acini), trabecular pattern (long strands of regular cells with double rows of nuclei close to the accompanying stroma and capillaries), and a papillary or intraductal pattern (778). These other patterns are accompanied by acinar or solid regions. Delicate vessels course through the tumors, sometimes associated with extravasation. When perivascular spaces are large, they cause a gyriform pattern of the epithelial elements. The neoplastic cells have nuclei that are round or oval, rather uniform, and medium sized. Clumps of chromatin often lie next to the nuclear membrane. Nucleoli are usually conspicuous, brightly eosinophilic, round or irregular, and centrally placed (783). Although they vary greatly in numbers from case to case, mitotic figures are easily identified (Fig. 35.102). Variable amounts of neuroendocrine elements in the form of scattered individual cells, large zones, and hybrid foci or even as separate well-established nodules are not

uncommon (771,784,785). If the neuroendocrine component comprises more than 25% (1) or 30% (742,786) of the tumor, by arbitrary convention, the case is classified as “mixed carcinoma” (see the following discussion) (742,787-790). Vascular invasion and perineural invasion are identified in many cases, and extension into peripancreatic tissue may be seen.

FIGURE 35.102 Acinar cell carcinoma, solid pattern. There is a delicate background vasculature. Note the prominent nucleoli. A mitotic figure is present in the center.

FIGURE 35.103 Acinar cell carcinoma, acinar pattern. The cells are columnar.

FIGURE 35.104 Acinar cell carcinoma, solid pattern. The cells vary in size, and some have large amounts of eosinophilic, granular cytoplasm.

Special Studies PAS after diastase digestion demonstrates the magenta zymogen granules, highlighting the acinar differentiation. However, the amount varies greatly from case to case and even within different areas of the same tumor. If present, intraluminal secretory concretions and crystals are also PAS positive (771,778). The neoplastic cells are immunoreactive with various pancreatic enzymes that, although specific, show different sensitivity for the diagnosis. Both trypsin (Fig. 35.105) and chymotrypsin are detectable in over 95% of cases and are the most diagnostically useful markers. Lipase is less commonly identified, seen in approximately 70% to 85% of cases (742,768,771,784,791). Other enzymes that are reportedly positive in ACC are (772,798,799) α1antitrypsin, α1-antichymotrypsin, amylase, phospholipase A2, and PSTI. Recently, monoclonal BCL10 antibody, which recognizes the COOH-terminal portion of carboxyl ester lipase, has also been suggested as a useful tool for detecting ACCs (766,792,793).

FIGURE 35.105 Acinar cell carcinoma. Immunohistochemical reactivity with trypsin.

ACCs are almost always positive for CAM 5.2, AE1/AE3, CK8, and CK18, whereas CK7, CK19, and CK20 are generally negative. Mucin-related glycoproteins and oncoproteins that are commonly

expressed in the DAs (MUC1, MUC5AC, B72.3, CA19-9, CEA, CA125, and DUPAN-2) are either negative or only very focally positive (261). Uncommonly, there is immunohistochemical positivity for AFP (774). The neuroendocrine component shows immunoreactivity for chromogranin or synaptophysin (742,771,778,794). Cytology smears show small- to moderate-sized loosely cohesive clusters along with a background of single cells; acinar formation is widespread; and the cells are cytologically bland but display prominent nucleoli and finely granular, eosinophilic cytoplasm (783,795,796). However, ACC is frequently misdiagnosed on cytology. Immunocytochemistry is useful for identifying acinar differentiation (783,797). Ultrastructural studies demonstrate dense-core zymogen granules, ample amounts of rough endoplasmic reticulum, and elongated membrane-bound bodies filled with filaments (771). By molecular analysis, ACCs rarely, if ever, show KRAS, TP53, SMAD4, CDKN2A (p16), or GNAS gene mutations, in contrast to PDACs. Thus, immunohistochemically, only rare cases exhibit abnormal nuclear accumulation of the p53 protein, and SMAD4 is retained. However, a high frequency of allelic loss on chromosomes 11p, 4q, and 16q has been identified (742,791,798-801). Recently, it has been reported that more than one-third of ACCs have potentially targetable genetic alterations, including mutations in BRCA2, BAP1, BRAF, and JAK1 (802). In addition, approximately 25% of ACCs have mutations in APC/β-catenin pathway, similar to those seen in pancreatoblastoma. Therefore, abnormal nuclear immunolabeling for β-catenin can be seen, usually in a patchy or mosaic pattern (742,791,798,799,800). Furthermore, recurrent gene rearrangements have been found in a subset of ACC and involve BRAF, RAF1, and RET (809-811). Clinical Outcome Although it is usually not as dismal as that of stage-matched DAs, the clinical course of ACC is aggressive, with 5-year survival figures of 20% to 50% depending on stage at diagnosis (766,767,769,772,773,805). Patients who present with localized disease have much better prognosis than those who present with metastases (767,806), which are most often found in regional lymph nodes and the liver, although, occasionally, in lung, cervical lymph nodes, and ovary (807). Although insufficient data have been accumulated on the pediatric ACCs, patients younger than 20 years may have better prognosis than adults (742). Surgical resection appears to be the most effective treatment for those patients with localized disease (772,773). For those patients with locally unresectable or metastatic tumors, chemotherapy and radiation may be considered in an attempt to downstage disease to achieve surgical resection (769,784,806,808,809). Of note, AJCC/TNM staging of ACCs is performed analogous to that of PDACs. Differential Diagnosis The main differential diagnosis of ACC is with other pancreatic neoplasms that are characterized by a solid cellular pattern, specifically PanNETs, pancreatoblastoma, and SPN (Table 35.10). Acinar Cell Carcinoma Versus Pancreatic Neuroendocrine Tumor. Although ACCs usually show an acinar growth pattern, at least focally, cases with predominantly solid or trabecular patterns are not infrequent and, with minimal stroma and cytologic uniformity, these can represent a significant diagnostic challenge. The characteristics that help distinguish ACCs from PanNETs include basal cytoplasmic basophilia and, although not always prominent, basal nuclear localization in acinar formations or at the interface of solid nests with the stroma, forming a palisading pattern. The cytoplasm is, at least focally, eosinophilic and granular, reflecting aggregates of zymogen granules in the apical cytoplasm, contrasting with the finely granular, nonpolarized amphophilic cytoplasm of most PanNETs. Despite the nuclear uniformity, ACCs lack the typical salt-and-pepper chromatin of PanNETs, and their large single nucleoli can provide an important clue to the diagnosis. Finally, the presence of readily identifiable mitoses (usually >10/10 hpf) in an otherwise uniform-appearing neoplasm suggests ACC (748). Immunohistochemical stains are invaluable in

establishing the diagnosis (Table 35.10). Markers for acinar enzymes (trypsin and chymotrypsin) are highly specific and sensitive. Scattered neuroendocrine cells or a focal neuroendocrine component are common in ACCs, so labeling (especially focally) for neuroendocrine markers is not sufficient to exclude an ACC unless enzyme markers are known to be negative. In addition, PanNETs may show focal immunoreactivity for acinar markers. Therefore, it is important to use an immunohistochemical panel that includes markers for both acinar and neuroendocrine differentiation. Acinar Cell Carcinoma Versus Pancreatoblastoma. Pan-creatoblastoma is a rare pancreatic tumor showing differentiation toward all three lineages (acinar, ductal, and neuroendocrine) in variable amounts. Microscopically, the tumors have acinar, solid, and nested growth patterns. A hallmark of the diagnosis is highly characteristic squamoid corpuscles composed of whorled, plump spindled cells that have optically clear nuclei (810,811). Acinar differentiation is the most common and the predominant pattern in the majority of the cases, which can cause diagnostic problems. In such cases, the patient’s age is helpful, suggesting the correct diagnosis if the histologic features are not well visualized (1). Careful microscopic examination and additional sampling may also be useful in revealing ductal and/or neuroendocrine components as well as squamoid nests and cellular stromal bands. Immunohistochemical labeling for markers of acinar, ductal, and neuroendocrine differentiation helps confirm the diagnosis (765). Abnormal nuclear and cytoplasmic β-catenin expression can also be found in pancreatoblastomas and cannot be used to help with the distinction of pancreatoblastoma from ACC and SPN. Acinar Cell Carcinoma Versus Solid Pseudopapillary Neoplasm. SPNs have a distinctive microscopic appearance characterized by a combination of solid, pseudopapillary, and hemorrhagic cystic areas. Foamy cells and eosinophilic globules might also be seen. The cells are very uniform, nuclei are grooved, and nucleoli are not prominent. SPNs generally lack mitoses (742). However, if an SPN displays a predominantly solid growth pattern without foamy cells and eosinophilic globules, it may be confused with ACC. Immunohistochemistry readily distinguishes these two neoplasms (Table 35.10). In contrast with ACCs, SPNs do not label with specific acinar markers (trypsin and chymotrypsin) but consistently express vimentin, CD10, CD56, and progesterone receptors; epithelial markers are usually either focal or weak (812). Furthermore, SPNs have βcatenin mutations and consistently show diffuse nuclear β-catenin staining, which is less common in ACCs (813). ACINAR CELL CYSTADENOCARCINOMA A cystic variant of ACC is well documented but is extremely uncommon, with only a handful of cases reported (809,814-816). Grossly, the lesions are large, circumscribed, and diffusely cystic, with individual locules ranging from a few millimeters to several centimeters. Microscopically, the cysts are lined by single or several layers of neoplastic acinar cells, sometimes forming minute lumina within the epithelial lining (Fig. 35.106). The cells often contain abundant apical, acidophilic (zymogenic) granules. There is usually minimal-to-mild nuclear pleomorphism. However, solid nests of neoplastic cells, areas of necrosis, and easily identifiable mitotic figures support a malignant diagnosis. Immunohistochemical markers are needed to document the presence of acinar differentiation. The clinical behavior of acinar cell cystadenocarcinomas does not appear to be different than that of ordinary ACCs (765).

FIGURE 35.106 Acinar cell cystadenocarcinoma. There is striking variation in the sizes of the locules, which are lined by acinar cells.

ACINAR CYSTIC TRANSFORMATION (ACINAR CELL CYSTADENOMA) Until recently, the conventional thought was that all acinar neoplasms in the pancreas are malignant, albeit solid or cystic. In the last decade, a rare, benign pancreatic cystic lesion of acinar origin has been described and termed acinar cystic transformation (also called acinar cell cystadenoma) (817-819). Acinar cystic transformation is often incidental; however, it may rarely produce a clinically detectable cystic mass, which can be unilocular or multilocular, characterized by innumerable cysts of varying sizes (817,818,820). Histologically, the cysts are lined by patches of bland acinar and ductal epithelium, and there is no nuclear atypia, necrosis, or mitotic figures. Although, to date, less than 30 cases have been described, there have been no reports of malignant transformation, (742) and the nonneoplastic versus neoplastic nature of this lesion remains unclear (820,821).

NEOPLASMS OF MULTIPLE LINES OF DIFFERENTIATION MIXED PANCREATIC CARCINOMAS Foci with neuroendocrine differentiation have been demonstrated within conventional PDAC (732,785,822) and ACCs (771,785,823,824). In contrast, entrapped nonneoplastic ductal epithelium (731) as well as focal true gland formation with luminal mucin and/or immunohistochemical staining for mucin-related glycoproteins have also been described in PanNETs (822). Moreover, focal ductal differentiation can be identified in ACCs by stains for mucins, mucin-related glycoproteins, or ductaltype cytokeratins (e.g., CK19). Beyond these examples, there are rare neoplasms in which significant components of more than one line of differentiation have been represented (731,732,785,788,789,790,794,822). Using an arbitrary definition, each component must comprise at least 30% (742,786) of neoplasm for a diagnosis of mixed carcinoma. Every combination of ductal, acinar, and neuroendocrine differentiation has been described. The best characterized and probably the most common is MANEC and more recently referred to as mixed neuroendocrine nonneuroendocrine neoplasm (MiNEN) (1,742,786). Although some mixed carcinomas have distinctive histologic features suggesting that more than one line of differentiation exists (Fig. 35.107), in many cases, mixed differentiation is only detectable by immunohistochemistry or electron microscopy (784,785), especially in MiNENs (785). For this reason, a thorough immunohistochemical evaluation is recommended for all acinar neoplasms.

FIGURE 35.107 Mixed acinar-neuroendocrine carcinoma or mixed neuroendocrine nonneuroendocrine. There is a well-developed neuroendocrine component in the center that blends with the surrounding acinar cell carcinoma (composite tumor).

Overall characteristics of MiNENs are highly similar to those of ACC (785,794,825,826); however, the distinction of ACC and MiNENs from PanNECs can be problematic. All three entities usually have a high proliferative index, ACCs and MiNENs can have a diffuse growth pattern and can lack the distinguishing features mentioned earlier, and large cell–type PanNECs can have prominent nucleoli. Moreover, although MiNENs are also aggressive carcinomas, survival of MiNENs seems to be significantly better than that of PanNECs (825). Therefore, this distinction is of clinical importance, and, given the rarity of PanNECs, it is recommended that a diagnosis of PanNEC should not be rendered unless acinar differentiation has been excluded immunohistochemically (759). Mixed acinar-ductal carcinoma is rare. Only a handful of these tumors had been reported (1,786). Stelow et al (782) published a series of tumors with significant acinar and ductal differentiation (some with neuroendocrine differentiation as well) and described two different architectural patterns: (a) “mucinous ACC” consists of intermingled conventional ACC and elements of intracellular or extracellular mucin production and (b) “combined acinar and ductal carcinoma” is characterized by separate, morphologically distinct DA components with individual glands surrounded by dense or desmoplastic stroma, alternating with typical ACC components. Regardless of the pattern, most cells expressing pancreatic enzymes (demonstrated by immunohistochemical labeling for trypsin and/or chymotrypsin) are negative for mucin stains and vice versa, but in a small population of cells, both enzyme production and mucin staining can be shown. Further, chromogranin and/or synaptophysin staining highlight the neuroendocrine component, if present, in tumors designated mixed acinar-ductal-neuroendocrine carcinoma. It has been shown that these mixed acinar-ductal carcinomas largely lack evidence of abnormalities in ductal carcinoma genes, although rare cases reveal KRAS mutations (787). Therefore, most of these tumors are probably biologically more closely related to ACCs than to DAs. The rarest of the mixed pancreatic carcinomas is the so-called mixed ductal-neuroendocrine carcinoma and similarly now referred to under the heading of mixed neuroendocrine nonneuroendocrine neoplasm (MiNEN) (732). The definition of mixed ductal-neuroendocrine carcinoma requires more than simple mucinous differentiation in an otherwise typical PanNET. True mixed ductal-neuroendocrine carcinomas must contain morphologically separate elements of both DA and pancreatic neuroendocrine neoplasm, each component having the histologic and immunohistochemical features of the corresponding entity. In the vast majority of these, the

neuroendocrine component is predominant and poorly differentiated (786). Thus, it may be appropriate to classify these as PanNECs with mixed adenocarcinoma component because any PanNEC component seems to be most significant (759). PANCREATOBLASTOMA Pancreatoblastoma is the ultimate example of a tumor with multiphenotypic differentiation. Clinical Findings Although very rare, pancreatoblastoma is the most frequent malignant pancreatic neoplasm of childhood (827). The majority of pancreatoblastomas occur in the first decade of life, with a mean age of 4 years (811,827). Rarely, pancreatoblastomas can be encountered in adults (1,810,828). There appears to be a slight male predominance (829), with patients often complaining of upper abdominal pain, frequently associated with a palpable mass. Rarely, hematemesis and vomiting may be seen. There is a known association with Beckwith-Wiedemann syndrome (830,831) and familial adenomatous polyposis (832). Elevated AFP levels are identified in some of the patients (833,834). Radiographic studies identify a heterogeneous mass lesion with well-defined margins but are otherwise nonspecific. There is no topographic predilection within the pancreas (810,811,829,835,836). Macroscopic Features At macroscopic inspection, they are generally encapsulated masses, often large (mean: 11 cm), and may extend outside the pancreas. The cut surfaces are described as yellow, white, gray, or tan; firm; and sometimes lobulated (Fig. 35.108). Hemorrhage, necrosis, calcification, and focal cystic change are rather frequent, may be extensive, and are usually identifiable on imaging studies (810,835,837). Lymph node metastasis may be seen.

FIGURE 35.108 Pancreatoblastoma. Note the lobulation on this macroscopic tumor.

Microscopic Features Microscopically, pancreatoblastoma is defined as an epithelial tumor exhibiting acinar and lesser degrees of endocrine and ductal differentiation, associated with squamoid corpuscles. Acinar differentiation, the most reproducible line of differentiation, is found in nearly all cases both in the form of microscopic acinar formations (Fig. 35.109) and in the production of pancreatic enzymes and the presence of zymogen-like granules by electron microscopy. Cells are medium sized and polygonal, have central round or ovoid nuclei with generally central nucleoli, and possess amphophilic-to-eosinophilic cytoplasm (Fig. 35.109). The amount of cytoplasm and the size and characteristics of the nuclei may vary in different parts of the neoplasm. Foci of necrosis may occur. Squamoid corpuscles are pathognomonic. These may be vague aggregates of spindled cells or may exhibit frank keratinization (Figs. 35.110 and 35.111). The nuclei are oval, and the chromatin is

cleared owing to intranuclear biotin (838). Although their exact nature is unclear, they do not appear to exhibit a reproducible line of distinct differentiation and are regarded simply as a peculiar growth pattern characteristic of this tumor (768,810,811,829). Both by morphology and immunophenotype, they show more striking similarities to morules seen in tumors such as endometrial carcinoma, pulmonary endodermal tumor, and cribriform-morular variant of papillary thyroid carcinoma, all of which are associated with β-catenin alteration. Neuroendocrine differentiation is commonly detected with immunohistochemical stains. When distinct ductular structures are present, the apical surfaces of the cells are positive for mucin; in occasional ducts, there are mucinous columnar cells (810,811,829). Especially in pediatric cases, pancreatoblastoma often have hypercellular stroma (Fig. 35.112). Occasionally, the stroma even appears neoplastic, and heterologous bone and cartilage formation has been observed.

FIGURE 35.109 Pancreatoblastoma. Acinar arrangement with enlarged cells showing prominent nucleoli.

FIGURE 35.110 Pancreatoblastoma. The epithelial cells alongside the stroma have eosinophilic cytoplasm and a tendency to form squamoid corpuscles.

FIGURE 35.111 Pancreatoblastoma. An island of squamous differentiation is noted within the acinar arrangement of a pancreatoblastoma. Note the prominent nucleoli and “secretions” within the lumens.

FIGURE 35.112 Pancreatoblastoma. Cellular stroma separates the tumor into nests and islands. Note the calcifications.

Cytology Findings FNA shows three-dimensional sheets of loosely cohesive epithelial cells and primitive spindled mesenchymal-like tissue. Squamoid and acinar differentiation and heterologous elements are often difficult to appreciate, although helpful when present (839,840). Immunohistochemical and Molecular Features The acinar component labels with antibodies to CAM 5.2, AE1/AE3, CK7, CK8, CK18, and CK19 (Fig. 35.113). Positivity for trypsin and chymotrypsin is found in nearly every case (261,811,841). The distribution pattern of the β-catenin immunolabeling is characteristic for pancreatoblastomas. The acinar/ductular elements show mostly membranous (normal) expression of β-catenin, whereas the squamous corpuscles display diffuse nuclear/cytoplasmic (abnormal) expression (842,899). The downstream target of β-catenin, cyclin D1, is also overexpressed in the squamoid nests (842).

Neuroendocrine markers chromogranin and synaptophysin are positive in majority of the cases in a highly variable proportion of the cells. In addition, the ductal elements, if present, express glycoprotein markers such as B72.3, CEA, and DUPAN-2 (811). Immunohistochemical positivity for AFP has been detectable in cases with elevations in the serum levels of AFP.

FIGURE 35.113 Pancreatoblastoma. A generous amount of immunoreactive keratin is visible, but note that some cells are negative for keratin.

The most common genetic alteration identified to date is loss of heterozygosity of the highly imprinted region of chromosome 11p (831,843). Alterations in the adenomatous polyposis coli (APC)/β-catenin pathway, usually involving β-catenin gene mutation, have also been reported in 50% to 80% of pancreatoblastomas (832,842). Unlike DAs, KRAS and TP53 mutations have not been detected (799,832,843-845). Differential Diagnosis The differential diagnosis of pancreatoblastoma includes other solid, cellular tumors of the pancreas (Table 35.10). In particular, pancreatoblastomas share many histologic features with ACCs (see earlier discussion). Because both tumors show similar lines of cellular differentiation, the presence of squamoid nests is helpful in distinguishing them. Clinical Outcome The prognosis for pancreatoblastoma in children is much better than pancreatic ACCs in adults (829). Overall prognosis is approximately 50% at 5 years, with metastatic disease seen in up to 20% of patients at presentation and a high risk of recurrence. The treatment of choice is complete surgical resection; unresectable tumors can be managed with neoadjuvant chemotherapy to reduce tumor volume; other patients have died because of recurrence and metastases (especially to lymph nodes and liver) (810,811,827,829). Age at presentation (>16 years), nonresectable disease, and metastases after resection are all unfavorable prognostic factors (829). A few pancreatoblastomas will develop in adults, where the prognosis is not as favorable (811,832).

NEOPLASMS OF INDETERMINATE DIFFERENTIATION SOLID PSEUDOPAPILLARY NEOPLASM Clinical Features

This neoplasm is rare, representing less than 2% of all exocrine pancreatic neoplasms (1,236). Many synonyms are used for this tumor, but solid pseudopapillary neoplasm is the WHO accepted term (846). It develops almost exclusively in adolescent girls and young women of all races, with a mean age of 33 years (range: 2-81 years) (379,846-854). It is rare in childhood (850,855), in older women, and in men (856-861). In fact, before a diagnosis of SPN is given in a child, the possibility of pancreatoblastoma should be ruled out. In a male patient, a PanNET or PanNEC ought to be excluded. Patients present with few nonspecific digestive-type symptoms, including abdominal discomfort or pain, dyspepsia, and bloating accompanied by an enlarging mass (848). Typical symptoms of conventional pancreas cancer such as obstructive jaundice, back pain, and rapid weight loss are not observed. There is no established association with other neoplasms, paraneoplastic syndromes, or serum tumor makers. Similarly, there is no established association with recognized clinical or genetic syndromes. Three patients in a single family, all carrying a protease serine 1 (PRSS1) mutation, have been reported, indicating the possibility of the involvement of a genetic background in the pathogenesis (862). The tumors have been found on routine physical examination or imaging study, and others have been found after abdominal trauma caused hemorrhage in the neoplasms. Imaging techniques will show a heterogeneous lesion with little vascularization (in contrast with endocrine tumors), often with cyst formation, blood, or degeneration, and usually without dilatation of the bile duct system (863-865). As such, SPNs may mimic pseudocysts. Calcification and ossification may be seen, usually in the wall of the tumor. There is no preferential localization within the pancreas (1,866). Origin outside the pancreas is uncommon, but SPNs have been reported in the mesocolon (867,868), retroperitoneum (869), and ovary (870,871). The tumors are commonly large, although tumors are detected at a smaller size with advanced radiographic techniques (872). Cytology Findings Percutaneous, endoscopic, or intraoperative FNA yields variable numbers of branching, “pseudopapillary” fragments of epithelial cells in a background of histiocytes, blood, psammoma bodies, and debris. The cells are aligned around central fibrovascular stalks, which may show myxoid stroma or fibrosis. The cells are uniform and bland, showing oval-to-elongated nuclei with grooves or folds. Eosinophilic inclusions within the cytoplasm are occasionally noted. The features are sometimes accentuated in cell block material (873-877). Macroscopic Features Macroscopically, the tumor is typically solitary; synchronous tumors are exceedingly uncommon. Most SPNs are deceptively round and circumscribed. The mean size is about 7 cm; however, SPNs may be up to 30 cm. The cut surfaces are typically soft, with solid, cystic, and solid cystic areas, frequently with degenerative cystic foci, hemorrhage, and necrosis (Fig. 35.114) (846-854).

FIGURE 35.114 Solid pseudopapillary neoplasm. Multifocal necrosis and extensive hemorrhage are visible in this neoplasm. The cut surface appears granular.

Microscopic Features The neoplasms appear to begin as solid masses in which there are many poorly supported tiny vessels; then, the cells farthest from the small vessels undergo swelling and degenerative changes, whereas the cells next to the vessels remain intact (Fig. 35.115). The result is a pseudopapillary pattern. Groups of foamy macrophages accumulate, and there are clusters of lipid crystals surrounded by foreign-body giant cells (Fig. 35.116). Myxoid connective tissue proliferates around the tiny blood vessels, and these regions of loose, hypocellular connective tissue may be mistaken for microcysts (Fig. 35.117). As collagen is deposited in the tumor along the blood vessels, a trabecular pattern of epithelial cells may emerge. Consequently, microscopic examination reveals a variety of patterns (solid, pseudopapillary, cystic, trabecular, etc.); however, true gland formation is not seen. Hemorrhagic zones are commonly noted. Eosinophilic globules composed of α1antitrypsin might also be seen (Fig. 35.118) (866,878,879). Although deceptively round and demarcated on macroscopic examination, many SPNs do show tonguelike projections into the adjacent pancreas, sometimes with no intervening stroma. For this reason, one often finds islets, ducts, and acinar units, either individually or in clusters, within the boundaries of the main lesion. In fact, entrapped pancreatic tissue may be found even in the inner aspects of the lesion, blending imperceptibly with the neoplastic cells. Conversely, tumor cell clusters in the pancreas away from the lesion may mimic islets. A fibrous pseudocapsule may be noted in some cases. Calcifications and even ossifications may be noted in this pseudocapsule as well as in the stroma within the tumor. It should be kept in mind that cystic change may be so extensive that it may create the false impression of a pseudocyst, not only clinically but also pathologically. In such cases, the tumor cells may be so attenuated that careful sampling of the “cyst wall” may be necessary to identify them (846,849-853).

FIGURE 35.115 Solid pseudopapillary neoplasm. The cells adjacent to the tiny blood vessels are attached to the stroma and to one another, but those that are farther away are detached from each other. Slitlike spaces separate the pseudopapillae.

FIGURE 35.116 Solid pseudopapillary neoplasm. The cells have degenerated with an increased number of foamy macrophages in the center.

FIGURE 35.117 Solid pseudopapillary neoplasm. Part of the tumor is solid. The remainder contain myxoid connective tissue producing the pseudomicrocystic pattern.

FIGURE 35.118 Solid pseudopapillary neoplasm. (Left) Large numbers of eosinophilic globules are present. (Right) α1Antitrypsin immunoreactive globules.

The cells are small to medium sized and polygonal to elongated, with ovoid nuclei that often are grooved or indented (Fig. 35.119). Rarely, a few cells have nuclei that are large or irregular in shape (Fig. 35.120). Nucleoli are inconspicuous. Mitotic figures are rare. The cytoplasm ranges from clear to eosinophilic and lacks glycogen and mucin (846,849-853).

FIGURE 35.119 Solid pseudopapillary neoplasm. Elongated cells, with small oval-to-round nuclei, surrounding small “fibrovascular” cores.

FIGURE 35.120 Solid pseudopapillary neoplasm. The large atypical nuclei present in this case are unusual.

Immunohistochemistry Results Despite intensive study, the line of differentiation of these neoplasms remains uncertain. Both ductal and acinar markers discussed previously are consistently negative in SPN. The tumors are also consistently negative for chromogranin. In fact, all experts agree that if a tumor shows substantial chromogranin expression, it is not an SPN (261). SPNs also fail to show any convincing neurosecretory granules by electron microscopy, which further corroborates these are not neuroendocrine neoplasms. However, these tumors commonly react with some of the so-called neuroendocrine markers: synaptophysin, NSE, as well as NCAM (or CD56) (812,866,882). Even the epithelial nature of SPN is dubious, although the tumor was referred to as carcinoma in the past. Cytokeratins (CAM 5.2 and AE1/AE3) and other epithelial markers are typically either negative or only very focally positive in rare cases. The neoplastic cells express vimentin and α1antitrypsin diffusely and strongly (812,866,880,881). 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 (713,882,883). Progesterone receptors are also expressed in SPNs, regardless of whether it is in women or men (866,881). Recently, a unique dotlike staining pattern for CD99, in contrast to membranous staining in PanNET and most of ACCs, along with negative immunostaining in DAs, has been reported in SPNs (884-886). Cytoplasmic CD117 and nuclear FLI1 reactivity is noted, but corresponding KIT/PDGFRA and EWS/FLI1 mutations/translocations, respectively, are not detected (887,888). Most cases studied show nuclear accumulation of β-catenin and LEF1 proteins immunohistochemically (756,882), along with activating gene mutations of β-catenin (CTNNB1, usually exon 3), a molecule participating in the Wnt signaling pathway and an element of cadherinmediated cell-cell adhesion (590,879,889-897). Nuclear cyclin D1 and E-cadherin are present, further supporting the β-catenin pathway abnormalities (879,880,889,890,895). New molecular analyses have shown that, in addition to the Wnt/β-catenin pathway, Notch, Hedgehog, and androgen receptor signaling pathways, as well as genes involved in epithelial mesenchymal transition, are activated in SPNs (552,898,899). KRAS mutations are not seen, and SMAD4 expression is intact (846,895,896), confirming a different histogenesis from DAs. The neoplasm probably arises from an uncommitted cell, similar to intercalated duct cells or centroacinar cells (895,900). Clinical Outcome

If completely resected, SPNs behave in a benign manner in the vast majority of patients. Extrapancreatic spread, usually to liver and/or peritoneum, is seen in only 15% of the patients and is usually present at the time of diagnosis as metastases seldom develop later in the course of disease. There are no reliable criteria to recognize the 15% that will spread. However, even patients with metastatic tumors often do well (847). Only a few patients have died of a metastatic SPN, mostly the ones whose tumors harbored an undifferentiated/sarcomatoid component (853). Because of this “low-grade” nature of SPN, adjuvant chemotherapy or radiotherapy is usually not considered in the management. Tamoxifen has been used on anecdotal cases with metastatic tumor, although the value of this approach is impossible to determine. Long-term clinical follow-up is recommended (490,846,848,854,864,901,902). Despite their enigmatic origins, the consensus is to AJCC/TNM stage SPNs similar to exocrine neoplasms, such as PDAC and ACC. Differential Diagnosis The main differential diagnosis of SPN is with the other stroma-poor (nonscirrhous) cellular neoplasia (Table 35.9), including PanNET, ACC, pancreatoblastoma, metastatic tumors, and the small blue cell tumors. In cases with a sheetlike growth pattern and those that lack the characteristic pseudopapillae, grooves, hyaline globules, or clusters of macrophages, the following clues may help in the differential. In SPN, the nuclei tend to be more ovoid than those in PanNET. Overlapping of nuclei is also more typical, possibly owing to its nonepithelial characteristics, in contrast to endocrine tumors. Immunohistochemistry is very helpful in this distinction because nuclear expression of β-catenin is consistent in SPN but very uncommon in PanNETs. In contrast, most PanNETs show diffuse strong labeling for chromogranin and keratins, whereas virtually all SPNs are negative for these markers (890). ACCs often have a distinct basophilia, and the nucleoli are prominent. Pancreatoblastomas often show foci of acinar or ductal differentiation, and more importantly, if present, squamoid corpuscles are diagnostic. In addition, expression of acinar enzymes, trypsin and chymotrypsin, coupled with diffuse keratin positivity are diagnostic of ACCs and also present in pancreatoblastomas. Small blue cell tumors such as PNETs/Ewing sarcoma and desmoplastic small cell tumors do rarely involve the pancreas. In fact, SPNs that have been reported to have t(11:22) translocation are most likely examples of small blue cell tumors mistaken for SPN.

OTHER CYSTIC LESIONS Cysts can be congenital or acquired (Table 35.7). Although their origins differ, the clinical manifestations, some imaging characteristics, and surgical treatment have similarities. Congenital cysts are rare and include (a) a simple, or solitary cyst apparently resulting from abnormal development of a duct; (b) multiple cysts associated with inherited polycystic diseases; (c) pancreatic and parapancreatic lymphoepithelial cysts; (d) dermoid cysts (mature teratomas); (e) and arteriovenous malformation. Cystic fibrosis is a genetic disorder, but the small cysts that develop are the result of viscid secretions, obstruction, and fibrosis (903,904). Acquired cysts consist of infection-related cysts (parasitic, etc.), pseudocysts (see section “Chronic Pancreatitis”), paraduodenal wall cysts, epithelial retention cysts, and cystic neoplasms. IPMN may present as a cyst. Further, usual DA, a sarcoma (such as leiomyosarcoma), and SPN may all undergo substantial cavitary necrosis or degeneration. DUPLICATION CYSTS An important variation to a congenital cyst is a duplication cyst, which can radiographically mimic IPMNs and may also harbor carcinomatous transformation. These can closely mimic IPMNs at the microscopic level if the muscular coat surrounding the cysts is missed on cursory examination.

These cysts can develop papillary nodules with high-grade dysplasia/CIS and can be of intestinal or pancreatobiliary patterns. Invasive carcinoma can be associated with these cysts. PARADUODENAL WALL CYST OF PARADUODENAL PANCREATITIS These cysts appear to occur as a consequence of chronic fibrosing inflammation in the periampullary region in which one or more of the accessory ducts form a cyst on the duodenal wall and mimic duodenal duplication. This usually occurs in the background of microcystic trabeculation of the duodenal wall by “myoadenomatosis”-type changes, which may also involve the adjacent pancreas in the groove area (also known as groove pancreatitis or cystic dystrophy of heterotopic pancreas) (201-203). In our experience, this process is often centered around the accessory ampulla and may be associated with the scarring of common bile duct, mimicking pancreas cancer (203,905). Paraduodenal wall cysts occur predominantly in males often with a history of alcohol abuse, who present in their 40s and in the context of severe abdominal symptoms. Some were interpreted to be associated with “pancreatic heterotopia” in the duodenal wall (906). The cyst wall may be partly lined by ductal epithelium and partly by inflammation as well as granulation tissue. Some cysts are devoid of epithelium. Instead, they are lined by more cellular fibroblastic tissue. On occasion, the lining fibroblasts may appear epithelioid and raise concern for a sarcomatoid carcinoma (203,208). The cyst contents may extravasate and lead to the development of a foreignbody giant cell reaction and stromal eosinophilia. LYMPHOEPITHELIAL CYST Lymphoepithelial cysts (907,908) occur more frequently in middle-aged men, usually in the sixth decade of life. Patients are usually asymptomatic, with the lesion found incidentally on imaging studies performed for unrelated reasons or at autopsy; abdominal pain has been described. No autoimmune disorder is identified, and there is no syndrome association, different from their salivary gland analogs. Association with HIV also appears to be coincidental and exceedingly uncommon (909). Lymphoepithelial cysts are typically unilocular or multilocular cystic masses that lie within or protrude from the pancreas, usually affecting the head or tail. However, imaging studies cannot consistently separate lymphoepithelial cysts from neoplastic mucinous cysts, such as MCN and IPMNs (910). FNA can support the diagnosis of a lymphoepithelial cyst when anucleated squamous cells, stratified squamous cells, amorphous keratinaceous debris, and cholesterol clefts and/or lymphocytes are present (911). However, FNA may be inconclusive, especially if atypical glandular cells are present. Using cyst fluid CEA as a discriminating test has its limitations as several case reports have noted elevated levels of CEA and/or CA19-9 in lymphoepithelial cysts (912,913). In contrast, molecular testing may helpful in preoperatively excluding a lymphoepithelial cyst, considering these lesions are devoid of genomic alterations (e.g., KRAS, GNAS) (263). Gross examination shows an encapsulated cystic lesion of medium size (mean: 5 cm). The cyst contents may vary from serous to cheesy/caseous appearing depending on the degree of keratin formation. Microscopic examination reveals an encapsulated lesion characterized by a dense band of mature lymphoid tissue with prominent, well-formed germinal centers subtending a cyst lining of mature stratified squamous epithelium occasionally containing keratinaceous debris (Fig. 35.121). Lymphoepithelial islands can be seen. It is uncommon for lymphoepithelial cysts to get infected or acutely inflamed; however, the adjacent pancreas may have granulomas, collections of foamy histiocytes, and fat necrosis. The uninvolved pancreas is unremarkable in most cases. Although lymphoepithelial cysts might contain sebaceous glands (914), they are distinct from dermoid cysts (cystic monodermal teratomas) or teratomas because of the large amount of organized lymphoid tissue present and the lack of hair, cartilage, and, occasionally, neural tissue. This benign lesion is managed by surgery.

FIGURE 35.121 Lymphoepithelial cyst. Keratinous material fills the cysts lined by squamous epithelium. Cholesterol clefts are noted. Lymphoid tissue is closely associated with the epithelium. Uninvolved pancreatic parenchyma is present in the lower left.

SQUAMOID CYST OF PANCREATIC DUCT Squamoid cysts of pancreatic ducts are relatively small cysts with a median size of 1.5 cm, perhaps explaining why they are only recently coming to clinical attention due to improved imaging technology. Most of the cases are detected at an older age (mean: 68 years), during workup for other conditions (474,915). These cysts typically result from unilocular cystic dilatation of the ducts and have variable lining ranging from attenuated, flat, nonstratified squamous, to transitional, to mucosal-type stratified squamous epithelium (without cornified layer or parakeratosis). Often, the cytoplasm also has an oncocytoid appearance. In most areas, the lining is multilayered with transitional features (Fig. 35.122). The wall of the cyst is composed of a thin band of fibrous tissue, focally showing islets or tributary ducts, which, along with the location and architecture, confirm the intraductal nature of the process. Neither acute nor chronic inflammation is a feature of this lesion.

FIGURE 35.122 Squamoid cyst of pancreatic duct. The cyst is unilocular and well circumscribed with a fibrotic wall. Note dense eosinophilic material, characteristic of enzymatic concretions, in the cyst lumen. Inset shows squamoid lining epithelium.

Immunohistochemically, nuclear p63 expression is present in all cases, a finding that is not seen in the normal pancreas or in nonsquamous cystic lesions of this organ (35), except in areas of squamous metaplasia due to obstructive chronic pancreatitis. Areas with a separate population of secretory-type (luminal) cells are negative for p63, as expected. Cytokeratin expression profiles are that of pancreatic ducts showing CK7 and CK19 positivity and lacking CK20. It is important to distinguish squamoid cysts of pancreatic ducts from other cystic lesions, in particular, from mucinous tumors clinically, because the latter often have malignant potential, whereas squamoid cysts of pancreatic ducts appear to be innocuous lesions. Preoperative differential diagnosis of these may be very difficult (816,817). Tumor markers (CEA and CA19-9) may be high in squamoid cysts of pancreatic ducts. Their distinction at the microscopic level, however, is fairly straightforward (815). EPIDERMOID CYSTS IN INTRAPANCREATIC ACCESSORY SPLEEN These are very rare lesions that are discovered in younger patients (second to third decades). They occur almost exclusively in the tail of the pancreas where accessory spleens are not uncommon. The cyst contains serous fluid or keratinaceous debris and is lined by attenuated squamous cells surrounded by unremarkable splenic tissue (Fig. 35.123) (907,918).

FIGURE 35.123 Epidermoid cyst within intrapancreatic accessory spleen. The cyst has a thin squamous epithelial lining surrounded by splenic red and white pulps.

LYMPHANGIOMA Lymphangioma of the pancreas is a rare neoplasm that presents clinically with nonspecific findings (vague abdominal pain or a palpable mass) or may be discovered incidentally during abdominal imaging studies or during abdominal surgery for unrelated diseases (653,919,920). There is a 2:1 female-to-male ratio, with patients of all ages affected. A rare association with blue rubber bleb nevus syndrome is reported (921). All topographic regions of the pancreas can be affected, with the tumor frequently reaching a large size (up to 20 cm). The mass may be unilocular or multilocular, closely resembling the other cystic neoplasms of the pancreas, especially if cavernous or cystic in type. The capillary lymphangioma might be mistaken for a solid tumor, although the capillary lymphangioma is exceedingly rare. Clear, serous, chylous, or hemorrhagic fluid occupies the spaces in the lesions. Lymphangiomas consist of dilated and/or interconnecting vascular channels (cysts) of variable size, separated by thin septa. The cystic spaces are lined by flattened to slightly elevated endothelial cells that do not contain glycogen and do not react with antibodies to keratin or EMA. The cyst walls contain various amounts of collagenous connective tissue, irregular smooth muscle fascicles, occasional adipocytes, and mature lymphocytes (Fig. 35.124). Phlebolith-like structures can be seen. The surrounding pancreatic parenchyma is often atrophic, with focal fibrosis. Factor VIII–related antigen, CD31, and CD34 are sensitive, specific, and reliable markers for endothelium (Fig. 35.125). Of the three histologic variants of lymphangiomas described (capillary, cavernous, and cystic), the differences between the cavernous and cystic lymphangiomas seem to be more quantitative than qualitative. The cystic nature of lymphangioma encompasses a broad differential diagnosis including lymphoepithelial cysts (especially those with denuded epithelium) because they also contain prominent lymphoid tissue often with follicles. However, the smooth muscle found in the wall of a cyst, in combination with the lymphocytes (both in the wall and in the vascular lumina), is most suggestive of the diagnosis.

FIGURE 35.124 Lymphangioma. Parenchymal atrophy, fibrosis, and lymphoid infiltration contrast with the spaces filled with coagulated lymph.

FIGURE 35.125 Lymphangioma. (Left) The cyst wall shows irregular smooth muscle fascicles. (Right) The smooth muscle is highlighted with actin (upper), whereas the endothelial lining cells are immunoreactive with factor VIII–related antigen (lower).

Hemangiomas in the pancreas are similar to these lesions elsewhere and are extremely rare. CYSTIC MESENCHYMAL NEOPLASMS

Some mesenchymal neoplasms that occur in the pancreatic region may present as cystic lesions. Schwannomas in the pancreas, especially, tend to be cystic (922,923). Further, some sarcomas, especially necrotic GI stromal tumors, may present as a cystic mass from the pancreas. SECONDARY NEOPLASMS THAT ARE CYSTIC Rarely, metastatic neoplasms (or neoplasms that secondarily involve the pancreas) can exhibit cystic change. We have seen examples of metastatic ovarian and RCC in the pancreas that presented as cystic masses. We have also seen a GI stromal sarcoma with marked cystic degeneration that presented as a cystic mass in the pancreatic region. Cystic lesions of the common bile duct and duodenum may also mimic pancreatic cysts. DIFFERENTIAL DIAGNOSIS OF CYSTIC LESIONS Alcoholism, biliary tract disease, or former abdominal trauma suggests a pseudocyst; most patients are young to middle-aged men. The pseudocyst is usually unilocular and may communicate with the ductal system. Fluid in pseudocysts typically is thin, contains necrotic debris or blood, and is rich in pancreatic enzymes (141,238,241) and has a low CEA level. The absence of an epithelial lining and specialized cellular stroma favors pseudocyst over MCN. In contrast, imaging studies of the mucinous tumors usually show multiple locules. Cyst fluid is often viscid, low in enzymes, and rich in CEA, CA19-9, and CA72.4 (241). However, there are exceptions (918,919). More importantly, hemorrhage, necrosis, and loss of epithelium may occur in any cystic neoplasm, thereby obscuring portions of its characteristic architecture. Critical evaluation of the history, the imaging and clinical laboratory data, and the pathologic specimen is essential with all cystic lesions. FNA or surgical biopsy may be insufficient for diagnosis. Close follow-up is needed; if a supposed pseudocyst does not disappear a few weeks after drainage, then a neoplasm must be considered as likely. Ductal carcinoma sometimes has a cystic component (cavitary necrosis) or is associated with a retention cyst or a pseudocyst. The same is true for PanNETs. Serous neoplasms should be differentiated from the MCNs (Table 35.8), clear cell PanNETs, the pseudomicrocystic pattern sometimes present in the SPNs, lymphangiomas, and RCC with clear cells. In SPNs, the cells lack glycogen, the nuclei are usually ovoid (some with a groove), and the pseudomicrocysts are actually multiple small foci of loose connective tissue; these tumors are usually invasive at their borders (850,852,854). In lymphangioma, the cells lining the spaces are positive for factor VIII–related antigen, CD34, and other endothelial markers; lymphoid follicles and occasional bundles of smooth muscle are present in the stroma (653,919,920). RCCs typically have larger cells with less regular nuclei and are not organized into a microcystic pattern (924).

MESENCHYMAL NEOPLASMS Lymphangiomas were discussed earlier (see section “Other Cystic Lesions”). Inflammatory myofibroblastic tumor (pseudotumor) is a rare, distinctive, and indeterminate neoplasm. With equal gender distribution, the pancreatic head is affected most frequently by an ill-defined and firm mass that is histologically characterized by a proliferative fibrous tissue with moderate to marked inflammation and obliterative phlebitis. Perineural proclivity can mimic adenocarcinoma. There is a known association with type 1 AIP, although ALK1 expression is also reported (although negative cases seem more aggressive), suggesting that long-term clinical follow-up is prudent, although to date, pancreatic cases seem not to progress (182,925-927). Solitary fibrous tumor (928-931), schwannoma (922,923,932,933), malignant paraganglioma (934,935), leiomyoma (936,937), leiomyosarcoma (938-940), liposarcoma (941), Ewing sarcoma (760,942), desmoplastic small round cell tumor (943,944), perivascular epithelioid cell neoplasm

(PEComa) (415,416,418,945,946), and pleomorphic sarcoma (182,947,948) are all primary mesenchymal tumors that have been rarely reported in the pancreas.

SECONDARY NEOPLASMS Malignant lymphoma may rarely arise primarily in the pancreas (949-951) but is more likely to affect the pancreas as part of systemic disease. Lymphomas of various types, usually of B-cell phenotype, have been described. Direct extension into the pancreas by neoplasms arising in the retroperitoneal tissues, nearby lymph nodes, or adjacent GI tract should be excluded before rendering a diagnosis of a primary pancreatic tumor. Metastatic neoplasms occur in the pancreas (757). They are often multifocal and lack the fibrosis so common in primary pancreatic adenocarcinoma. Carcinomas of the lung (Fig. 35.126), GI tract, kidney (Fig. 35.127) , and breast are among the most common (924,952-955).

FIGURE 35.126 Metastatic lung adenocarcinoma. The patient had a known history of lung adenocarcinoma. The adenocarcinoma demonstrates gland formation with prominent perineural invasion. However, the cells are strongly and diffusely immunoreactive with TTF-1 (right), confirming a metastatic lung tumor.

FIGURE 35.127 Metastatic renal cell carcinoma to the pancreas. (Left) Note the clear cells and pseudoglandular architecture filled with erythrocytes. (Right) Epithelial membrane antigen highlights the membranes of the neoplastic cells (upper), whereas renal cell marker also yields a positive reaction (lower).

MISCELLANEOUS LESIONS PANCREATIC HAMARTOMA The term hamartoma refers to an excessive, focal overgrowth of cells and tissues native to the organ in which it occurs. Thus, hamartoma seems to be a malformation rather than a true neoplasm. Pancreatic hamartomas are rare and present as well-demarcated masses with a solid or solid and cystic appearance (956-959). Microscopically, these lesions are composed of small- to medium-sized ductal structures surrounded by disorganized acini and embedded in various amounts of fibrous stroma, forming a well-demarcated lesion, within an otherwise unremarkable pancreatic parenchyma. Although scattered neuroendocrine cells might be identified by immunohistochemistry, pancreatic hamartomas lack well-formed islets of Langerhans, peripheral nerves, and concentric elastic fibers in their duct walls, all of which exist in both the normal and atrophic pancreas. Immunohistochemically, both acinar cells and ductal cells are positive for epithelial markers, and the acinar cells are positive for exocrine markers (trypsin, chymotrypsin, etc.). The stromal spindle cells reportedly express CD34 and CD117 but are negative for S100 protein, SMA, desmin, and Bcl-2 (271,930,956-959).

ACKNOWLEDGMENTS

The opinions or assertions contained herein are the private views of the authors and are not to be construed as official or as reflecting the views of Southern California Permanente Medical Group, Memorial Sloan Kettering Cancer Center, Koc University and American Hospital, or University of Pittsburgh Medical Center.

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867. Yu MH, Lee JY, Kim MA, et al. MR imaging features of small solid pseudopapillary tumors: retrospective differentiation from other small solid pancreatic tumors. AJR Am J Roentgenol. 2010;195(6):1324-1332. 868. Kosmahl M, Seada LS, Jänig U, et al. Solid-pseudopapillary tumor of the pancreas: its origin revisited. Virchows Arch. 2000;436(5):473-480. 869. Hibi T, Ojima H, Sakamoto Y, et al. A solid pseudopapillary tumor arising from the greater omentum followed by multiple metastases with increasing malignant potential. J Gastroenterol. 2006;41(3):276-281. 870. Ishikawa O, Ishiguro S, Ohhigashi H, et al. Solid and papillary neoplasm arising from an ectopic pancreas in the mesocolon. Am J Gastroenterol. 1990;85(5):597-601. 871. Junzu G, Yanbin S, Suxia W, et al. A case of extrapancreatic solid pseudopapillary tumor in the retroperitoneum. Jpn J Radiol. 2012;30(7):598-601. 872. Stoll LM, Parvataneni R, Johnson MW, et al. 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36

Nonneoplastic Liver Disease Ryan M. Gill ■ Sanjay Kakar

INTRODUCTION AND BASIC PRINCIPLES OF NONNEOPLASTIC LIVER DISEASE The architecture of liver microanatomy has evolved through several models, beginning with the hepatic vein at the center in the hexagonal lobule (1,2) followed by the liver acinus model proposed by Rappaport et al. (3), with the portal tract (particularly the portal venule) at the center and now with more recent models again favoring a lobular unit based on blood flow (4,5), or even smaller microanatomic units (6,7) (as well as other models based on metabolic (8) and exocrine function (9,10) with description of the choleohepaton liver unit [the smallest vascular “unit” in a lobule]). Although the primary lobule model, or some variation, appears to be most accurate, in practice, both lobular and acinar terminologies are useful in describing pathologic processes (Table 36.1). TABLE 36.1 Lobular and Acinar Terminology Lobular

Acinar

Description

Centrilobular, central, centrizonal

Zone 3

Area around the central venule

Midzonal

Zone 2

Parenchyma between central venule and portal tract

Periportal

Zone 1

Area around the portal tract

Blood draining from the gastrointestinal (GI) tract flows through the portal veins and eventually into terminal portal venules and then sinusoids, where it mixes with arterial blood (which enters the sinusoids from arteriosinusoidal twigs branching off of the ~10 μm in diameter terminal hepatic arteriole) (11). The hepatic arteriole represents a key structure in identification of portal tracts by light microscopy (Fig. 36.1). The complex architecture of the liver lobule leads to structural and functional heterogeneity, such as variable density of fenestration (12) and central ammonium metabolism with associated glutamine synthetase expression (Fig. 36.2) (13). Even the extracellular matrix components of the space of Disse may vary from periportal to pericentral regions (14).

FIGURE 36.1 Normal portal tract with hepatic arteriole (arrows). Trichrome stain, ×200 magnification. BD, bile ductule; PV, portal venule.

FIGURE 36.2 Glutamine synthetase expression in normal liver. Glutamine synthetase immunostain, ×100 magnification. CV, central vein; PT, portal tract.

Intrahepatic biliary epithelium is thought to develop from limiting plate hepatoblasts (15) through formation of a ductal plate, which envelops the mesenchyme of early portal tracts. Duct anastomosis progresses from the extrahepatic biliary structures in the porta hepatis to the ramifying smaller intrahepatic septal ducts, in association with portal vein and hepatic artery branches, which eventually form interlobular bile ducts that drain bile from hepatocytes through the canals of Hering and associated bile ductules. This process is often not complete at birth and contributes to physiologic jaundice (16). Formation of the biliary tree normally involves dismantling of the ductal plate, a physiologic process that may be disrupted/damaged in intrahepatic biliary atresia (17). Liver is the dominant site for hematopoiesis from about 12 weeks’ to 5 months’ gestation (followed by the bone marrow), but hematopoiesis is usually no longer detectable outside the neonatal period. Hepatocytes are not only interconnected through both tight and gap junctions along their lateral membrane (18) but also interfaced

with the perisinusoidal space of Disse (without an intervening basement membrane) and a specialized canalicular space. At the limiting plate, the space between hepatocytes and periportal connective tissue is termed the space of Mall, which is in continuity with the space of Disse. The bile canaliculus forms from adjacent hepatocyte canalicular membranes (diameter 1-2.5 μm); like the sinusoidal surface, it is covered by microvilli. Synthesis of glycogen represents a key function of the hepatocyte, which also coordinates glycogen breakdown and release as glucose. Smooth endoplasmic reticulum (ER) uses glucoronyltransferase to convert bilirubin to a water-soluble form and uses the p450 system to eliminate toxins in bile (19). Smooth ER may be prominent in some drug-induced liver injury (DILI) and appear as pseudo–ground-glass change (also termed polyglucosan bodies) (Fig. 36.3), which may be mistaken for hepatitis B cytoplasmic inclusions. A minority of hepatocytes are normally binucleate (20). Mitotic activity is very low, and usually, no mitotic figures are present on a normal core biopsy. Lipofuscin, iron (i.e., ferritin), and copper (as in cholestasis) may accumulate in lysosomes (Fig. 36.4). Mitochondria account for a substantial portion of hepatocyte cytoplasmic volume and may be visible on light microscopy as giant mitochondria in a variety of settings (e.g., steatohepatitis, Wilson disease [WD], glycogenic hepatopathy) (Fig. 36.5). Hepatocytes, like many other epithelial cells, contain two predominant cytokeratins (CKs), CK8 and CK18, in contrast to biliary epithelial cells, which express CK7 and CK19 (21,22), although hepatocytes may also express CK7 in the setting of cholestasis (see section IV). Intermediate filaments are also present and are typically not seen on light microscopy, unless there is network collapse and Mallory hyaline formation.

FIGURE 36.3 Pseudo–ground-glass change in hepatocytes (polyglucosan bodies). Hematoxylin and eosin stain, ×400 magnification.

FIGURE 36.4 Lipofuscin (ceroid) pigment in pericentral hepatocytes. Hematoxylin and eosin stain, ×400 magnification.

FIGURE 36.5 Giant mitochondria (arrow) in a ballooned hepatocyte with Mallory hyaline. Hematoxylin and eosin stain, ×400 magnification.

Additional cell types include the hepatic stellate cells (HSCs) (i.e., Ito cells), which contain fat droplets, store vitamin A, and play a role in inflammation, regeneration, and fibrosis (23-27). They are only observed on light microscopy in the setting of Ito cell hyperplasia or lipidosis (e.g., as may be due to hypervitaminosis A). HSC may express glial fibrillary acidic protein (GFAP) and α smooth muscle actin when activated (28). Kupffer cells are specialized macrophages with diverse functions that modulate cytokine networks in the liver (29). Kupffer cells are likely derived from both bone marrow–derived monocytes (30) and precursor cells in the yolk sac (31) and have substantial phagocytic activity, which may be readily observed in the setting of hemophagocytosis (Fig. 36.6) (as may be seen due to infection or lymphoma). A substantial normal compliment of lymphocytes (mostly T cells, natural killer [NK] cells, and NK T cells) reside in the liver (32) and likely play a major role in innate and adaptive immunity (33,34).

FIGURE 36.6 Kupffer cell hemophagocytosis (arrow). Hematoxylin and eosin stain, ×400 magnification.

BASIC PRINCIPLES OF NONNEOPLASTIC LIVER DISEASE Direct toxic effects, immune response to foreign or self-antigen, ischemia, metabolic disturbance, or bile duct obstruction may all injure the liver. Hepatic response to injury can lead to regeneration, which can return the liver to its normal structure and function, even after extensive injury, or, in the case of ongoing injury, the hepatic response may result in fibrosis, which may progress to cirrhosis with its associated vascular flow changes and systemic effects. As with inflammation elsewhere in the body, a network of cytokine signaling plays a key role in direction of an immune response within the liver, with some cytokines generally accepted to promote a cellmediated immune response (e.g., Th1 cytokines such as tumor necrosis factor-α and interferon-γ) and others with a role in amplifying the humoral response (e.g., Th2 cytokines such as interleukin [IL]-10 and IL-4), which may serve to downregulate an overall inflammatory response. Under direction of proinflammatory cytokines, activated macrophages may aggregate to form a

granuloma (Fig. 36.7), which is thus a relatively nonspecific finding. A subset of T cells (i.e., regulatory T cells) clearly plays a role in inhibiting cytotoxic T cells and thus protect against autoimmune disease in the liver (35). Recruited lymphocytes may travel to the site of hepatocyte injury in the lobule, but with chronic infection, for example, will also aggregate in portal regions. Neutrophils may extravasate directly through periductal capillaries in response to biliary damage (36-39).

FIGURE 36.7 Granuloma in lobular parenchyma. Hematoxylin and eosin stain, ×200 magnification.

Liver injury most obviously manifests as morphologic evidence of hepatocyte death (Fig. 36.8). Hepatocyte death may occur through multiple pathways (ischemic/toxic, necrosis, apoptosis, autophagy, etc.), but isolated damaged/dying hepatocytes will show overlapping morphologic features and thus related terms, such as apoptosis, spotty necrosis, and acidophil bodies, are used interchangeably. Hepatocyte swelling (Fig. 36.9) may be seen in a variety of settings, but the term ballooning specifically refers to hepatocyte injury in the

setting of steatohepatitis and has three morphologic features: (a) large swollen hepatocyte, (b) rarefaction of cytoplasm, and (c) clumping of intermediate filaments into eosinophilic globular areas, which may form Mallory hyaline (also referred to as Mallory-Denk bodies) with loss of CK8/18 (40). A variety of systems are available for grading inflammation/liver injury (Table 36.2), with the BattsLudwig system being one of the most commonly used schemes (grade 1, Fig. 36.10; grade 2, Fig. 36.11; grade 3, Fig. 36.12; grade 4, Fig. 36.13).

FIGURE 36.8 Spotty hepatocyte necrosis (arrow). Hematoxylin and eosin stain, ×200 magnification.

FIGURE 36.9 Hepatocyte swelling, diffuse. Hematoxylin and eosin stain, ×400 magnification.

FIGURE 36.10 Portal inflammation with minimal to no interface hepatitis, grade 1 (scales 0-4, Batts-Ludwig methodology). Hematoxylin and eosin stain, ×400

magnification.

FIGURE 36.12 Portal inflammation with moderate interface hepatitis, grade 3 (scales 0-4, Batts-Ludwig methodology). Hematoxylin and eosin stain, ×200 magnification.

FIGURE 36.11 Portal inflammation with mild interface hepatitis, grade 2 (scales 04, Batts-Ludwig methodology). Hematoxylin and eosin stain, ×200 magnification.

FIGURE 36.13 Panacinar necrosis, grade 4 (scales 0-4, Batts-Ludwig methodology). Hematoxylin and eosin stain, ×100 magnification.

TABLE 36.2 Grading Inflammation/Liver Injury Batts-Ludwig Grade

Classifier of Activity

Interface Hepatitis (Piecemeal Necrosis)

Lobular Activity

0

None

No significant inflammation

None

1

Minimal

Minimal to no interface hepatitis

Rare spotty necrosis

2

Mild

Portal inflammation with mild (1-3 foci) interface hepatitis

Spotty necrosis

3

Moderate

Portal inflammation with moderate interface hepatitis (~3 or more foci) in multiple portal tracts

Confluent necrosis

4

Severe

Severe interface hepatitis (diffuse/continuous along interface)

Bridging or extensive necrosis

Adapted with permission from Batts KP, Ludwig J. Chronic hepatitis. An update on terminology and reporting. Am J Surg Pathol. 1995;19(12):1409-1417.

Many metabolic derivatives may accumulate in hepatocytes, but fat droplets, or steatosis, are particularly common in the United States, because this finding is the diagnostic feature of fatty liver disease (discussed in the section “Fatty Liver Disease”). Steatosis may be separated into microvesicular (Fig. 36.14) and macrovesicular forms. Microvesicular steatosis is characterized by very small/fine fat globules and is a result of mitochondrial injury. Macrovesicular steatosis may show multiple fat droplets that do not displace the nucleus (small droplet steatosis) or coalescent fat vacuoles that displace the nucleus (large droplet steatosis). In older descriptions, small droplet steatosis has been classified as microvesicular steatosis; however, based on morphology and

pathogenesis, it is best considered as a form of macrovesicular steatosis.

FIGURE 36.14 Microvesicular steatosis, diffuse. Hematoxylin and eosin stain, ×200 magnification.

Cholestasis refers to deficient secretion of bile constituents, which may occur from bile duct obstruction or hepatocellular injury and which may manifest with a variety of morphologic changes. Bile may accumulate in hepatocytes, or in canaliculi, and hepatocytes may show swelling and degenerative changes. Bile leaks may form large destructive lakes/infarcts. Mallory hyaline may form in periportal hepatocytes, which, along with swelling in this zone, has been termed cholate stasis. Portal tracts may show edema and ductular reaction (41). The latter term encompasses both an increase in the number of well-formed duct lumina in a portal tract and any apparent metaplastic change of limiting plate hepatocytes into smaller ductular structures. Inspissated bile may be seen, particularly in sepsisassociated bile duct obstruction (i.e., cholangiolar cholestasis).

Although some injury may be completely resolved through progenitor cell–driven regeneration, other injuries result in chronic long-lasting changes, namely, fibrosis. Collagen fibers (including types I, III, and IV) collect at sites of chronic injury, most likely as a result of activated HSCs (42-44). Fibrosis affects blood flow and leads to sinusoidal capillarization (which can be highlighted on CD34 immunohistochemical staining) (Fig. 36.15) (45,46). Elastin deposition typically precedes collagen fiber deposition (47) and thus can be used as a marker of early fibrosis and aids in distinguishing necrosis from fibrosis on trichrome stain (Figs. 36.16 and 36.17). Several systems for staging liver fibrosis (48) have been developed (Table 36.3); Brunt-Kleiner methodology is most commonly used for steatohepatitis (49) (see later discussion in the section “Fatty Liver Disease”), whereas several systems such as Scheuer, Batts-Ludwig, Metavir, and Ishak methodologies are used for chronic viral hepatitis (50) (portal/periportal fibrosis, stage 1, Fig. 36.18; periportal/incomplete septal fibrosis, stage 2, Fig. 36.19; bridging fibrosis, stage 3, Fig. 36.20; cirrhosis, stage 4, Fig. 36.21). In the commonly used Batts-Ludwig methodology, fibrosis progresses from stage 1 “portal fibrosis” (similar to “enlarged fibrotic portal areas” in Scheuer methodology) to stage 2 “periportal fibrosis” (similar to “periportal or portal-portal septa” formation in Scheuer methodology) and then bridging septal fibrosis (stage 3) and cirrhosis (stage 4). Thin periportal fibrous extensions are considered part of stage 1 fibrosis, whereas better developed incomplete fibrous septa are classified as stage 2 (48,51,52). The result of long-standing chronic injury is cirrhosis, which is defined by diffuse involvement and regenerative nodules isolated by fibrous septa. Fibrous bands may span portal-portal, portal-central, or central-central regions, and nodule size may be uniformly small, less than 3 mm (micronodular), or variable (macronodular). Advanced fibrosis, including cirrhosis, can be remodeled and even reversed in some scenarios in which the underlying etiology of hepatocellular injury has been eliminated (the so-called incomplete septal cirrhosis) (Fig. 36.22) (53,54). Coincident with the architectural changes of advanced fibrosis, liver vasculature undergoes remodeling and shunting, which is likely to have more

impact on liver function than fibrosis itself. Sclerosis around vessels and in the space of Disse leads to vascular shunts, which, along with sinusoidal capillarization, allows hepatic arterial blood to bypass parenchyma on its way to the hepatic vein (39). Vascular obstruction in cirrhosis can lead to large zones of parenchymal loss, termed parenchymal extinction, which may form a mass-like lesion on imaging. Parenchymal extinction commonly has extensive ductular reaction, which may mimic a biliary neoplasm (Fig. 36.23).

FIGURE 36.15 Sinusoidal capillarization highlighted by CD34 immunostain (normal sinusoidal endothelial cells do not label with anti-CD34 antibody), ×100 magnification.

FIGURE 36.16 Necrosis versus early fibrosis. This distinction cannot be made on some trichrome stains. Trichrome stain, ×200 magnification.

FIGURE 36.17 Necrosis versus early fibrosis. Lack of elastin fiber deposition in much of the area that stains blue in Figure 36.16 allows for correct identification of

necrosis without significant fibrosis. Elastin fibers within the confines of the adjacent portal tract are highlighted (arrows) for comparison. Orcein stain, ×200 magnification.

FIGURE 36.18 Portal/periportal fibrosis, stage 1 (scales 0-4, Batts-Ludwig methodology). Trichrome stain, ×200 magnification.

FIGURE 36.19 Periportal septal fibrosis, stage 2 (scales 0-4, Batts-Ludwig methodology). Trichrome stain, ×200 magnification.

FIGURE 36.20 Bridging fibrosis, stage 3 (scales 0-4, Batts-Ludwig methodology). Trichrome stain, ×100 magnification.

FIGURE 36.21 Cirrhosis, stage 4 (scales 0-4, Batts-Ludwig methodology). Trichrome stain, ×100 magnification.

FIGURE 36.22 Cirrhosis with remodeling effect (incomplete septal cirrhosis). Trichrome stain, ×100 magnification.

FIGURE 36.23 Parenchymal extinction with benign ductular reaction. Hematoxylin and eosin stain, ×200 magnification.

TABLE 36.3 Staging Liver Fibrosis Stage

Scheuer

Batts-Ludwig

0

No fibrosis

Normal connective tissue

1

Enlarged fibrotic portal areasa

Fibrous portal expansiona

2

Periportal septa or portal-portal septa but intact architecture

Periportal, or rare portal-portal, septa

3

Fibrosis with architectural distortion but no obvious cirrhosis

Fibrous septa with architectural distortion, no obvious cirrhosis

4

Probable or definite cirrhosis

Cirrhosis

aSmall,

thin periportal extensions are considered part of stage 1. Adapted by permission from Nature: Theise ND. Liver biopsy assessment in chronic viral hepatitis: a personal, practical approach. Mod Pathol. 2007;20(Suppl 1):S3-S14.

ACUTE HEPATITIS, AUTOIMMUNE HEPATITIS, AND INFECTIOUS LIVER DISEASE ACUTE HEPATITIS Acute hepatitis is defined clinically as at least a doubling of alanine transaminase (ALT) or aspartate transaminase (AST) (without chronic liver disease history). Alkaline phosphatase (ALP) is either normal, or ALT/ALP is increased (≥5). By definition, acute hepatitis is characterized by lobular hepatocellular injury, which manifests as hepatocellular swelling, apoptosis, or dropout. Lobular injury may be random or zonal but, in severe cases, will form confluent clusters of apoptotic cells or bridge between zones. Bridging necrosis denotes a higher risk of permanent injury in the form of fibrosis. In its most severe presentation, acute hepatitis may lead to acute liver failure manifested by widespread confluent or panacinar necrosis. Massive necrosis refers to loss of almost all hepatocytes (~60%-70% loss), whereas submassive necrosis (30%-70%) has more viable hepatocytes, often with periportal sparing. This distinction cannot be made on core liver biopsy, as necrosis is variable throughout the liver, although it is helpful to indicate the extent of injury (e.g., focal, confluent, bridging, panacinar). As the acute phase of liver injury may last up to 6 months, regenerative features are commonly encountered (e.g., numerous binucleate hepatocytes, mitoses, thick plates, prominent Kupffer cells) and active injury may be minimal, especially in a resolving phase. It is important to note that cholestasis (and occasionally ductular reaction) may be present in acute hepatitis (the so-called “cholestatic hepatitis”), if bile secretion is inhibited, and thus is not indicative of a chronic biliary obstructive process in this setting. Lobular inflammation is commonly encountered, typically lymphocytic, and may be a predominant feature; bile ducts are usually intact, allowing for exclusion of chronic biliary disease. Portal inflammation is variable. Multinucleated hepatocytes (giant cells or syncytial cells) may be present. By definition, there is no fibrosis; however, distinction between necrosis and fibrosis represents a common diagnostic dilemma. Areas of

necrosis show pale staining on trichrome stain as compared to dense coarse collagen in the portal tracts and fibrous septa, but this distinction can be challenging on a poorly stained trichrome stain (Fig. 36.16). Orcein, or other elastic stains, may be used to demonstrate early elastin fiber deposition as a first manifestation of established fibrosis (Fig. 36.17). Acute hepatitis can be divided into six morphologic subpatterns, each with its distinct morphologic features and differential diagnoses (Table 36.4). The inflammation-predominant pattern is the most common and shows hepatocellular injury accompanied by a prominent inflammatory infiltrate. The differential diagnosis includes acute viral hepatitis (AVH), DILI, autoimmune hepatitis (AIH), WD, and celiac disease (CD). Serology is not reliable in excluding AVH; instead, nucleic acid testing (typically with polymerase chain reaction [PCR]–based assays) could be considered to allow for window period detection. DILI has variable histologic manifestations but may have prominent eosinophils, granulomas, centrizonal necrosis, and cholestasis out of proportion to lobular activity. Careful clinical evaluation of drug/herbal history and correlation with temporal profile of disease onset is necessary. Transaminitis typically subsides with cessation of the offending agent but may persist for months. Histologic and clinical findings for specific drugs are summarized and updated on a website maintained by the National Institutes of Health (NIH) (www.livertox.nih.gov). Most cases of AIH will have fibrosis, but up to 10% lack fibrosis at initial presentation. High necroinflammatory activity, interface inflammation, hepatocyte rosette formation, and abundant plasma cells (Fig. 36.24) are the most suggestive histologic features, but these findings may be less prominent at various phases of disease or with therapy. Elevated serum immunoglobulin G (IgG) and demonstration of autoantibodies (antinuclear, smooth muscle, liver-kidney microsomal, soluble liver antigen) are helpful. Definitive diagnosis is based on a scoring system proposed by the International Autoimmune Hepatitis Group (55). In addition to steatosis, chronic hepatitis, and cirrhosis, WD can present with acute hepatitis and liver failure (Fig. 36.25) (described in more detail in the section “Metabolic, Genetic, and Developmental

Disorders”). CD is commonly associated with a mild transaminitis, which may normalize after a gluten-free diet and shows mild nonspecific reactive hepatitis on biopsy. Rare instances of acute hepatitis have been reported in CD, and liver involvement can also manifest as chronic hepatitis or nodular regenerative hyperplasia (NRH). Serologic testing for CD should be considered in all cases of unexplained liver dysfunction. Inflammation-predominant hepatitis can show zonation, and when injury is confined to the centrizonal region, special consideration should be given to AIH and DILI, specifically drugs such as minocycline, nitrofurantoin, and methyldopa. Idiosyncratic reactions to isoniazid, monoamine oxidase (MAO) inhibitors, phenytoin, valproate, and various antibacterial (e.g., sulfonamides) and antifungal agents (e.g., ketoconazole) may result in inflammation-predominant acute hepatitis that progresses to liver failure. Herbal agents should be carefully evaluated for known hepatotoxins, which include chaparral leaf, germander, pennyroyal oil, Jin Bu Huan, and kava kava, among others (56).

FIGURE 36.24 Acute hepatitis with abundant portal plasma cells in an untreated case of autoimmune hepatitis (steatosis represents unrelated fatty liver due to obesity and hyperlipidemia). Hematoxylin and eosin stain, ×200 magnification.

FIGURE 36.25 Bridging necrosis demonstrated in a patient with Wilson disease presenting in acute liver failure. Hematoxylin and eosin stain, ×100 magnification.

TABLE 36.4 Patterns of Acute Hepatitis 1. Inflammation-predominant pattern (with variable necrosis) → acute viral hepatitis, idiosyncratic drug reaction, autoimmune hepatitis, Wilson disease, or celiac disease 2. Cholestatic pattern → nonspecific, most commonly with acute hepatitis due to drug, hepatitis A, or hepatitis E 3. Toxic pattern → direct hepatocellular injury (halothane, acetaminophen, cocaine, MDMA, organic solvents, toxic mushrooms, some herbal medications, HSV, and adenovirus) or acute vascular injury/ischemia 4. Resolving pattern → nonspecific but common in idiosyncratic drug reaction and in systemic autoimmune and inflammatory disorders 5. Giant cell pattern → nonspecific in adults, most common in autoimmune hepatitis 6. Mild nonspecific pattern → raises consideration for early acute hepatitis vs. early biliary injury, requires clinical and laboratory correlation HSV, herpes simplex virus; MDMA, 3,4-methylenedioxy-N-methylamphetamine.

In cholestatic hepatitis (Fig. 36.26), the ALP is typically elevated but is less than five times normal, and ALT/ALP is less than five. The morphologic features are similar to inflammation-predominant acute hepatitis and are accompanied by cholestasis. The differential diagnosis includes all entities described earlier, but in particular, viral hepatitis A, hepatitis E, and DILI. Commonly implicated drugs include chlorpromazine and macrolide antibiotics (e.g., erythromycin) (56).

FIGURE 36.26 Acute hepatitis with cholestasis, the so-called “cholestatic hepatitis,” with spotty necrosis and canalicular bile plugs. Hematoxylin and eosin stain, ×400 magnification.

The toxic pattern manifests as necrosis with essentially no inflammation (Fig. 36.27), which indicates a direct toxic effect rather than an immune-mediated event. The differential diagnosis is, therefore, limited to exposures with known liver toxicity (e.g., halothane, acetaminophen, cocaine, 3,4-methylenedioxy-Nmethylamphetamine [MDMA or “ecstasy”], organic solvents, toxic mushrooms [e.g., Amanita phalloides], some herbal medications, herpes simplex virus [HSV] 1/2, and adenovirus). Acute vascular

injury, as in ischemia, and acute-onset vascular outflow obstruction, as in Budd-Chiari syndrome (BCS), can also show this pattern of injury.

FIGURE 36.27 Acute hepatitis, toxic pattern with zone 3–centered necrosis without significant inflammation. Hematoxylin and eosin stain, ×100 magnification.

The resolving hepatitis pattern of injury is characterized by mild portal and lobular inflammation with scattered pigmented macrophages in sinusoids (Fig. 36.28), often clustered around central veins. There is usually only mild hepatocellular necrosis manifested by rare/spotty acidophil bodies. The differential diagnosis includes a resolving phase of any acute hepatitis, but it is especially common in DILI, as the offending agent has often been stopped prior to biopsy. A similar mild hepatitis pattern of injury, with or without prominent sinusoidal macrophages, is seen as a nonspecific response in systemic autoimmune and inflammatory disorders (e.g., systemic lupus erythematous, rheumatoid arthritis, and CD) and systemic or abdominal infections such as appendicitis and cholecystitis.

FIGURE 36.28 Resolving hepatitis with prominent pigmented macrophages (arrow). Hematoxylin and eosin stain, ×400 magnification.

Giant cell hepatitis (i.e., postinfantile giant cell hepatitis, syncytial giant cell hepatitis) is not only similar to inflammation-predominant acute hepatitis but also shows multinucleate syncytial hepatocytes (defined as numerous hepatocytes with four or more nuclei (57)). Although not specific, this pattern in adults is most commonly seen in AIH. Syncytial giant cell hepatitis is common in neonatal hepatitis (see section “Metabolic, Genetic, and Developmental Disorders”). In mild acute hepatitis, it may be difficult to reliably distinguish hepatitis versus biliary pattern of injury. Correlation with serologic findings, liver enzymes, and autoantibodies is important in establishing an underlying etiology. Demonstration of periportal hepatocyte copper, by copper stains, and staining of periportal hepatocytes with CK7 is helpful in establishing a biliary etiology. ACUTE LIVER FAILURE Acute liver failure manifests as onset of hepatic encephalopathy and coagulopathy within 8 to 26 weeks of symptom onset (in patients

without prior chronic liver disease). Drugs, such as acetaminophen overdose, are the most frequent etiology, but many of the etiologies of acute hepatitis can progress to acute liver failure. The histologic pattern can help in determining the etiology: inflammation dominant (e.g., DILI, AIH, WD), necrosis dominant (e.g., acetaminophen overdose, other toxins, HSV, vascular causes), and microvesicular steatosis (e.g., drugs such as valproate and tetracycline, Reye syndrome). Extensive sinusoidal involvement by amyloidosis or metastatic disease can rarely present as acute liver failure. Liver biopsy–associated bleeding is more common in the setting of severe coagulopathy and so transjugular biopsy may be performed (although biopsy may also be entirely contraindicated). The etiology may remain undetermined in at least 15% of adult acute hepatitis cases that progress to liver failure (58). CHRONIC HEPATITIS Chronic hepatitis is a form of liver injury that persists for at least 6 months after initial presentation. Common etiologies include AIH, WD, α1-antitrypsin deficiency (A1ATD) (both WD and A1ATD are described in more detail in the section “Metabolic, Genetic, and Developmental Disorders”), and some hepatotropic viruses. Drugrelated chronic hepatitis is uncommon but is well described with certain drugs. AUTOIMMUNE HEPATITIS AIH may present as acute hepatitis, including a fulminant form, chronic hepatitis, and cirrhosis. AIH is more common in women (peak incidence at 35-40 years) and often shows hypergammaglobulinemia (IgG >1.5× normal) and/or autoantibodies (antinuclear antibodies [ANAs], smooth muscle antibodies [SMAs], and liver-kidney microsomal antibody [LKM]). AIH patients commonly express human leukocyte antigen (HLA) DR3 or DR4. Bilirubin levels may be elevated, ALP is usually not significantly elevated, and transaminitis may be mild to severe. The clinical course may “wax

and wane,” but patients will progress to cirrhosis if untreated. AIH usually responds to immune suppression, but a prolonged period of treatment may be needed and relapse is possible. The predominant histologic finding is interface and lobular hepatitis, often with prominent plasma cells. Hepatocyte rosette formation may be seen. The bile ducts are largely intact, but focal lymphocytic infiltration and mild epithelial injury can be seen in areas of intense portal inflammation. Definitive diagnosis of AIH requires correlation with clinical parameters such as IgG level and autoantibodies: ANA (type 1 AIH), SMA (type 1 AIH), LKM (type 2 AIH, younger patients), soluble liver antigen/liver-pancreas antibodies (SLA/LP) (type 3 AIH), perinuclear antineutrophil cytoplasmic antibodies (p-ANCAs), and liver enzyme findings (59), and as scored in the International Autoimmune Hepatitis Group (55). Histopathologic findings remain important in establishing a diagnosis of AIH, as serologic autoimmune markers are notoriously nonspecific, being noted in hepatitis C virus (HCV) infection (60,61) and in fatty liver disease (62). Overlap syndromes represent AIH overlap with primary biliary cirrhosis (PBC) or primary sclerosing cholangitis (PSC) and may be concurrent or sequential (see section “Chronic Biliary Obstructive Disease”). Autoimmune Hepatitis—Primary Biliary Cirrhosis Overlap Syndrome PBC is characterized by bile duct destruction (nonsuppurative), cholate stasis in a periportal/periseptal distribution, absence of canalicular bile, florid duct lesions, and less prominent lobular and/or interface activity. Antimitochondrial antibodies (AMAs) are usually positive, and there is evidence of cholestasis on copper and CK7 stains. ANA may be positive in PBC or autoimmune cholangiopathy (AMA-negative PBC) and does not suggest overlap syndrome. By definition, clinical, biochemical, and/or histologic features of both PBC and AIH are present simultaneously or consecutively in the same patient. The diagnosis should be considered if two of three typical features of AIH (ALT elevation five times above normal, IgG elevation twice normal [or SMA], or moderate-to-severe lymphocytic

interface or lobular activity) and PBC (ALP elevation two times above normal [or γ-glutamyl transpeptidase or GGT elevation five times above normal], AMA, or florid duct lesion) are present (63,64). The diagnosis of AIH-PBC overlap syndrome has significant impact on treatment. Immunosuppressive agents such as steroids are not indicated in PBC because they are ineffective and can induce or worsen osteoporosis, whereas failure to treat with steroids can lead to rapid progression in AIH-PBC overlap syndrome (65-67). The pathologist’s primary role is to highlight significant interface and lobular activity in a case that otherwise resembles PBC and highlight bile duct injury in a case that otherwise resembles AIH. This will enable the hepatologist to integrate the overall features for a diagnosis of overlap syndrome. Autoimmune Hepatitis—Primary Sclerosing Cholangitis Overlap Syndrome AIH/PSC overlap is most often diagnosed in the pediatric setting and is more likely to require transplantation than AIH (68). Definitive diagnosis of AIH/PSC requires histologic evidence of AIH and cholangiographic evidence of PSC (69). Periductal sclerosis is unlikely to be present, but other evidence of biliary obstructive disease (e.g., bile duct injury, duct loss, periportal cholestatic effect) should prompt cholangiography to exclude a PSC component. HEPATOTROPIC VIRUSES Hepatitis A (HAV), B (HBV), and E (HEV) viruses can lead to acute hepatitis, whereas acute presentation of hepatitis C is extremely rare and is subclinical in nearly all the cases. Hepatitis E may persist in immunocompromised patients. Hepatitis A Hepatitis A clinical disease results from fecal-oral contamination and is generally mild and easily diagnosed by serology. Sewage contamination can result in concentration of virus in shellfish such as oysters and is a source of sporadic infection in the United States.

Newly infected patients are positive for IgM anti-HAV antibodies; IgG antibodies signify past exposure and do not indicate acute hepatitis A if IgM antibodies are absent. Hepatitis A typically follows a short benign course and does not cause chronic hepatitis. Rare cases of hepatitis A can pursue a variant course and are more likely to be biopsied: 1. Fulminant hepatitis: Rare cases of acute hepatitis A can be associated with massive or submassive necrosis and clinically present with acute hepatic failure. 2. Hepatitis A, cholestatic variant: These patients present with cholestatic features that may persist for several months. The cholestatic features include hyperbilirubinemia, pruritus, and a cholestatic liver enzyme pattern (i.e., elevated ALP with relatively mild elevation of ALT and AST). Acute hepatitis A should be considered in all cases of unexplained hepatitis with cholestasis. 3. Hepatitis A, relapsing variant: Although hepatitis A does not lead to chronic hepatitis, rare patients can present with recurrence of symptoms after complete recovery. Relapse usually occurs within 3 weeks, is milder than the initial illness, and often shows cholestatic features. Immune manifestations such as purpura, nephritis, and arthralgia are common. Uneventful recovery occurs in nearly all cases. Hepatitis B and D Hepatitis B presents as acute hepatitis in 20% to 25% of the patients exposed to the virus, which can occur through blood or body fluid exposure. In Asia and Africa, where HBV infection is endemic, vertical transmission is common. A majority of the patients (~65%) remain asymptomatic after acquisition of HBV infection. Chronic disease develops in less than 5% of infected individuals. The histologic features are similar to other causes of acute hepatitis, and diagnosis is based on serologic findings. Hepatitis B surface antigen (HBsAg) appears in the serum 4 to 26 weeks (average 8 weeks) after the infection. In self-limited cases, HBsAg may disappear before the onset of symptoms or persist during the symptomatic

phase. The disappearance of HBsAg is followed by anti-HBs antibody after a window period of a few weeks. The presence of IgM anti–hepatitis B core (HBc) antibody (or nucleic acid testing) can be useful for diagnosis during this window period. Conversion of IgM (anti-HBc) to IgG occurs in a few months and is the hallmark of prior hepatitis B exposure. In chronic infections, HBsAg persists, and antiHBs antibodies do not appear. Hepatitis D virus (HDV) relies on HBV for effective infection and can be present as part of initial bloodborne coinfection with HBV or as a secondary infection with new exposure to HBV/HDV, which is more common in Africa and the Middle East. Anti-HDV IgM is an indicator of recent infection, and anti-HDV IgG will be present in chronic HBV/HDV coinfection. Hepatitis D superinfection of a patient with HBV may result in fulminant hepatitis (70). Hepatitis C Among hepatotropic viruses, hepatitis C is the most commonly encountered etiology of chronic hepatitis in the United States, with the majority of infections progressing to a chronic stage. Like HBV, HCV is a blood-borne pathogen, but risk of transmission with other body fluids is lower than that with HBV. HCV RNA appears in the blood within a few weeks, and nucleic acid testing is the earliest option for detection and is also performed to determine viral load. HCV antibody does not appear in the serum until 2 to 26 weeks (average 6-12 weeks), and because the clinical course is mild, it is unusual for acute HCV to manifest clinically. HCV genotyping is clinically useful (71), and genotype 3a is notably associated with significant steatosis. Hepatitis E Hepatitis E is a pathogen transmitted via the fecal-oral route that is endemic in Asia, India, Africa, and Mexico. In the United States, there are reports of infection following exposure to infected pigs. In healthy people, the disease is often self-limited and can lead to acute liver failure in pregnant women. Immunosuppressed patients, specifically transplant patients, may develop chronic liver disease

(72). Anti-HEV IgM signifies recent infection, whereas anti-HEV IgG signifies past exposure. Viral RNA can also be detected in blood or stool with nucleic acid testing. Liver biopsy is needed to assess for stage of fibrosis and need for antiviral therapy in chronic viral hepatitis. Plasma-based testing for markers of fibrosis has so far proven imprecise in defining fibrosis severity (73-76), but advanced imaging techniques (e.g., transient elastography) have more accuracy (77), especially in advanced fibrosis. Biopsy remains the gold standard for staging and is particularly important when there are competing etiologies for liver injury (e.g., HCV and steatohepatitis). Chronic viral hepatitis, in native liver tissue, most commonly has a predominant portal lymphocytic infiltrate often with germinal centers and variable interface and lobular activity (Figs. 36.10 to 36.13). Endotheliitis and bile duct damage may also be seen (Fig. 36.29), which can complicate distinction between recurrent viral hepatitis and acute cellular rejection in the posttransplant setting (see section “Transplant Pathology”). The inflammatory activity is graded using one of several commonly used schemes (Table 36.2). Ideally, a core biopsy for grading and staging of chronic viral hepatitis will be taken from the right lobe, away from the subcapsular region, and will be of sufficient length and thickness to obtain greater than 10 portal tracts (78-81). Mild fatty change may occur entirely because of viral hepatitis and not in relation to metabolic risk factors for fatty liver (most typically with HCV genotype 3a) (82) (Fig. 36.30).

FIGURE 36.29 Hepatitis C virus infection may demonstrate portal venule endotheliitis (arrowheads) and bile duct damage (arrow), which can complicate distinction of recurrent viral hepatitis from acute cellular rejection in a posttransplant setting. Hematoxylin and eosin stain, ×400 magnification.

FIGURE 36.30 Hepatitis C virus infection, genotype 3a, with mild portal inflammation and severe large and small droplet macrovesicular steatosis. Hematoxylin and eosin stain, ×200 magnification.

Alcoholic liver disease often coexists with HCV infection, and there may be an increased risk of progression to cirrhosis (83). On iron stain, hepatocellular iron raises suspicion for hereditary hemochromatosis (HH), which may have variable penetrance (see the following texts). Reticuloendothelial iron, on the other hand, may be seen after transfusion or perhaps in relation to antiviral treatment (e.g., ribavirin) (84) (Fig. 36.31). Patients with chronic viral hepatitis are at risk for dysplasia and carcinoma. Large cell change (common in HBV) and small cell change should be identified and included in the report because they portend an increased risk (or in the case of small cell change, a precursor) of carcinoma (see Chapter 37).

FIGURE 36.31 Hepatitis C virus infection treated with ribavirin with subsequent reticuloendothelial iron deposition. Iron stain, ×400 magnification.

Viral inclusions can be seen only in HBV (and HDV), which manifest as ground-glass cytoplasmic inclusions (HBsAg) and

sanded nuclear change (HBcAg or HδAg) (Fig. 36.32). These inclusions can be highlighted on immunostaining (Fig. 36.33), which can allow distinction from mimics (e.g., cyanamide toxicity, Lafora disease, fibrinogen storage disease, drugs such as barbiturates, glycogen inclusions, and polyglucosan bodies) (Fig. 36.3). Absence of nuclear HBcAg could be owing to sampling or suggest suppression of HBV replication by coinfection with HCV or HDV, which could be excluded by serologic testing (85,86).

FIGURE 36.32 Hepatitis B virus infection with subtle pale hepatitis B surface antigen cytoplasmic inclusions (arrow) and hepatitis B core antigen nuclear inclusions (i.e., sanded nuclei) (arrowheads). Hematoxylin and eosin stain, ×400 magnification.

FIGURE 36.33 Hepatitis B virus infection with obvious hepatitis B surface antigen (HBsAg) cytoplasmic inclusions on HBsAg immunostaining, ×200 magnification.

It is relatively common for HCV (less often in the setting of HBV infection) patients to have low-titer autoantibodies, and it can be challenging to distinguish AIH with false-positive HCV serology from true HCV infection (87). Some studies have shown that autoantibodies have no influence on the natural history of hepatitis C, whereas others have shown that these patients may have more aggressive disease with higher necroinflammatory activity and fibrosis (88,89). Recent reports indicate that there is no difference in treatment response and disease progression in hepatitis C with or without antibodies (90,91). If the biopsy shows high necroinflammatory activity, viral nucleic acid testing and serologic testing for additional antibodies, such as p-ANCA, that are rare in HCV infection can be considered (92,93). NONHEPATOTROPIC VIRUSES Several nonhepatotropic viruses may also cause significant liver disease (e.g., cytomegalovirus [CMV], adenovirus, HSV, Epstein-

Barr virus [EBV]) and should be considered in the appropriate clinical context. Viral inclusions may be seen with CMV (Fig. 36.34), HSV, and adenovirus (Fig. 36.35) infection, but viropathic change is relatively insensitive, especially if the patient has received antiviral therapy. HSV often affects immunocompromised individuals, but herpetic hepatitis can occur in immunocompetent hosts, and both scenarios may present with prominent hepatocellular necrosis. EBV causes infectious mononucleosis that usually occurs in adolescence in Western countries. Liver involvement is mild but occurs frequently. Liver transaminases can be elevated, but jaundice is rare. The typical histologic feature is a diffuse infiltrate of lymphocytes in the sinusoids (Fig. 36.36), often with nonnecrotizing granuloma formation. Hepatocellular damage is usually not prominent, and apoptotic bodies are rare. Adenovirus, as with HSV, may demonstrate large zones of hepatocellular necrosis. CMV hepatitis in the immunocompetent host is typically mild; as with EBV, the liver shows sinusoidal lymphocytosis, and hepatocellular injury is mild or absent. Severe involvement with necrosis can occur in the immunocompromised settings and rarely in immunocompetent individuals. CMV hepatitis is well described in allograft liver and is often associated with neutrophil microabscesses in the lobule (Fig. 36.34). Hemophagocytosis (Fig. 36.6) may also be noted with EBV infection. Immunohistochemical or in situ hybridization (ISH) (e.g., EBV-encoded RNA [EBER] ISH [Fig. 36.37]) staining for the respective viruses can be used to confirm the diagnosis.

FIGURE 36.34 Cytomegalovirus nuclear viral inclusion (arrow) and adjacent neutrophil microabscess. Hematoxylin and eosin stain, ×400 magnification.

FIGURE 36.35 Adenovirus nuclear inclusion (arrow). Hematoxylin and eosin stain, ×400 magnification.

FIGURE 36.36 Epstein-Barr virus hepatitis with prominent sinusoidal lymphohistiocytic inflammation. Hematoxylin and eosin stain, ×200 magnification.

FIGURE 36.37 Epstein-Barr virus (EBV) hepatitis with EBV virus detection in sinusoidal B-lymphocyte nuclei (arrows). EBV-encoded RNA in situ hybridization

stain, ×400 magnification.

Other less common viral infections that can cause acute hepatitis with widespread necrosis include yellow fever, dengue fever, and Ebola fever. Parvovirus B19 can cause fulminant hepatitis in children. Mild nonspecific liver inflammation can be seen in many infectious processes, such as human herpesvirus 6 (HHV-6), Coxsackie virus, rubella, and measles virus infection. OTHER INFECTIONS Many systemic bacterial and fungal infections can involve the liver, which often demonstrates a nonspecific reactive hepatitis; other manifestations include abscess (e.g., with bacteria, fungal organisms, or ameba infections), granulomas (e.g., with bacteria, fungal organisms, or parasite infections), fibrin-ring granulomas (e.g., with Q fever infection), peliosis (Fig. 36.38) (e.g., Bartonella sp. infection, typically in the setting of severe immune deficiency, as in HIV infection/AIDS) (94), cysts (e.g., with Echinococcus granulosus infection), intracellular organisms (e.g., with visceral leishmaniasis or histoplasmosis), or bile duct involvement by parasites (e.g., with Clonorchis sinensis infection). Culture is usually most helpful in definitive organism identification. Special stains may be useful for Mycobacteria sp. (Ziehl-Neelsen stain), Bartonella sp. (WarthinStarry stain) (Fig. 36.39), fungal infections (Grocott methenamine silver [GMS] or periodic acid–Schiff with diastase [PASD] stains), and Fusobacterium sp. (Warthin-Starry stain), which can lead to liver abscess and may be missed on routine culture (95). A number of parasitic organisms may also present with abscess, granulomas, or fibrosis. Common parasitic diseases of the liver include schistosomiasis, amoebiasis, and visceral leishmaniasis (Fig. 36.40), as well as infection with E. granulosus (see Chapter 37) or C. sinensis (Fig. 36.41). Hepatic schistosomiasis is caused by Schistosoma mansoni (Africa, Middle East, and Latin America) or Schistosoma japonicum (East Asia) and is contracted by exposure to cercaria released by infected fresh water snails. Schistosome eggs

may be identified within hepatic granulomas, and the host immune response may lead to portal fibrosis without regenerative changes (i.e., “pipe-stem fibrosis”) and portal hypertension.

FIGURE 36.38 Peliosis due to Bartonella henselae infection in a patient with AIDS. Hematoxylin and eosin stain, ×200 magnification.

FIGURE 36.39 Bartonella henselae (same case as shown in Fig. 36.38) organisms (arrowheads) identified on Warthin-Starry stain, ×400 magnification.

FIGURE 36.40 Visceral leishmaniasis involving liver, with amastigotes identified within Kupffer cell cytoplasm (arrow) with a subtle rod-shaped kinetoplast noted in

one organism (arrowhead), allowing distinction from Histoplasma capsulatum. Hematoxylin and eosin stain, ×1000 magnification.

FIGURE 36.41 Clonorchis sinensis within a denuded intrahepatic bile duct. Hematoxylin and eosin stain, ×40 magnification.

FATTY LIVER DISEASE Nonalcoholic fatty liver disease (NAFLD) is defined as evidence of fat in the liver in the absence of a significant alcohol consumption history or other reasons to have secondary fat accumulation (Table 36.5) (96). NAFLD may be subclassified as nonalcoholic fatty liver (NAFL) and nonalcoholic steatohepatitis (NASH). NAFL represents steatosis without histologic liver injury, whereas NASH represents steatosis with histologic evidence of liver injury (as defined in the following texts). Progression to cirrhosis and/or development of hepatocellular carcinoma (HCC) may occur in NASH (97), but is not expected in NAFL. Fibrosis may regress in NASH and is linked to disease activity (98). NASH cirrhosis is defined as cirrhosis with current or previous evidence of NAFLD. NAFLD and

NASH prevalence are estimated at 20% and 3% to 5% in the United States, respectively (96,97). Risk factors for NASH include metabolic syndrome, dyslipidemia, type 2 diabetes mellitus, and obesity (96) (Table 36.6). Metabolic syndrome manifests with at least three of the following: blood pressure greater than 130/85 mm Hg, increased waist circumference (>102 cm in men and >88 cm in women), fasting blood sugar greater than 110 mg per dL, triglycerides greater than 150 mg per dL, and low high-density lipoprotein (HDL) (i.e., 250 μg/g dry tissue in untreated cases), although this assay requires most of the tissue present in a typical core liver biopsy, and thus, special stains should be ordered up front in such cases to preserve tissue. Histochemical stains for copper may be negative in small needle biopsies from WD patients, especially if cirrhosis is already present, because the copper is often redistributed to other tissues in the body. A1ATD may cause inflammation and/or fibrosis in the liver. A1AT is a proteinase inhibitor that inhibits proteases, such as neutrophil elastase. The normal allele is designated as PiM. The PiZ allele is the most common variant associated with A1ATD. The abnormal allele has a point mutation that leads to defects in polymerization. The abnormal protein accumulates in the ER of hepatocytes. Homozygous state for PiZZ is associated with chronic hepatitis and cirrhosis. Some studies have shown that heterozygous PiMZ patients can also develop chronic liver disease and cirrhosis. The heterozygous PiMZ state may also play a role in progression of other concurrent liver disease, such as hepatitis C and NAFLD (142,143). Serum levels of A1AT are not reliable for ruling out either homozygous or heterozygous disease because normal levels may be seen with both phenotypes under stimulatory responses from inflammation, surgery, chronic liver disease, or malignancy. The cytoplasmic globules of A1AT are typically seen in periportal or periseptal hepatocytes. In homozygous disease (PiZZ), the globules are large and numerous. However, in heterozygous disease, the globules can be small and inconspicuous. Hence, PASD stain should be evaluated in all cases. A1AT immunohistochemistry may reveal globules in some cases that were not detected on PASD stain. The presence of globules is not specific for A1ATD and can occur with acute inflammation and alcoholic liver disease, and diagnosis must be confirmed by Pi typing (e.g., isoelectric focusing or molecular techniques such as single-strand conformational polymorphism [SSCP] and DNA sequencing) (144). HSCs (Ito cells) are modified fibroblasts that store lipids and vitamin A in the normal liver. They are located in the space of Disse,

between the sinusoidal endothelium and the hepatocytes, but are generally not easily visible. In certain conditions, especially hypervitaminosis A, excessive lipid gets stored in the stellate cells (Ito [stellate] cell lipidosis). The nuclei of stellate cells are crescent shaped, dark staining, and indented by the lipid droplets. Thin strands of cytoplasm separate the lipid droplets. These lipid-laden cells can easily be mistaken for hepatocytes with steatosis (145). Hypervitaminosis A results from excess vitamin A intake in diet or supplements or use of oral/topical retinoids. Stellate cell lipidosis can also occur in cholestasis, alcoholic liver disease, hepatitis C, and in association with some other drugs (e.g., methotrexate, valproic acid, steroids). Early recognition can prompt reduced intake of vitamin A to avert progression and fibrosis. DEVELOPMENTAL DISORDERS WITH DUCTAL PLATE MALFORMATION Ductal plate formation represents a normal step in liver formation, but this structure is normally dismantled early in life and, when persistent, is termed ductal plate malformation. The presence of liver cysts or multiple von Meyenburg complexes should raise suspicion for ductal plate malformation disorders. The classification is based on morphologic features and size of duct involved (Table 36.18). Congenital hepatic fibrosis represents a biliary pattern of fibrosis with multiple von Meyenburg complexes. Cystic change and large duct involvement are not present. Patients can present with recurrent biliary infections, or portal hypertension, and progression to cirrhosis can occur. There is a strong association with autosomal recessive kidney disease. Autosomal dominant polycystic liver disease occurs either in isolation or in association with autosomal dominant kidney disease (146). Cysts can be a few in number to numerous, with total involvement of the liver, and are lined by benign epithelium. Von Meyenburg complexes are often present, but are not required for diagnosis. Potential complications include infection, hemorrhage, and rupture of cysts. Progression to cirrhosis is uncommon. Caroli disease manifests as cystic dilatation of intrahepatic bile ducts and

can be associated with congenital hepatic fibrosis (Caroli syndrome) and is often associated with autosomal recessive polycystic kidney and nephronophthisis. Complications include biliary infections, stone, and increased risk for cholangiocarcinoma (147). TABLE 36.18 Ductal Plate Malformation Size of Involved Ducts

Disease

Small

Congenital hepatic fibrosis Biliary hamartomas

Medium

Autosomal dominant polycystic liver disease

Large

Caroli disease (intrahepatic) Choledochal cyst (extrahepatic)

IRON OVERLOAD IRON METABOLISM AND THE LIVER There are three sources of iron influx into plasma: intestinal absorption, recycled iron from senescent red blood cells (RBCs) from macrophages, and hepatocellular iron. Ferric iron in the diet is reduced to its ferrous form, and uptake into intestinal epithelial cells is mediated by the ferrous iron transporter, divalent metal transporter-1 (DMT1). Iron transport from epithelial cells into plasma is mediated by ferroportin. This process is regulated by hepcidin, an iron regulatory hormone produced predominantly by hepatocytes (148). If body iron stores are adequate, hepcidin binds to ferroportin, leading to degradation of both proteins. This prevents iron from being transferred from epithelial cells into plasma. Macrophage iron, from senescent RBCs, is also transported into plasma by ferroportin 1 (FPN1) and is regulated by hepcidin. In the setting of anemia and

hypoxia, stimulation of erythropoiesis leads to decreased hepcidin production and resultant increase in iron absorption. Plasma iron is transported by transferrin and is taken up by cells using transferrin receptor 1. Its homolog, transferrin receptor 2, is restricted to hepatocytes and plays a role in hepcidin regulation. The principal storage form of iron is ferritin (apoferritin and iron oxyhydroxide), which is found in virtually all cells and also in trace amounts in plasma. Hemosiderin (aggregates of iron oxyhydroxide crystals without apoferritin) is found in the reticuloendothelial system, including Kupffer cells. HFE HEMOCHROMATOSIS HFE mutation leads to low hepcidin level and consequent excess iron absorption and increased mobilization from macrophages. It is the most common cause of HH in people of Northern European descent but is less common or absent in other ethnic groups. The two most commonly described mutations are C282Y and H63D. Most cases of HH are related to C282Y homozygosity. Compound heterozygosity of C282Y and H63D has also been implicated in HH; homozygosity for H63D is not sufficient to cause iron overload, but the less common H65C mutation can cause iron overload when present with C282Y (but does not lead to HH). The allelic frequency of C282Y and H63D is estimated to be 6% and 14%, respectively (149). Disease penetrance is low with iron overload in only 30% to 50%, and symptomatic disease in only 10% to 30%, of C282Y homozygotes. Evidence of increased iron overload along with C282Y homozygosity is necessary to establish the diagnosis of HFEassociated HH (HFE-HH). Evidence of iron overload can be assessed by serum iron indices, magnetic resonance imaging (MRI), and/or liver biopsy (150). Serum ferritin is a highly sensitive index of elevated iron, and a normal serum ferritin essentially excludes iron overload. However, ferritin has low specificity and can be elevated in fatty liver disease, metabolic syndrome, alcohol intake, and inflammatory diseases. MRI provides an accurate assessment of liver iron. Liver biopsy is not necessary for the diagnosis of HFE-HH.

Age greater than 40 years, serum ferritin greater than 1000 ng per L, elevated ALT, and hepatomegaly are risk factors of fibrosis, and liver biopsy can be considered in these patients to evaluate fibrosis (149). Iron deposition can also occur in the pancreas (leading to diabetes), heart (leading to cardiomyopathy), joints (leading to arthritis), and skin (leading to bronze pigmentation). Liver involvement manifests as iron overload that is almost exclusively hepatocellular in distribution. The periportal hepatocytes show preferential accumulation early in the disease (Fig. 36.81). With progression, siderosis becomes diffuse and can also be seen in Kupffer cells, sinusoidal endothelial cells, fibrous septa, and bile duct epithelial cells. Inflammation is typically mild or absent. Fibrosis, and eventually cirrhosis, can occur. HH cirrhosis is characterized by broad fibrous septa, with preservation of vascular structures until late in the disease. This accounts for the rarity of portal hypertension and liver failure in HH (150). Patients with HH and cirrhosis have high risk for HCC. Iron is typically absent in the tumor. Localized areas of hepatic parenchyma with minimal or no iron, in an iron overloaded liver, are referred to as iron-free foci. These areas may show small cell change, large cell change, and high proliferation. Patients with iron-free foci in liver biopsy are at higher risk for development of HCC. Intrahepatic cholangiocarcinoma can also occur in HH cirrhosis and may be accompanied by numerous von Meyenburg complexes (150).

FIGURE 36.81 Hemosiderin deposition in periportal hepatocytes is a common finding in early HFE-associated hereditary hemochromatosis. Prussian blue stain, ×200 magnification.

NON-HFE HEMOCHROMATOSIS Juvenile hemochromatosis (JH, type 2 HH) occurs in two forms, one characterized by hemojuvelin (HJV) mutation (type 2A) and the other by mutations in hepcidin (type 2B) (Table 36.19). HJV is a protein located on the cell surface of hepatocytes that plays a key role in hepcidin upregulation. HJV mutation leads to low hepcidin level and resultant increased iron mobilization from intestine and macrophages. Most JH cases are caused by HJV mutation, whereas hepcidin mutations are rare. Iron overload in JH equally affects males and females and is more severe compared to HFE-HH; presentation in the first or second decade with cardiomyopathy and hypogonadism is common (151,152). Type 3 HH is associated with mutations in transferrin receptor 2 and tends to be similar to HFE-HH (151,152). Ferroportin disease (type 4 HH) is autosomal dominant and is associated with mutations in FPN1. There are two subtypes depending on the type of mutation involved. One subtype is similar to HFE-HH, whereas the other subtype shows normal transferrin and

iron overload in the reticuloendothelial system (151,152). Hereditary aceruloplasminemia is an autosomal dominant disorder that is characterized by iron accumulation in hepatocytes. It is not considered a form of HH, and cirrhosis does not occur. TABLE 36.19 Types of Hereditary Hemochromatosis Genetics

Liver Biopsy

Clinical Presentation

Type 1 (HFEassociated HH)

Autosomal recessive C282Y homozygous, C282Y and H63D double heterozygous

Iron overload in hepatocytes, most prominent in periportal region

Third or fourth decade Liver, pancreas, heart, skin, and joints can be involved

Type 2 (juvenile HH)

Autosomal recessive mutations involving hemojuvelin (2A) or hepcidin (2B)

Iron overload in hepatocytes

First three decades More severe disease compared to HFEassociated HH

Type 3

Autosomal recessive transferrin receptor type 2 mutation

Iron overload in hepatocytes

Similar to HFEassociated HH. Severity is intermediate between type 1 and type 2 HH.

Type 4

Autosomal dominant ferroportin mutation

First subtype: iron overload in hepatocytes Second subtype: iron overload in Kupffer cells

Fourth or fifth decade Severity varies with type of mutation.

HH, hereditary hemochromatosis.

SECONDARY IRON OVERLOAD Unlike HH, iron deposition is Kupffer cell dominant or demonstrates a mixed hepatocellular-Kupffer cell distribution in secondary iron overload. The most common causes are hemolytic anemia, excess iron intake, chronic inflammatory conditions, fatty liver disease, metabolic syndrome, frequent blood transfusions, cirrhosis of any etiology, WD, and alcoholic liver disease (150). The high erythropoietic activity in hemolytic anemia suppresses hepcidin production, despite high tissue iron derived from RBC destruction. In chronic diseases, inflammatory cytokines such as IL-6 stimulate hepcidin production, leading to anemia; the concomitant deficient mobilization of iron from macrophages leads to iron deposition in the reticuloendothelial system. Patients with cirrhosis, especially cirrhosis due to alcoholic steatohepatitis, can develop marked secondary accumulation of iron in both Kupffer cells and hepatocytes. Coexistent HH should be excluded in these cases. The heterogeneity of iron distribution and absence of iron deposition in biliary epithelial cells and fibrous septa favor secondary iron overload in these cases. Mixed or mild hepatic periportal siderosis along with mild steatosis is often present in porphyria cutanea tarda, a disorder of porphyrin metabolism. Needlelike crystalline inclusions may be identified in hepatocytes and are birefringent under polarized light. The inclusions may be better seen in unstained sections and can be highlighted under fluorescent light because of their autofluorescence. IRON ASSESSMENT IN THE LIVER The amount of iron is often underestimated based on hematoxylin and eosin (H&E) stain, and routine use of iron stain is recommended. Hepatic iron may contribute to disease progression in chronic liver diseases, such as hepatitis C and fatty liver disease. Scheuer system is one of many systems used for semiquantitatively grading liver iron (Table 36.20). Alternatively, a simplified subjective four-scale system of minimal, mild, moderate, and severe siderosis can be followed based on the extent of liver iron, with separate assessment of hepatocytes and Kupffer cells. Perls Prussian blue

stain is most commonly used and demonstrates only the ferric form of iron. The scoring should be based on granular staining, which represents hemosiderin, whereas the smooth blue blush attributed to ferritin should not be considered (Fig. 36.82). Quantitative iron can be determined from fresh or paraffin-embedded liver tissue. Iron level is less than 400 μg per g of liver tissue in normal states (150). Hepatic iron index is considered a more accurate indicator of iron overload because it takes both age and liver iron into account, with values greater than 1.9 being characteristic of HH. However, this score is not specific for HH, and quantitative iron testing has become uncommon with the advent of genetic testing.

FIGURE 36.82 Cytoplasmic blue blush owing to ferritin should not be considered for scoring hepatic iron. Prussian blue stain, ×400 magnification.

TABLE 36.20 Scheuer System of Grading Liver Iron Grade 0

Granules absent or barely discernible at ×400 magnification

Grade 1

Granules barely discernible at ×250, easily seen at ×400

Grade 2

Discrete granules seen at ×100

Grade 3

Discrete granules seen at ×25

Grade 4

Masses visible at ×10 or naked eye

VASCULAR DISORDERS The major vascular disorders in the liver involve the hepatic artery, portal vein, sinusoids, or obstruction to venous outflow. Ischemic injury to the liver is uncommon owing to its dual blood supply but can occur in systemic conditions, such as shock, sepsis, and following variceal bleeding. Apart from rare cases of vasculitis, most instances of hepatic artery flow problems are related to iatrogenic injury. Common clinical settings for this injury include transarterial chemotherapy or embolization, liver transplantation, and cholecystectomy (153). Injury to the arterial microcirculation can lead to ischemic injury to the biliary tree (ischemic cholangiopathy), which manifests as cholestasis and ductular reaction. The late stage of ischemic cholangiopathy is characterized by strictures and can be distinguished from PSC only based on the clinical setting. Hepatic artery thrombosis is a rare complication of liver transplantation that can lead to ischemic injury to liver parenchyma and the biliary tree and result in graft loss (154). Portal vein obstruction can occur as a result of tumor invasion or thrombosis (153,155). The etiology of thrombosis may be local, such as with cirrhosis, abdominal infections, or other inflammatory conditions (e.g., diverticulitis, appendicitis, pancreatitis, inflammatory bowel disease) or systemic, such as with prothrombotic states (e.g., factor V Leiden mutation, protein C or S deficiency, or in association with myeloid neoplasms). Numerous collateral channels may develop in chronic portal vein thrombosis (cavernous transformation). Injury to small portal veins can occur in systemic autoimmune diseases (e.g., rheumatoid arthritis and systemic lupus erythematosus), biliary diseases (e.g., PBC and PSC), granulomatous diseases (e.g., sarcoidosis), or due to cytotoxic drugs (e.g., azathioprine). Portal hypertension can occur in these settings without cirrhosis. Hypoplasia of portal veins, as in congenital hepatic

fibrosis, can also lead to portal hypertension. Portal hypertension with patent portal vein, absence of cirrhosis, and no discernible etiology is referred to as idiopathic portal hypertension (also referred to as noncirrhotic portal fibrosis, hepatoportal sclerosis, or obliterative portal venopathy). Most cases probably result from portal vein thrombosis, which cannot be demonstrated by the time the disease becomes clinically apparent. Extension into the portal vein branches leads to narrowing or obliteration of small- and mediumsized portal veins with resultant portal hypertension (153,156). This diagnosis should be considered in patients with portal hypertension that show mild nonspecific changes and no evidence of cirrhosis on biopsy. This disease has high prevalence in India and Japan. Several cases have been reported in association with antiretroviral drugs used to treat HIV (157). The liver biopsy in portal vein disease may be normal or show portal fibrosis, thin bridging fibrous septa, and patchy pericellular fibrosis. Some portal tracts may not have any discernible portal vein radicles, whereas dilated portal veins are present in others. These findings are difficult to evaluate because portal vein branches are often not identified in some portal tracts in normal livers. Intimal thickening and webs can be present in medium and larger portal veins, but are often not sampled on biopsy. Portal vein radicles can extend into the periportal parenchyma (“herniation”), or multiple thinwalled vascular channels may be seen in the periportal region (“angiomatosis”) (158). Hepatocellular atrophy, sinusoidal dilatation, and NRH can be present. In the setting of ascites and splenomegaly, the portal vein should be examined at autopsy for thrombosis. NRH is an uncommon condition characterized by the diffuse transformation of hepatic parenchyma into small regenerative nodules with little to no fibrosis (159). It is a consequence of alterations in blood flow resulting from obliteration of small portal vein radicles in a wide variety of vascular, hematopoietic, and systemic diseases (160). The nodules probably represent a hypertrophic response to normal or slightly increased blood flow. Symptomatic NRH clinically manifests as portal hypertension and its sequelae: variceal bleeding, ascites, and splenomegaly. Liver

transaminases and serum bilirubin levels are usually normal, whereas ALP is often elevated. The nodules are present throughout the hepatic parenchyma and are typically 1 to 3 mm in size and lack fibrous septa (Fig. 36.83) (Table 36.21). Focal sinusoidal or periportal fibrosis can be seen. The hepatocytes within the nodule are arranged in one- to two-cell thick plates. The hepatocytes between the nodules are small and atrophic and may appear as thin parallel plates. Sinusoidal dilatation and slitlike central veins are often present. Absence of significant fibrosis in cases suspected to be cirrhotic, based on imaging, should raise consideration for NRH. The diagnosis can be challenging on needle biopsy, and the nodular architecture is best demonstrated on reticulin stain (Fig. 36.84).

FIGURE 36.83 Nodular regenerative hyperplasia featuring small nodules, intervening areas of hepatic plate atrophy, and no fibrosis. Hematoxylin and eosin stain, ×40 magnification.

FIGURE 36.84 Nodular architecture in nodular regenerative hyperplasia with intervening atrophic areas. Reticulin stain, ×40 magnification.

TABLE 36.21 Wanless Criteria for Diagnosis of Nodular Regenerative Hyperplasia Histologic Feature

Grading

Hepatocellular nodules 20 liver cysts, although smaller numbers of cysts are acceptable in patients with known family histories of autosomal-dominant polycystic kidney disease (ADPKD). PCLD is divided into autosomal-dominant forms, ADPKD, and autosomal-recessive forms: congenital hepatic fibrosis (CHF) Caroli disease and Caroli syndrome. Autosomal-dominant polycystic kidney disease. ADPKD is the most common hereditary renal disease, affecting from 1 per 400 to 1 per 1,000 live births (37); hepatic cysts are rarely evident at birth but eventually develop in more than 90% of patients (38). Approximately 95% of cases of ADPKD have been linked to mutations in one of two genes. PKD1 is mutated in 85% of patients and encodes an integral membrane glycoprotein, polycystin-1. The second gene causing ADPKD, PKD2, is responsible for 5% to 10% of cases and encodes an integral membrane protein known as polycystin-2. Both polycystin-1 and polycystin-2 form part of a mechanosensitive Ca2+ signaling pathway in renal tubule primary cilia; one function of this pathway is to inhibit renal tubular growth (39). Patients with PKD2 mutations are clinically similar to patients with PKD1 mutations, but they develop clinically apparent renal disease later in life (40). Although ADPKD is inherited in a dominant fashion, the disease is recessive on a cellular level, in that loss of the wild-type allele in renal or biliary epithelial cells is necessary for cyst formation. PCLD that is not associated with cystic kidney disease is much less common than ADPKD and is called isolated autosomal-dominant PCLD or autosomal-dominant PCLD. While most cases do not have a known genetic cause to date, two genes have been associated with this pattern of liver cysts: PRKCSH, which encodes the protein hepatocystin (the beta-subunit of glucosidase II), and SEC63, which encodes a component of the protein translocation apparatus located in the endoplasmic reticulum (41). It remains unclear how aberrations in these proteins, which are involved in protein trafficking in the endoplasmic reticulum, lead to cyst formation (42). In both ADPKD and isolated autosomal-dominant PCLD, multiple unilocular cysts resembling simple biliary cysts are scattered diffusely throughout the liver or, more rarely, are limited to one lobe (Figs. 37.2 A, B). They range in size from a few millimeters to more than 10 cm in diameter. The background liver shows numerous microscopic von Meyenburg complexes that are histologically identical to their sporadic counterpart. The cysts usually do not compromise hepatic function but may lead to hepatomegaly (sometimes massive) and abdominal discomfort. Women are more likely to be symptomatic from the cysts and morbidity is related to the number of pregnancies and the use of oral contraceptive steroids (43). The severity of renal involvement in ADPKD is generally a more important determinant of patient outcome than the hepatic manifestations.

FIGURE 37.2 Polycystic liver disease (PCLD). (A) Multiple thin-walled cysts of varying sizes have distorted the liver, but the intervening parenchyma is relatively normal. (B) The cysts are lined by flattened to cuboidal biliary-type epithelium. Reprinted from Moreira RK, Washington K. Liver neoplasms. In: Iacobuzio-Donahu CA, Montgomery EA, eds. Gastrointestinal and Liver Pathology. 2nd ed. Elsevier; 2012:626-677. Copyright © 2012 Elsevier. With permission.

Hepatic complications of PCLD include cyst infection, intracystic hemorrhage, Budd-Chiari syndrome, biliary obstruction, portal hypertension, and adenocarcinoma. Treatment of symptomatic cysts includes fenestration, aspiration and sclerosis, or resection. In severe, refractory cases, liver transplantation can be performed. Congenital Hepatic Fibrosis. CHF is also associated with cystic renal disease, most commonly autosomal-recessive polycystic kidney (ARPKD), caused by mutations in the PKHD1 gene (44). The portal tracts are enlarged by fibrous tissue, often with portal-to-portal bridging, but the most distinctive feature is the presence of malformed biliary channels, often with inspissated bile, which are located at the periphery of the portal tracts. The biliary changes result from malformation of the ductal plate at the level of the interlobular bile duct. The portal vein branches are also affected, generally being decreased in number and smaller than usual. The changes of CHF may be mistaken for cirrhosis, but the characteristic patent biliary channels and lack of hepatocyte regeneration with well-defined parenchymal nodularity are features that help separate CHF from conventional cirrhosis. Four clinical forms of CHF are recognized depending on the clinical presentation: latent, cholangitic, portal hypertensive, and mixed cholangitic/portal hypertensive. Rarely, cholangiocarcinoma arises in the setting of CHF (45). Caroli Disease. Caroli disease is characterized by saccular dilation of the larger intrahepatic bile ducts, alternating with segments of normal caliber bile ducts. When CHF is also present, the disorder is termed Caroli syndrome. Caroli disease and Caroli syndrome are most strongly associated with ARPKD but have rarely been reported with ADPKD (46). Although the entire liver is usually involved, lobar or segmental disease, particularly with involvement of the left lobe, is not uncommon (47). The cystic areas are lined by biliary epithelium, which may be hyperplastic or attenuated or ulcerated in the setting of cholangitis. Inspissated bile and biliary stones are common in the dilated ducts, especially when cholangitis is present. Cholangiocarcinoma is an important complication (48). Multiple Hilar Cysts (Peribiliary Gland Cysts) The walls of the large intrahepatic bile ducts and extrahepatic bile ducts contain peribiliary glands that are lined by mucinous or biliary type epithelium. Microscopic cystic dilation of these glands is not uncommon in autopsy or resection specimens, but is generally an incidental finding. The cysts are usually multiple and range in size from 1 mm up to 2 cm and occasionally result in obstructive jaundice (49). They are lined by a single layer of columnar to cuboidal or flattened epithelium and are typically associated with mild, chronic inflammation in the pericystic fibrous tissue (50). Pancreatic metaplasia/heteropias can also be associated with the cysts (50). Other Nonparasitic Cysts

Hepatic pseudocysts are similar to their pancreatic counterparts; they lack an epithelial lining and consist of a cystic space surrounded by fibrous tissue. They are generally the result of trauma (51) or hepatic involvement by a pancreatic pseudocyst (52). They may be quite large, often contain blood or bile, and often are secondarily infected. Other rare nonparasitic nonneoplastic, cystic lesions of the liver include intrahepatic ileal and duodenal duplication cysts (53). Rare cases of hepatic endometrioma have also been reported (54). NEOPLASTIC CYSTS Mucinous Cystic Neoplasms MCNs were formally called mucinous cystadenomas or, when malignant, mucinous cystadenocarcinomas. The terminology was changed to parallel the analogous MCN of the pancreas. MCNs account for approximately 5% of solitary hepatic cysts. These uncommon tumors can be mistaken for simple cysts, but it is important to make the correct diagnosis, given that the treatment of choice for MCN is complete excision to prevent recurrence. Essentially all MCNs occur in women with a mean age at diagnosis of approximately 45 years (55), although a wide age range is reported, including rare cases in children. Common presenting symptoms include abdominal discomfort or pain, abdominal distension, and less commonly, nausea and vomiting (56). Most MCNs are intrahepatic (84%), although they can rarely arise within the common bile duct, hepatic ducts, cystic duct, or gallbladder (55). Internal septations on radiographic imaging of a solitary hepatic cyst can suggest an MCN (57), but calcifications, present in roughly 20% of cases, may lead to confusion with echinoccocal cysts. Elevated levels of CA19-9 and carcinoembryonic antigen (CEA) in cyst fluid may be helpful in distinguishing MCN from simple biliary cysts; reported CA19-9 levels in cyst fluid range from roughly 2,200 to more than 1 million U per mL (normal, 5 cm

Absent, except for in areas of old hemorrhage

Absent

Absent

Absent, except for fibrolamellar carcinomas

Mitoses

Absent

Absent

Absent

Rare in HG DN

Common

Intralesional (naked) arteries

Common

Common

Absent or rare

Common

Common

Nuclear atypia

Absent

Absent

Absent

Mild

Mild to marked

Large cell change

Can be present if similar changes are found in the background liver

Can be present in inflammatory and androgenrelated adenomas

Can be present if similar changes are found in the background liver

Can be present if similar changes are found in the background liver

Can be present

Small cell change

Absent

Absent

Absent

Absent, but there can be small vaguely outlined clone-like subpopulations

Can be present

Nodule-withinnodule growth

Absent

Absent

Absent

Absent

Can be present

Stromal invasion

Absent

Absent

Absent

Absent

Useful when present, most biopsies absent

Reticulin frame work

Intact

Intact

Intact

Intact

Disrupted with loss of reticulin

Glypican 3

Negative

Negative

Negative

Rare focal positivity in HG DN

50% of tumors positive

Ki-67

Same as background liver

Same as background liver

Same as background liver

Can be minimally elevated in HG DN

Low to high

Other key stains

Glutamine synthetase shows a map-

Immunostains for subtyping are used after a

None

Positive staining for beta-catenin, glutamine synthetase

Stains to confirm hepatic

like staining pattern

diagnosis of adenoma is made

(strong diffuse) or HSP70 favor HCC

differentiation are sometimes useful

aRarely,

MRN can develop in noncirrhotic livers following massive hepatic necrosis. FNH, focal nodular hyperplasia; DN, dysplastic nodule; HA, hepatocellular adenoma; HCC, hepatocellular carcinoma; HG, high grade; MRN, macroregenerative nodule.

Most FNHs are asymptomatic and discovered incidentally (80%) at surgery or autopsy. An abdominal mass is the most common complaint in symptomatic patients (10%-15%). Serum alpha-fetoprotein (AFP) levels are within the normal range. Macroscopic Features FNHs are usually solitary (>80% of cases) and subcapsular, ranging in size from 1 cm to over 10 cm, although most nodules measure 5 cm or less. The lesion is sharply circumscribed (Fig. 37.5A) but unencapsulated and may bulge from the surface of the liver, but is rarely pedunculated. On cut surface, FNH is tan and lighter in color than the adjacent liver, which is essentially normal. A central stellate scar with radiating fibrous septa is a characteristic finding and divides the mass into multiple smaller nodules. Smaller FNHs, however, often lack a central scar. FNHs rarely demonstrate intratumoral hemorrhage or necrosis. Green discoloration from bile accumulation is uncommon.

FIGURE 37.5 Focal nodular hyperplasia (FNH). (A) FNH arises in a noncirrhotic liver and is characterized by a central stellate scar and tan nodular parenchyma subdivided by fibrous septa. (B) At low power, the parenchyma is nodular. The background liver showed steatohepatitis and the FNH also shows fatty change. (C) The fibrous septa contain a variable

number of biliary channels, without well-formed interlobular bile ducts (Masson trichrome). (D) A vessel with eccentrically thickened vessels is located within a centrally located fibrous scar.

Microscopic Features FNH is composed of nodules of bland hepatocytes (Fig. 37.5B) surrounded by bands of fibrosis that contain artery branches, bile ductules (a variable but key feature), variable lymphocytic/neutrophilic inflammation, and decreased or absent interlobular bile ducts and portal vein branches. The parenchymal nodularity and bands of fibrosis are best developed in larger lesions. There is a range of bile ductular proliferation (Fig. 37.5C), from a few inconspicuous biliary channels to numerous proliferating structures. Normal portal tracts and central veins are lacking in the nodule. The central scar is composed of dense collagen and contains numerous thick-walled arteries many of which display fibromuscular hyperplasia (Fig. 37.5D). The hepatocytes in FNH are similar to those in the surrounding liver. Mallory hyaline and other features of chronic cholestasis such as feathery degeneration and accumulation of excess copper may be found in hepatocytes adjacent to the fibrous septa. Nuclear pleomorphism, prominent nucleoli, and mitotic figures are not found in FNH. The hepatocyte plates are one to two cells thick and are supported by an intact reticulin framework. Intralobular hepatic arterioles (naked arteries) can be found (65) and do not distinguish FNH from true neoplasms such as hepatic adenoma or HCC. If the background liver shows steatosis or steatohepatitis, similar findings can also be seen in the FNH (Fig. 37.5B) (66). A map-like distribution of glutamine synthetase expression is very helpful to confirm an H&E impression of FNH (Figs. 37.6A,B) (67,68).

FIGURE 37.6 Immunostains and FNH. (A) At low power, FNHs show a map-like staining pattern with glutamine synthetase. (B). At higher power, the hepatocytes that are negative for glutamine synthetase tend to be located along fibrous septae. (C) CRP often shows nonspecific patchy staining in FNH, in contrast to strong and diffuse staining seen with inflammatory hepatic adenomas.

Differential Diagnosis, Treatment, and Outcome FNH is a nonneoplastic lesion that has little risk of hemorrhage, and if the diagnosis can be established with certainty by radiology, asymptomatic lesions can be followed. When followed by imaging, most FNH are stable or regress (69). However, in some cases, the imaging is not typical of FNH and the radiological differential includes hepatic adenomas, HCC or fibrolamellar carcinoma (FLC); in this situation, a biopsy is recommended. In resection specimens, the diagnosis of FNH is usually straightforward because of the characteristic features of the lesion. In biopsy specimens, the diagnosis of FNH can also be established with confidence when radiologists are sure they sampled the mass and the lesion contains the characteristic pattern of nodules of bland hepatocytes surrounded by fibrous bands with ductular reaction and prominent arteries. The fibrous septa and bile ductules make the distinction from cirrhosis problematic when it is unclear whether the biopsy is from a cirrhotic or a noncirrhotic liver. In addition, rarely, the adjacent liver parenchyma next to an unsampled liver mass can have changes that resemble FNH (70), so correlation with imaging is important to ensure the lesion was adequate sampled. Of note, FNH-like lesions can also develop in cirrhotic livers (71), a diagnosis, however, that can only be firmly made on full resection specimens. The differential for FNH can also include an inflammatory hepatic adenoma. Immunostains for glutamine synthetase and for CRP/SAA are very helpful in this distinction, with a map-like staining pattern by glutamine synthetase favoring FNH and strong and diffuse staining for CRP indicating a diagnosis of inflammatory hepatic adenoma. As a potential diagnostic pitfall, patchy nonspecific staining for CRP is not uncommon for FNH (Fig. 37.6C). SEGMENTAL ATROPHY/NODULAR ELASTOSIS Segmental atrophy/nodular elastosis develops from thrombosis of blood vessels feeding a small subsegment of the liver, usually in a subcapsular location (72). The affected region of the liver then transitions through a series of sequential histological changes: (1) parenchymal collapse with bile ductular proliferation and mixed neutrophilic and lymphocytic inflammation; (2) reduction and eventual loss of the bile ductular proliferation, reduced inflammation, and increasing deposition of elastosis. This stage can also have small biliary retention cysts that result from dilated bile ducts; (3) nodular lesion composed of elastosis with occasional islands of hepatocytes (Figs. 37.7 A,B); (4) replacement of elastosis by fibrosis, leading to a nodular scar of dense collagen, often with still a few islands of hepatocytes. A biopsy or resection can show any of these different stages, depending on the time interval since the initial vascular injury.

FIGURE 37.7 Nodular elastosis. (A) At low power, there are a few islands of residual benign hepatocytes embedded in dense elastosis. (B) A VVG highlights the elastosis.

REGENERATIVE HEPATIC PSEUDOTUMORS

RHPs are also associated with vascular flow changes (73). The lesions have ill-defined borders on both gross examination and on low-power histological examination. Histologically, they show the normal architectural components of the liver, including portal tracts and central veins, but with additional reactive changes that appear to result from localized vascular flow abnormalities. Key findings include (1) a mass lesion seen on imaging, (2) portal vein or central vein thrombi (Fig. 37.8A), (3) patchy sinusoidal dilatation (Fig. 37.8A), (4) portal tracts with portal vein abnormalities such as muscular hyperplasia, herniation, atrophy, or absence (Fig. 37.8B), and (5) often various subtle arterial changes (hyperplasia within portal tracts, arterialization of the lobules, abnormal CD34 staining in the sinusoids). In some cases, the blood flow abnormalities lead to absence of the normal zone 3 staining for glutamine synthetase (Fig. 37.8C). RHPs lack the fibrosis of FNH as well as the changes of segmental atrophy/nodular elastosis.

FIGURE 37.8 Regenerative hepatocellular pseudotumor. (A) A remote fibrotic central vein thrombus is seen in a background of sinusoidal dilatation. (B) Portal vein abnormalities can include muscular hyperplasia with narrowing of the lumen. (C) This RHP has lost complete expression of glutamine synthetase, even though central veins are present.

OTHER PSEUDOTUMORS OF THE LIVER Other benign hepatocellular proliferations that should be considered are compensatory segmental or lobar hyperplasia (36,74), manifested as enlargement of one hepatic lobe following atrophy of another lobe secondary to ischemic injury or prolonged biliary obstruction; accessory lobes, a developmental anomaly; and regenerative nodules occurring in livers with massive necrosis. The “Reidel lobe” is a common anatomic variant of the liver that leads to a caudal projection from the right lobe, which can often be palpated on physical examination, simulating a mass lesion (75). Hepatic ectopia refers to a portion of hepatic parenchyma that is not contiguous with the liver (74,75). Heterotopias of the adrenal gland, thyroid, and spleen have been described, and pancreatic acinar heterotopia is found in up to 4% of liver explants (76). The heterotopic tissues may be present entirely within the liver parenchyma or directly adjacent.

Splenosis, the heterotopic implantation of splenic fragments following trauma or splenectomy, may occur in association with the liver, both on the surface of the liver capsule and within the hepatic parenchyma (77). When biopsied, the ectopic spleen can closely mimic a vascular neoplasm, but the endothelial cells will be strongly CD8 positive. Macroregenerative Nodules The differential for a well-differentiated hepatocellular lesion in a cirrhotic liver includes macroregenerative nodules (MRNs), which are larger than the nodules in the background liver, but typically less than 1.5 cm in diameter. MRNs can mimic a tumor on imaging and gross examination, but are composed of benign regenerative hepatocytes that look cytological the same within the MRN as in the background liver outside the MRN. Focal Fatty Change Steatosis may occur as a multiacinar foci within otherwise normal parenchyma, mimicking a tumor on imaging studies. These lesions are seen in association with the usual risk factors for fatty liver disease, such as diabetes mellitus, obesity, and alcoholic hepatitis, as well as peritoneal dialysis (77,78). Focal fatty change can be multifocal and is grossly yellow-white in color and may measure up to 10 cm in diameter. On microscopic examination, there is macrovesicular steatosis that is typically panacinar; however, sparing of zone 1 or zone 3 may be seen. The structural elements of the liver are otherwise normal. Over time, the lesions may grow or regress in response to the underlying associated condition.

HEPATIC ADENOMA Hepatic adenomas (HA) (Table 37.4) are uncommon, benign hepatocellular neoplasms that arise in normal or nearly normal livers. The four most common subtypes, based on molecular and immunohistochemical findings, are described—HNF1A-inactivated, beta-catenin–activated, inflammatory, and unclassified adenomas (Table 37.5) (79,80). TABLE 37.5 Common Subtypes of Hepatic Adenoma Subtype

Molecular Features

Microscopic Features

Clinical Associations

Immunohistochemistry

HNF1αinactivated

Biallelic inactivation of HNF1α

Fatty change in many; not specific

Most cases sporadic but rare cases in patient with MODY3

LFABP negative

Beta-catenin activated

Mutation in CTNNB1

No specific features

Higher rate of malignant transformation and hemorrhage

Nuclear beta-catenin positive Strong diffuse glutamine synthetase staining

Inflammatory

Activation of IL6/JAK/STAT pathway

Sinusoidal dilation; patchy inflammation; variable number of faux portal tracts

Obesity, alcohol Higher rate of hemorrhage More frequently develops secondary beta-catenin mutations, compared to HNF1alpha

Cytoplasmic positivity for serum amyloid A– associated protein (SAA) for C-reactive protein (CRP) There can be nonspecific patchy staining in FNH, so staining should be strong and diffuse

Unclassified

No specific genetic abnormalities

No specific features

No specific features

NF1α, hepatocyte nuclear factor 1α; LFABP, liver fatty acid–binding protein.

CLINICAL FEATURES

Important risk factors in the development of HA include excess estrogen exposure (oral contrcepative pill [OCP] use, fatty liver disease) excess androgen exposure (medical therapy such as danazol, or illicit steroid use by body builders), vascular flow disease such as Budd-Chiari or portal vein agenesis, or a variety of rare genetic disorders including glycogen storage diseases and mature-onset diabetes of the young type 3 (MODY 3) (80). Patients with HA can present with an abdominal mass (25%-35% of patients), chronic or intermittent abdominal pain (20%-25%), or rarely with acute abdominal pain resulting from hemorrhage into the tumor or into the peritoneal cavity. HA can also be an incidental finding discovered on imaging studies. Serum AFP levels are in the normal range. MACROSCOPIC FEATURES HA is usually a single, sharply circumscribed nodule (70%-80%), often subcapsular and usually unencapsulated, arising in a noncirrhotic liver. In 10% of cases, the tumor is pedunculated. HAs range in size from less than 1 cm to 38 cm. The cut surface can show areas of hemorrhage and yellow necrotic areas. Fibrous septa are usually a result of previous infarct. Rare HAs appear slate-gray or black because of excessive lipofuscin accumulation, termed a pigmented HA (81); green discoloration due to bile staining is uncommon. Ruptured HA may be obscured by blood clot and difficult to recognize on gross examination, but the tumor is usually paler or more yellow than the adjacent liver. Rare cases of coexistent FNH elsewhere in the liver can be seen but are etiologically unrelated. HISTOPATHOLOGY HAs are composed of virtually normal hepatocytes growing in cords that are one to two cells thick and are separated by sinusoids lined by inconspicuous Kupffer cells. However, portal tracts are absent. While duct-like structures are present in many inflammatory adenomas, normal bile ducts are not present. Another common feature of HA is the presence of haphazardly distributed arteries and thinwalled veins. The reticulin framework is intact. The proliferation rate by Ki-67 is very low, usually about 1% (82) and is indistinguishable from the background liver at low power examination. Extramedullary hematopoiesis and noncaseating epithelioid granulomas are rarely found. Mild patchy chronic inflammation is often found in inflammatory HAs, which also typically displays dilated sinusoids (Fig. 37.9). Areas of necrosis, infarct, and hematoma may also be seen. Organization of these foci may lead to the accumulation of hemosiderin-laden macrophages and the formation of fibrous scars.

FIGURE 37.9 Hepatic adenomas. (A) HNF1A inactivated adenomas often show mild steatosis and/or glycogenosis. (B) HNF1A inactivated adenomas show loss of LFABP expression, while the background liver is strongly positive. (C) An inflammatory adenoma shows sinusoidal dilatation. (D) Inflammatory adenomas often have structures that can mimic portal tracts, with a bile-ductular-like proliferation. (E) A CRP is strongly and diffusely positive. Even though the background liver shows some patchy staining, there is clearly a difference between it and the tumor. (F) An unclassified hepatic adenoma has no distinguishing histological or immunohistochemical findings.

After a diagnosis of HA is made using H&E, reticulin, and Ki-67 stains, HA are then subclassified (Fig. 37.9). It is important to make the diagnosis of HA first, as the stains used to subtype HA do not distinguish HA from HCC (83). HAs are subclassified in order to provide prognostic information on their risk of hemorrhage and malignant transformation (84). The four most common subtypes of HA are HNF1A inactivated HA (lowest risk of hemorrhage and malignant transformation), inflammatory HA (increased risk for hemorrhage), beta-catenin activated (increased risk for hemorrhage and malignant

transformation), and unclassified. Additional rare subtypes include androgen-related HA (85), pigmented HA (81), and myxoid HA (86); these latter three all have an increased risk for malignant transformation. For HA with beta-catenin mutations, some authors have suggested expanding the subtypes of betacatenin-activated HA to include their specific mutations, but this has not been widely adopted for clinical care, as it is impractical at most medical centers. Other rare subtypes have been proposed, such as sonic hedgehog activated HA (84) and argininosuccinate synthetase 1 (ASS1) HA (87). The ASS1 subtype overlaps with inflammatory HA and sonic hedgehog activated HA (88); currently, neither of these have been widely adopted for clinical care. The classification schemas to subtype hepatic adenomas have some overlap (80). For example, in many and probably most cases, beta-catenin activation appears to be a secondary event that occurs within an inflammatory HA or an otherwise unclassified HA (or very rarely in HNF1A inactivated HA). Likewise, androgen-associated HA and pigmented HA can be of the inflammatory subtype, HNF1A inactivated type, and/or have beta-catenin activation. Furthermore, myxoid HA always shows HNF1A inactivation and, as mentioned previously, many of the ASS1 HA can also be classified as inflammatory HA (87). This can be confusing to clinicians and patients and pathologists. A reasonable approach is to emphasize in the pathology report those findings that are of the highest risk for malignancy: betacatenin activation, heavy pigmentation, androgen-associated changes, myxoid change. HNF1A Inactivated Subtype Tumors exhibiting biallelic inactivation of the HNF1A gene account for up to 40% of HAs (89). The histological features of the HA are not specific but can include diffuse steatosis and mild glycogenosis (Fig. 37.9A). These tumors are defined in surgical pathology by the loss of expression of liver fatty acid– binding protein (LFABP) on immunostain studies (Fig. 37.9B). LFABP is a downstream target of HNF1A and expression is lost when HNF1A genes are mutated. Overall, this group of HA seems to have the lowest risk of malignant transformation. Inflammatory Adenomas. The inflammatory subtype comprises up to 50% of HA and is characterized by activation of the JAK/STAT pathway. This subtype is associated with fatty liver disease from excessive alcohol consumption, metabolic syndrome, and obesity (90), although not all patients will have these risk factors. Many but not all inflammatory HA have patchy sinusoidal dilatation (Fig. 37.9C), mild patchy lymphocytic infiltrates, and faux portal tract-like structures with a plump artery embedded in a cuff of connective tissue, with a ductular-like proliferation at the edges (Fig. 37.9D). No true portal veins or true bile ducts are seen, however. The ductular-like proliferation at the edges of the faux portal tracts can mimic changes seen in FNH. In fact, cases previously classified as the telangiectatic variant of FNH now appear to be inflammatory HAs (91). Inflammatory HAs also commonly have occasional scattered hepatocytes with large cell change. Immunohistochemistry for C-reactive protein (CRP) and/or serum amyloid A–associated protein is required for diagnosis (unless there is molecular classification) and shows granular cytoplasmic staining. CRP immunohistochemistry is more sensitive than SAA but also tends to have more background staining (Fig. 37.9E). Unclassified Adenomas Unclassified adenomas show retained LFABP expression, and are negative for CRP/SAA, are negative for beta-catenin nuclear accumulation, lack a strong and diffuse glutamine synthetase staining pattern, and have no distinctive histological findings. They comprise approximately 10% of HAs (Fig. 37.9F). Beta-Catenin-Activated Adenomas This subtype of HA is characterized by beta-catenin–activating mutations and comprises 10% to 15% of HAs (89). These tumors are defined by either nuclear positivity for beta-catenin (Fig. 37.10A) or by strong and diffuse staining for glutamine synthetase. Glutamine synthetase staining serves as an excellent surrogate for beta-catenin activation when it is strong and diffuse (Fig. 37.10B). Glutamine synthetase staining in some cases can be weak and patchy, but still more than in the background liver (Fig. 37.10C). This pattern is less strongly linked to beta-catenin activation and, when mutations are present, they tend to be those leading to lower levels of beta-catenin activation (92).

FIGURE 37.10 Atypical hepatic adenomas. (A) Beta-catenin shows nuclear positivity; any number of positive nuclei, even 1, is sufficient to be scored as positive. (B) A hepatic adenoma shows diffuse and strong glutamine synthetase staining; the background liver in the bottom of the image shows the normal staining pattern of zone 3 hepatocytes. (C) A hepatic adenoma shows heterogeneous but diffuse glutamine synthetase staining; this pattern is less specific but can be associated with betacatenin mutations. (D) This androgen adenoma shows large cell change. (E) A pigmented adenoma has abundant cytoplasmic lipofuscin. (F) A myxoid hepatic adenoma has sinusoids distended by abundant myxoid material.

If other immunostains for subtyping HA are also positive in HA with beta-catenin activation, then they are also reported. For example, a HA may be of the inflammatory type (i.e., it is CRP/SAA positive) with beta-catenin activation. Hepatic Adenomatosis

The term adenomatosis is used for those cases with more than 10 adenomas (93) and has been reported for HNF1A inactivated adenomas, inflammatory adenomas, and unclassified adenomas. Since beta-catenin activation is usually a secondary event (80), adenomatosis with beta-catenin activated adenomas does not occur. Atypical Adenomas HA can be atypical for clinical and/or histological findings. Atypical clinical findings include female age >50, male gender, androgen-associated adenomas, and no identifiable risk factors. These findings warrant careful evaluation to rule out HCC. Histologic features of atypia include focal equivocal reticulin loss, cytological atypia, bile production, and pseudogland formation (94). Additional forms of atypia include heavy lipofuscin pigmentation, beta-catenin activation, and myxoid changes. The presence of advanced fibrosis/cirrhosis in the background liver indicates a diagnosis other than HA. While rare outlier papers have suggested otherwise, a diagnosis of HA should not be made in a cirrhotic liver for purposes of clinical care; the natural history of HAs has been sufficiently well studied to allow appropriate patient care, while this is not true for so-called HAs in cirrhotic livers. Conventional HAs should not have cytological atypia, with the cells within the tumor indistinguishable from those in the background liver, although mild large cell change is acceptable for both inflammatory HAs and androgen-associated HAs. Histological findings that would indicate transformation to HCC include nodule-within-nodule growth patterns, definite reticulin loss (usually multifocal), a proliferative rate clearly higher than the background liver, and expression of various oncofetoproteins such as AFP or glypican 3. Not all HCC arising in HA will have all of these findings, but HA should have none of them. Androgen Adenomas Androgen adenomas are more likely than other types of HA to show bile production, pseudogland formation, and large cell change (Fig. 37.10D) (85). Most are beta-catenin activated, with 60% showing beta-catenin nuclear staining and 80% having strong, diffuse GS staining; 20% do not show betacatenin activation by either stains (85). About 20% also stain for CRP or SAA, while 10% show loss of LFABP (85). Pigmented Adenomas Pigmented adenomas are defined as HAs showing heavy and usually diffuse lipofuscin deposition in the tumor cytoplasm (Fig. 37.10E). By way of contrast, lipofuscin deposition that is light and patchy is common and nonspecific in HAs and does not qualify as a pigmented HA. The largest series of pigmented HAs to date reported a 27% frequency of malignant transformation (81). About 2/3 of pigmented HAs show LFABP loss, with most of the remaining positive for SAA and/or CRP (81). Betacatenin activation can also be seen. Myxoid Adenomas Myxoid HAs are a rare variant that shows a distinctive deposition of extracellular myxoid material between thin and often attenuated cords of cytologically bland hepatocytes (Fig. 37.10F). The percent of tumor that shows myxoid change can range from 20% to 80% (86). Myxoid HAs have a high risk of malignant transformation (86,95). Rare cases of multiple myxoid adenomas have been reported, while in other cases, only 1 of several adenomas in the liver show myxoid change (86). Myxoid HAs also consistently show loss of LFABP1 by immunocytochemistry (86) and have HNF1A mutations (96). At a practical level, they can be reported as myxoid HAs or as HNF1A inactivated adenomas with myxoid change, but the high risk for malignancy should be indicated in the pathology note/comment. DIFFERENTIAL DIAGNOSIS, TREATMENT, AND OUTCOME Radiographic features of HA include decreased or absent uptake on scintigraphy, centripetal hypervascularity with a central hypovascular region on angiography, and the absence of a central scar. Histologic overlap of the inflammatory variant of HA with FNH can be a source of diagnostic difficulty on imaging and histology. Distinct parenchymal nodularity with well-developed bands of fibrosis favors FNH. Immunohistochemical studies are very helpful and demonstrate map-like expression of glutamine

synthetase in FNH but not inflammatory HAs. Likewise, strong and diffuse cytoplasmic positivity for SAA and or CRP indicates a diagnosis of inflammatory HA, with the caveat that there can be patchy nonspecific staining for both of these stains in FNH. Thus, comparing the distribution and intensity of CRP and SAA staining between the tumor and background liver is particularly helpful. Copper positivity is more common in FNH than inflammatory HA, but does not distinguish between these two tumors (97). Although HA may regress after withdrawal of OCPs, the lesions often persist. The likelihood of rupture and bleeding cannot be predicted from size of the tumor alone, but the risk is higher with tumors greater than 5 cm. Malignant transformation of HA also occurs most often in tumors larger than 5 cm and in those with histological risk factors discussed above. For HAs greater than 5 cm or those with atypical clinical or histological findings, complete surgical excision, if technically feasible, is an important management option because it eliminates the risk of bleeding and malignant transformation. However, more conservative approaches can be appropriate for small lesions without histological or clinical atypia.

MACROREGENERATIVE AND DYSPLASTIC NODULES IN CIRRHOSIS Small nodular lesions, some of which represent precursors to HCC, can develop in cirrhotic livers and often pose diagnostic challenges for the pathologist if the lesions are biopsied. In addition, these atypical hepatic nodules that do not meet histologic criteria for malignancy are commonly found in explanted livers. Although the natural history of these nodules in cirrhosis is still not fully understood, it is now clear that dysplastic nodules (DNs) are grossly recognizable precursor lesions of HCC (98,99). Nodular lesions arising in the setting of cirrhosis show a spectrum of histologic changes, from large MRNs without atypical features to nodules falling just short of the morphologic criteria diagnostic of HCC. Generally, they are well circumscribed and have a rim of fibrous tissue, similar to other cirrhotic nodules. On histologic evaluation, there are two types of nodules: macroregenerative nodules (considered nonneoplastic) and DNs (those with atypical features). Because imaging studies and serum AFP levels (normal or within the range seen in chronic liver disease) cannot readily distinguish these nodules from early HCC, microscopic evaluation of these lesions is necessary for definitive classification. At a practical level, a firm diagnosis of MRN or DN is ill-advised in biopsy specimens, due to sampling concerns. Instead, these diagnoses are reserved for the differential diagnosis in the note/comment section of surgical pathology reports for those difficult biopsies with findings that are atypical but do not reach the level of HCC. A definite diagnosis of MRN or DN can be made in fully resected specimens. In this setting, these diagnoses no longer change clinical management but are still important for understanding tumor biology. In resection specimens, including liver explants, it is acceptable for clinical care to group them as MRN/DN in the pathology report and to not provide a more specific classification if you are uncertain of the diagnosis, which can be somewhat subjective even amongst experts; of course, HCC should be carefully excluded. MACROREGENERATIVE NODULES There is no single minimum size criterion for MRN. Instead, they are identified as nodules that stand out from the background cirrhotic nodules in the liver based on their noticeably larger size; generally, MRNs measure at least 0.5 cm, but minimum size criteria of 0.8 to 1.0 cm have also been used (100). MRNs are usually multiple and most measure less than 1.5 cm in diameter (Fig. 37.11A). They are found in about 15% of cirrhotic livers (depending on how they are defined) and are three to four times more common than DNs.

FIGURE 37.11 Macroregenerative nodule. (A) The MRN is distinguished grossly by its large size relative to other nodules in the cirrhotic liver. It cannot be distinguished grossly from dysplastic nodules or hepatocellular carcinoma (HCC). (B) Microscopically, the MRN is cytologically bland and is indistinguishable from the background cirrhotic nodules.

Microscopically, hepatocytes within MRNs are identical to those in the surrounding liver and typically demonstrate histologic abnormalities characteristic of the underlying disease (Fig. 37.11B). The hepatocellular plates are one or two cells thick and the reticulin framework is intact. Portal tracts are scattered throughout the nodule and may be relatively normal in morphology or show obliteration of portal vein branches and a mild bile ductular reaction (101). Portal tracts are frequently fewer in number compared with the background liver. Solitary or unpaired intranodular arteries are absent or rare, and diffuse sinusoidal capillarization is uncommon (conversion of fenestrated sinusoidal channels to continuous capillary vessels, characterized by the acquisition of sinusoidal immunoreactivity for factor CD34). Nonspecific changes such as steatosis, feathery degeneration, hemosiderin deposition, or accumulation of Mallory hyaline are not sufficient to classify the nodule as dysplastic. DYSPLASTIC NODULES DNs are nodular lesions displaying some degree of cytological or architectural atypia but lacking definitive histologic features of malignancy. On macroscopic examination, they cannot be distinguished from MRNs. Although they may be slightly larger than MRNs, DNs are rarely larger than 2 cm. DNs are further subdivided by histological examination into low-grade DNs and high-grade DNs, depending on the degree of atypia. There are two primary categories of cytological atypia: small cell change and large cell change. Small cell change is characterized by hepatocytes that show smaller cell size, a greater nuclear-to-cytoplasmic ratio, cytoplasmic basophilia, and denser cellularity, in comparison with the surrounding hepatocytes (Fig. 37.12A). Hepatocytes in small cell change have been shown to have a higher proliferative rate and a lower apoptotic rate compared to hepatocytes in the surrounding liver and is considered a precursor lesion for HCC (102,103). Large cell change, characterized by cellular enlargement, nuclear pleomorphism, and hyperchromasia, but with a normal or near-normal N:C ratio (Fig. 37.12B), is a heterogeneous lesion and can be a precursor lesion for HCC in some cases, while in other cases is reactive.

FIGURE 37.12 (A) Small cell change is recognized by the denser packing of smaller hepatocytes in the dysplastic nodule compared to background liver. (B) Large cell change is characterized by cellular enlargement, nuclear hyperchromasia, and pleomorphism, with irregular nuclear membranes. (C) High-grade dysplastic nodule. This high-grade DN exhibits nodulewithin-nodule growth and a few foci of pseudoglandular architecture. Stromal or portal invasion, which would indicate frank malignancy, is not identified.

In low-grade DNs, the hepatocytes show minimal to mild nuclear atypia and there is no mitotic activity (75). Scattered portal tracts can be present, although their frequency diminishes from MRN to low-grade DN to high-grade DN. Areas suggestive of a clonal population may be seen, such as a uniform population of hepatocytes with a map-like growth pattern, a finding useful in making the distinction between low-grade DN and MRN. Another useful factor is the presence of cytological atypia that is mild, yet more than seen in the background liver. Large cell change is acceptable. In high-grade DNs, the architectural and cytological abnormalities are more pronounced, and may include large cell change with more striking atypia, small cell change, or nodule-within-nodule formation (Fig. 37.12C) suggestive of subclone evolution. Rare mitotic figures and focal pseudogland formation may be identified. Stromal invasion, where tumor cells extend into the portal tracts/fibrous septae, is not seen. Unpaired (naked) arteries are often more conspicuous than in low-grade DN. Capillarization of the sinusoids is more common in DNs than in MRNs. A diagnosis of DN cannot be made confidently on biopsy specimens because of sampling issues, so can be included in the differential diagnoses, but should not be the final diagnosis. Association With Hepatocellular Carcinoma DNs are an important precursor to HCC (98,99). Longitudinal studies of MRNs indicate these lesions have a low risk of transformation to HCC and are usually stable in size and can even regress by imaging studies (98). At the molecular level, MRNs are similar to ordinary cirrhotic nodules (104). In contrast, DNs do not regress and often increase in size (99). In one study of cirrhotic patients, where patients were followed longitudinally, roughly 80% of high-grade DNs, 36% of low-grade DNs, and 12% of MRNs

progressed to HCC within 5 years (99). Therapeutic intervention (ablation or resection) must be seriously considered when the histological differential diagnosis includes high-grade DN because of the high risk for development of HCC and the possibility of undersampling leading to a false-negative diagnosis of HCC. Patients with apparent MRNs should be followed up with an appropriate imaging study at more frequent intervals than the usual patient with cirrhosis. DIFFERENTIAL DIAGNOSIS AND REPORTING The differential diagnosis for well-differentiated hepatic lesions depends on whether or not the background liver shows cirrhosis. For noncirrhotic livers, the differential is mostly that of FNH, hepatic adenoma, and well-differentiated HCC. In cirrhotic livers, the differential is MRNs, DNs, and welldifferentiated HCC. Separation of low-grade DNs from MRNs, and of high-grade DNs from HCC, can be challenging, but features useful in the differential diagnosis are summarized in Table 37.4. The most reliable diagnostic criteria for the distinction of HCC from high-grade DN are convincing reticulin loss or a Ki-67 proliferative rate that is clearly above that of the background liver. Glypican 3 or AFP is positive in less than half of cases, but when positive supports a diagnosis of HCC. Of course, vascular, capsular, or stromal invasion would indicate HCC, but these features are rarely present in welldifferentiated HCC, especially on needle biopsies. The presence of residual portal tracts within a nodule does not rule out early HCC, as early HCC can arise within DN.

HEPATOCELLULAR CARCINOMA EPIDEMIOLOGIC FACTORS Malignant epithelial tumors account for about 98% of all primary hepatic malignancies, with HCC representing by far the single most common histologic type (roughly 90%). Although it is relatively uncommon in North America and Western Europe (incidence 4.1-5.8/100,000 male population), HCC is one of the most prevalent malignant tumors worldwide, accounting for roughly 5% of all human cancers and is the third most common cause of cancer death globally (105). Men are at higher risk than women, with a ratio of about 4:1 (106). In Western countries, patients usually develop HCC after age 50, whereas it is not uncommon to find HCC in patients aged 20 to 35 in areas with high incidences of HBV infection. ETIOLOGY AND PATHOGENESIS HCC is strongly associated with chronic liver disease, especially those leading to cirrhosis. The annual risk of HCC developing in a cirrhotic liver is estimated at 1% to 7% (107-109), with the risk generally highest in the context of viral hepatitis. Historically, the most important etiological factors were chronic hepatitis B, chronic hepatitis C, and alcohol cirrhosis, but fatty liver disease from the metabolic syndrome (obesity, hypertension, diabetes mellitus, and dyslipidemia) has recently become an additional major driver of HCC risk. Other risk factors include aflatoxin contamination of food, most commonly seen in sub-Saharan Africa, and coinfection with hepatitis D virus in patients with chronic hepatitis B. Although the strong association between HCC and cirrhosis is well known, the pathogenetic sequence leading to malignancy is less well elucidated. Liver cell proliferation is increased during chronic hepatitis but is often decreased in cirrhosis. Limitation of the regenerative reserve of the liver has been attributed to accelerated telomere shortening in hepatocytes in chronic liver disease, leading to telomere dysfunction and susceptibility to chromosomal alterations (110). The most common mutations in HCC are in the TERT promoter, CTNNB1, TP53, AXIN1, ARID1A, CDKN2A, and CCDN1 (111). HBV infection promotes carcinogenesis by at least four mechanisms: integration of viral DNA into the host genome leading to chromosomal instability (112); insertional mutations at specific sites leading to activation of genes involved in cell proliferation, such as the TERT promoter (112); the ability of HBX viral protein to modulate cell proliferation and inactivate p53 (113); and activation of host methylation

pathways in an attempt to downregulate the virus by methylating CpG islands located within the HBV genome (114,115), but also leading to aberrant host DNA methylation as a bystander effect (116). CLINICAL FINDINGS Surveillance of patients with chronic liver disease and cirrhosis has led to increased detection of small, treatable, asymptomatic HCC, but many tumors still present with advanced disease. Common presenting symptoms are abdominal pain, fullness or a mass, or worsening of symptoms attributed to cirrhosis. HCC may invade hepatic veins and spread to the inferior vena cava or, rarely, even into the right atrium, producing right heart failure. The most common metastatic sites are lymph nodes, bone, and lung. Although most HCC patients die from cancer progression, the comorbidity of cirrhosis can hasten death. Overall, up to 20% of HCCs arise in a noncirrhotic liver. HCC in the noncirrhotic liver generally presents at a later stage and with a larger mass, in comparison to HCC arising in patients with cirrhosis. Patients without cirrhosis can still have chronic liver disease such as HBV or HCV (117). Screening and early detection programs for HCC rely on a combination of ultrasonography or other imaging methods and serum levels of AFP and have led to the diagnosis of many asymptomatic HCCs. Although it is difficult to demonstrate a decrease in disease-specific or all-cause mortality, due to the need for large cohorts and randomization to screening or no screening, screening for HCC is considered appropriate (118) because the cure rate for symptomatic cancers is very low (0%-10% 5-year survival) and early-stage tumors that are amenable to resection or liver transplantation have 5-year survival rates of up to 50% (119). AFP, a glycoprotein produced primarily by fetal liver, remains the most useful serologic marker for HCC, although its limitations are well recognized; in particular, AFP is not a good marker for early HCCs (105). Sensitivity ranges from 40% to 65% and specificity from 76% to 96% (120). A cutoff of 20 ng per mL is commonly used to define the upper limit of the normal range, but values of 400 ng per mL or greater are needed to be considered diagnostic for HCC. Progressive increase in serum AFP is also suggestive of HCC and warrants further investigation. A variant of AFP called AFP-L3, which differs in its sugar chains, appears to be more specific for HCC than total AFP. Serum levels of des-γ-carboxy prothrombin (DCP) is another useful marker of HCC, with sensitivities ranging from 28% to 89% and specificities from 87% to 96%. Because DCP and AFP serum levels do not correlate, combination of both markers improves accuracy in HCC diagnosis (120). Although serum AFP levels may be nonspecifically elevated in nonneoplastic conditions, levels higher than 400 ng per mL are rarely seen. Other malignant neoplasms often associated with very high levels of serum AFP (>1,000 ng/mL) include hepatoblastoma, germ cell tumors containing a yolk sac component, and hepatoid adenocarcinomas arising in various sites, such as the stomach or ovary. MACROSCOPIC FEATURES General Gross Findings Most tumors grow as a single large mass, often with satellite nodules. The tumors are occasionally pedunculated. Rarely, HCC grows as numerous small nodules that may be indistinguishable from cirrhosis (diffuse type or cirrhotomimetic type). Staging criteria for HCC are listed in Table 37.6; they principally depend on the size and number of the tumor nodules and the presence or absence of vascular invasion. TABLE 37.6 pTNM Staging of HCCa T – Primary tumor TX – Primary tumor cannot be assessed T0 – No evidence of primary tumor T1 – Solitary tumor ≤2 cm or ≥2 cm without vascular invasion T1a – Solitary tumor ≤2 cm T1b – Solitary tumor ≥2 cm without vascular invasion T2 – Solitary tumor >2 cm with vascular invasion, or multiple tumors, none >5 cm T3 – Multiple tumors, at least one of which is >5 cm

T4 – Single tumor or multiple tumors of any size involving a major branch of the portal vein or hepatic veins, or tumor(s) with direct invasion of adjacent organs, other than the gallbladder, or with perforation of the visceral peritoneum N – Regional lymph nodes NX – Regional lymph nodes cannot be assessed N0 – No regional lymph node metastasis N1 – Regional lymph node metastasis M – Distant metastases M0 – No distant metastases M1 – Distant metastases Stage groupings Stage IA

T1a

N0

M0

Stage IB

T1b

N0

M0

Stage II

T2

N0

M0

Stage IIIA

T3

N0

M0

Stage IIIB

T4

N0

M0

Stage IVA

Any T

N1

M0

Stage IVB

Any T

Any N

M1

From AJCC Cancer Staging Manual. Liver. 8th ed. Springer; 2017:291; reproduced with permission of SNCSC.

Tumor nodules are typically fleshy and variegated. Some are bile-stained, fatty tumors are often pale yellow, while most HCC are tan-brown. Areas of hemorrhage and necrosis can be seen. Multiple tumor nodules may be due to synchronous primaries (multicentric growth), which is more common when tumors are small and widely separated in cirrhotic livers. Most multinodular tumors, however, have this pattern: one large dominant nodule along with smaller satellite nodules located within several cm of the dominant nodule. This pattern typically represents intrahepatic metastases from tumor spreading through portal vein branches. Early Hepatocellular Carcinoma Early (or small) HCCs are defined as tumors in cirrhotic livers measuring less than 2 cm in diameter (101); this distinction is primarily for research purposes and does not need to be included in clinical reports. Small HCCs often bulge above the cut surface, are rarely necrotic, and may be variegated, with green areas corresponding to bile staining and yellow areas reflecting fat accumulation (Fig. 37.13A). In small tumors, a diagnosis of HCC is made in the usual fashion, based on histological atypia, loss of reticulin (Fig. 37.13B, C), and aberrant expression of oncofetoproteins such as AFP or glypican 3.

FIGURE 37.13 Small HCC. (A) This small HCC shows a variegated cut surface due to bile accumulation and hemorrhage. However, many small HCCs cannot be distinguished from large regenerative nodules or dysplastic nodules based on gross

appearance. (B) Mallory hyaline and globular cytoplasmic proteinaceous accumulations may be conspicuous in HCC, including small lesions. (C) Reticulin stain demonstrates loss of reticulin fibers and highlights thickened liver cell plates.

Early HCC is further subdivided into two types: the distinctly nodular type and the indistinctly (vaguely) nodular type. The latter is ill-defined on gross examination, with a vaguely nodular appearance and indistinct borders. A fibrous capsule is commonly present in the distinctly nodular type of early HCC, but not in the vaguely nodular growth pattern. Overall, vaguely nodular HCC tends to be well differentiated (>90%), versus distinctly nodular HCC, where 60% are moderately differentiated (121). They also differ in their frequency of vascular invasion: 5%, vaguely nodular; 40%, distinctly nodular (121). MICROSCOPIC FEATURES The cytology and microarchitecture of the tumor can range from almost normal appearing liver to highly anaplastic malignancies that show little evidence of hepatocellular differentiation. In 1954, Edmondson and Steiner (122) devised a four-tiered grading system based on autopsy data that was subsequently modified in a large series reported from the Armed Forces Institute of Pathology (AFIP) (Table 37.7) (123,134). The World Health Organization (WHO) classification divides tumors into well, moderately, and poorly differentiated tumors (Table 37.8) (124). Most HCCs are moderately differentiated (grade 2 in the WHO system), but this will vary on the study population. For example, resected tumors are biased toward better differentiation, while autopsy-based studies are enriched for poorly differentiated HCC. More than one histologic grade is often present within any given tumor, in which case the worse grade tends to drive prognosis (125). A common rule of thumb is that any grade that is less than 5% of the tumor is not counted. The prognostic power of tumor grade has been well studied, and while not entirely consistent, the center mass of the data indicates there is prognostic power in essentially all clinical settings, including for HCC resections in cirrhotic livers (126,127), noncirrhotic livers (128), and after liver transplantation (126,129). TABLE 37.7 Modified Edmondson and Steiner Grading of HCC Grade

Nuclear Changes

Cytoplasm Changes

1

No or minimal atypia

Abundant, eosinophilic

2

Mild atypia, prominent nucleoli, hyperchromasia, and nuclear irregularity

Not specified

3

Moderate nuclear atypia, greater hyperchromasia, and nuclear irregularity

Not specified

4

Marked nuclear pleomorphism, marked hyperchromasia, and anaplastic giant cells

Not specified

TABLE 37.8 WHO Grading of HCC Numerical Grade

Descriptive Grade

Global Assessment

Criteria

1

Well differentiated

Tumor cells resemble mature hepatocytes

Cytoplasm: ranges from abundant and eosinophilic to moderate and basophilic Nuclei: minimal to mild nuclear atypia

2

Moderately differentiated

Clearly malignant on H&E; morphology strongly suggests hepatocellular differentiation

Cytoplasm: ranges from abundant and eosinophilic to moderate and basophilic Nuclei: moderate nuclear atypia; occasional multinucleated tumor cells are acceptable

3

Poorly differentiated

Clearly malignant on H&E, but morphology is consistent with broad spectrum of poorly differentiated carcinomas

Cytoplasm: range from moderate to scant, usually basophilic

Nuclei: Marked nuclear pleomorphism, may include anaplastic giant cells 1. Although not part of the WHO grading system, well-differentiated tumors can be divided into grades 1A and 1B or can be grouped together into a single grade; data at this point is limited as to the best approach. In grade 1A, the diagnosis is often made only by loss of reticulin or by aberrant expression of immunostains such as glypican 3; the cytology and morphology would otherwise be compatible with a hepatic adenoma in a noncirrhotic liver or a large regenerative nodule or a dysplastic nodule in a cirrhotic liver. 2. In grade 3, immunostains are often necessary to confirm hepatocyte differentiation. 3. Sarcomatoid and anaplastic morphology is classified as grade 3.

The term primary undifferentiated carcinoma is reserved for primary liver malignances (i.e., metastatic disease has been excluded as much as reasonably possible) that are histologically proven to be carcinomas but lack any convincing evidence of biliary or hepatocellular differentiation on morphology and immunostain analysis (75). Growth Patterns Histological growth patterns seen within HCC are different from HCC subtypes, which are discussed further in their own section. Multiple histologic growth patterns are often found in the same tumor and most subtypes show a variety of different growth patterns. In most studies, a cutoff of 5% is used. These patterns are helpful for pathologists in recognizing HCC, but do not need to be included in pathology reports. Trabecular (sinusoidal, plate-like). This common pattern (~70%) resembles normal hepatic architecture with tumor cells growing in cords or plates that are separated by vascular channels lined by endothelial cells and Kupffer cells (Fig. 37.14A), with little or no supporting stroma. The trabeculae vary in thickness, from only a few cells thick (microtrabecular) to thicker trabeculae. Trabeculae 10 cells or greater in thickness are classified as macrotrabecular (see below). Solid (compact). The solid pattern (Fig. 37.14B) is seen in about 20% of HCC and shows sheets of cells without well-defined trabeculae at low power (e.g., 4X or 10X) and appears to reflect a compressed trabecular growth pattern, as trabecular can be observed, at least focally, on high power in many cases. This pattern is typically admixed with the conventional trabecular pattern. Pseudoglandular (acinar). This pattern (Fig. 37.14C) is found in about 10% to 20% of HCC, depending on the cutoff used, and shows pseudogland formation that ranges from diffuse to focal. The pseudoglands represent greatly dilated bile canaliculi and are lined by a single layer of tumor cells that stain with ordinary hepatic markers. In a subset of cases, pseudogland formation is further associated with a well-differentiated morphology that also has eosinophilic tumor cells growing in thin trabeculae, often with bile production; this composite pattern is associated with CTNNB1 mutations (130-132). In other cases, pseudogland formation is seen in moderately to poorly differentiated HCC with solid or thick trabecular growth patterns and without CTNNB1 associations. When prominent, pseudoglands may be mistaken for metastatic adenocarcinoma, cholangiocarcinoma, or HCC combined with cholangiocarcinoma (Fig. 37.14C). Macrotrabecular This pattern (Fig. 37.14D) is uncommon (6 or >20 tumor cells in thickness; there is no objective data to determine the best cutoff, but >6 or >10 are the most common in use.

FIGURE 37.14 HCC, microscopic features. (A) Trabecular pattern. (B) Compact (solid) pattern. (C) Pseudoglandular (pseudoacinar) pattern. (D) Macrotrabecular pattern. (E) Hyaline bodies; these have been associated with a worse prognosis. (F) This HCC shows clonal progression, with two distinct tumor morphologies.

Cytological Appearance The tumor cells of HCC are usually polygonal but may be cuboidal. The cytoplasm in well-differentiated HCC can be almost normal in amount and appearance, with finely granular eosinophilic cytoplasm. Moderately and poorly differentiated HCC tend to have less cytoplasm with increasing basophilia. When considering primary carcinomas of the liver, bile production is pathognomonic of HCC, but it is found in less than one-third of cases, most commonly in well-differentiated HCC. The presence of bile canaliculi is also diagnostic of hepatic differentiation and can be identified on special stains (see below). Of note, however, bile and bile canaliculi can also be seen in rare cases of hepatoid adenocarcinomas arising in other organs and metastatic to the liver (133). The nuclei of HCC tumor cells are usually round to oval,

with coarse chromatin, single prominent nucleoli, and thickened or irregular nuclear membranes. Multinucleation, often with bizarre nuclei, increases in frequency with worse tumor grades. Intranuclear cytoplasmic invaginations are a common and nonspecific finding. A variety of cytoplasmic inclusions may be identified in HCC cells (75): hyaline bodies (Fig. 37.14E), Mallory-Denk bodies, and pale bodies. These inclusions are not diagnostically useful and are not sufficiently prognostic that they need to be mentioned in the pathology report. Other cytoplasmic changes include fatty changes, clear cell changes, and occasionally abundant lipofuscin deposition. Hemosiderin is rarely found in HCC, even in the context of hereditary hemochromatosis. About 40% of HCCs show multiple distinctive morphologies, a finding called clonal progression (Fig. 37.14F). Sarcomatoid changes can be seen in moderately or poorly differentiated HCC, typically as a distinct nodule of poorly differentiated spindled cells. The spindled cells retain keratin positivity, which is very helpful to distinguish this pattern from a carcinosarcoma. The keratin staining, however, is often patchy and broad-spectrum keratin stains may be needed. Markers of hepatocyte differentiation are either focally positive or are negative. In addition, the spindle cell areas of sarcomatoid carcinomas, by definition and in contrast to many cases of carcinosarcoma, cannot be further subclassified into specific types of sarcoma, by either morphology or by immunostains. HEPATOCELLULAR CARCINOMA SUBTYPES HCC can be further subtyped by morphology (Table 37.9) (134). The terms subtype and variant are interchangeable. Morphological subtypes are important so that pathologists recognize the variable histological findings of HCC and because different subtypes tend to have their own diagnostic pitfalls. The current definition of a morphological subtype for HCC includes four key components (135) as detailed below, with the recognition that it takes some time for all of these elements to be fully developed. On the other hand, if a proposed morphologic HCC subtype can be clearly shown to not meet these criteria over time, then it can be abandoned. 1. Morphology. There should be distinctive histological findings consistently present in the tumor. This criterion is usually how a morphological subtype is first recognized, but is insufficient in isolation to establish it as a distinct variant, requiring the next 3 criteria to provide specificity and clinical meaning. 2. Immunohistochemistry or other confirmatory testing. Histological findings alone are seldom sufficient to reliably classify all cases. When combined with morphology, however, this criterion of immunostain or other confirmatory tests (e.g., FISH or molecular) provides powerful specificity, making the subtype more meaningful for clinical care and research studies. 3. Clinical correlates. This criterion can include clinical presentation, risk factors, laboratory findings, treatment response, and prognosis. Prognosis correlates can take time to develop, as most HCCs have bad outcomes. 4. Unique molecular findings. This criterion can lead to potential treatment targets. In addition, strong molecular correlates can feed back into criterion 2 for use as molecular or immunohistochemically based confirmatory tests. TABLE 37.9 HCC Morphological Subtypes Subtype

Frequency (%)

Prognosisa

Unique Histology

Confirmatory Immunohistochemistry or FISH

Unique Clinical Findingsb

Steatohepatitic

20

Similar

Steatohepatitis in at least 50% of tumor

No

Risk factor of fatty liver disease

Subtype

Frequency (%)

Prognosisa

Unique Histology

Confirmatory Immunohistochemistry or FISH

Unique Clinical Findingsb

Macrotrabecular massive

10

Worse

Macrotrabecular growth pattern in at least 50% of tumor

No

Elevated serum AFP

Clear cell

7

Better

Clear cell in at least 50% of tumor

No

No

Scirrhous

5

Similar to better

Dense fibrosis in at least 50% of tumor

No

No

Chromophobe

5

Similar

Light eosinophilic to clear cytoplasm with scattered foci of anaplastic nuclei, in a background of low-grade nuclei

ALT FISH

No

Cirrhotomimetic

1

Worse

None; but unique growth pattern with >30 small nodules that mimic cirrhosis in size and morphology

No

Amount of tumor is consistently underestimated by imaging

Fibrolamellar carcinoma

1

Similar to better

Large eosinophilic cells with prominent nucleoli and prominent intratumoral fibrosis

CD7, CD68 FISH for PRKACA deletion

Young age, no underlying liver disease

Granulocyte-colonystimulating-factor producing

2

Uniformly poor outcome, few cases studied; other patterns needed for ddx with small round cell tumors, CK +

High

Variable

Minimal

–

“Osteoblasts” + CK. Mixed HBL may be favorable prognostic factor

Mixed epithelial/mesenchymal type (44%) Nonteratoid (34%)

Teratoid (10%)

Spindle oval cells; frequent osteoid, rarely other differentiated sarcomatoid elements

Problem of overlap with germ cell tumors

CK, cytokeratin; ddx, differential diagnosis; EMH, extramedullary hematopoiesis; HBL, hepatoblastoma; HCC, hepatocellular carcinoma; hpf, high-power field; N:C, nuclear-to-cytoplasmic ratio.

About 40% of HBs will have mesenchymal and epithelial components. An epithelial component is required for the diagnosis of HB. The epithelial components vary in their morphology, depending on their resemblance to mature hepatocytes, the most common being the embryonal and fetal patterns. In fact, the fetal pattern is seen at least focally in about 80% of cases and the embryonal pattern at least focally in about 30% of cases. Each of the remaining patterns is rare, found in 5% or less of cases. Most HBs show a mixture of epithelial patterns, but single patterns are found in about 20% of cases, usually representing one of the fetal variants (36,322). The surgical pathology report should indicate whether the epithelial component is mixed or pure and what type(s) of epithelial component are present; any mesenchymal component should also be reported. The most important patterns for clinical care are the pure fetal pattern with low mitotic activity and the small cell undifferentiated pattern. The pure fetal pattern with low mitotic activity can be cured by full resection alone, without need for chemotherapy, while more aggressive chemotherapy is often used if there is a component of the small cell undifferentiated pattern. Neoadjuvant therapy can result in necrosis and a fibrohistiocytic response. Some HBs show extensive osteoid after treatment. Small cell undifferentiated The tumor cells grow in discohesive nests and sheets, lacking pseudoglands, and trabeculae The cells are small (5-10 microns) with scant, pale to amphiphilic cytoplasm. The small cell undifferentiated pattern is usually less than 10% of the tumor but can be more substantial (323,324). There are unanswered questions as to the prognostic significance of very small foci of small cell undifferentiated morphology, but until further clarification, any component should be mentioned in the pathology report. Immunostains are negative for HepPar-1 (325) and Arginase-1 but glypican-3 can be focally positive (326). The tumor cells are usually negative for beta-catenin nuclear accumulation, but can be very focally positive. INI-1 immunostaining should be performed to rule out a

rhabdoid tumor, which can closely mimic HB; if there is diffuse loss of nuclear staining, the best diagnosis is a rhabdoid tumor. Embryonal pattern The tumor cells grow in solid sheets and commonly form pseudoglands or tumor rosettes. They have basophilic cytoplasm, angulated nuclei, and a high N:C ratio. Immunostains are positive for Hepar-1, Arginase, and glypican 3 (326). Beta-catenin shows nuclear accumulation often with abnormal cytoplasmic staining. Fetal pattern This is the most common pattern in HB. The tumor cells grow in solid sheets or in trabeculae. The tumors cells show morphological evidence for hepatic differentiation on H&E. The tumor cells can produce bile and sometimes show clear cell changes that result from the accumulation of glycogen. In some cases, the clear cell changes form irregular patches, leading to alternating areas of “light and dark” tumor cells at low power, a finding that is distinctive but not specific for HB. As befitting the morphological evidence for hepatic differentiation, immunostains are positive for Hepar-1, Arginase, ALB-ISH, while showing a negative or weak stippled cytoplasmic staining pattern for glypican-3; a strong diffuse granular staining pattern for glypican-3 is more commonly seen with the embryonal pattern. Betacatenin shows scattered positive nuclei, but less nuclear positivity than is seen in the embryonal pattern. The fetal growth pattern is further subdivided into three groups based on mitotic counts and on cytological atypia. The pure fetal with low mitotic activity pattern has no mesenchymal component and no other epithelial component. There is no more than 1 mitotic figure in 10 high power fields. This pattern of HB has an excellent prognosis and is cured by resection alone in most cases. Of course, these patterns when present on a biopsy are not always representative of the full tumor, so this diagnosis is not used in biopsy specimens or if the patient received therapy prior to resection. HBs are classified as mitotically active fetal hepatoblastoma (or crowded fetal hepatoblastoma) when they show a pure fetal growth pattern but have 2 or more mitoses in 10 high power fields. Rarely, HBs have a pure fetal growth pattern but show striking cytological atypia that resembles a conventional HCC, a pattern called pleomorphic fetal hepatoblastoma. Atypical mitotic figures can also be seen in the pleomorphic variant. To distinguish this pattern from conventional HCC, look for other HB patterns in the tumor, as they are present in almost all cases of pleomorphic HB. Macrotrabecular pattern The tumor cells grow as thick macrotrabecula (10 or > cells in thickness) and show fetal type morphology. This pattern is usually a minor component of the tumor. The tumor cells are positive for Hepar-1 and arginase and often glypican-3. If a tumor is composed mostly or entirely of this pattern, then conventional HCC should be considered in the differential; in fact, if no other HB patterns are present, then conventional HCC is the most likely diagnosis, especially in older children. AFP levels can be substantially elevated in both settings, so the clinical findings (age, underlying liver disease) and the presence or absence of other more common HB growth patterns are the important distinguishing features. Cholangioblastic pattern (sometimes called cholangiocellular) This pattern is very rare and usually focal when present, although it seems to be a bit more common in resection specimens if the patient was treated with chemotherapy. The tumor shows duct-like structures embedded in a mesenchymal component. Treated specimens often have benign bile ductular reaction at the edges of the tumor bed, but the tumor cells in the cholangioblastic pattern show mitotic figures and significantly more atypia than is seen in the epithelia cells of a benign ductular proliferation. Nuclear accumulation of beta-catenin can sometimes be helpful and invariably supports a diagnosis of malignant cells. Mesenchymal component About forty percent of fully resected HB will have a mesenchymal component, which usually consists of nondescript spindle cells. The spindle cell component is usually relative cytologically bland and varies in composition from loose and myxoid to dense and cellular. Osteoid formation can be seen and, rarely, skeletal muscle or cartilage. A teratoid hepatoblasoma is very rare but shows multiple lines of differentiation including neural cells, glial cells, melanin-containing cells, neuroendocrine differentiation, gland formation, mucinous, or squamous differentiation Differential Diagnosis The most difficult differential diagnoses for small cell undifferentiated HB are metastatic small round cell tumors such as Wilms tumor, neuroblastoma, and rhabdomyosarcoma, as well as lymphoma. In this setting, immunohistochemical findings aid in the diagnosis. Both the pleomorphic fetal patterns and the

macrotrabecular patterns of HB can closely resemble HCC, but they are almost always accompanied by other areas with more typical appearances for HB (Table 37.19). TABLE 37.19 Hepatoblastoma and Childhood HCC: Differential Diagnosis Feature

Hepatoblastoma

HCC

Age

90% 5 year

Sex (male:female ratio)

1.5-2.1

1.7-11.1

Single mass

70%-80%

15%-40%

Cirrhosis

Absent

5%-25% (hepatitis B virus, metabolic disease)

Trabeculae

Usually 2-3 cells thick; occasionally macrotrabeculae

Often >2-3 cells thick

Cell size versus nonneoplastic hepatocytes

Same size or smaller

Usually larger, well-differentiated HCC can be smaller

Tumor giant cells

Rarely present

Common

Intranuclear inclusions

Absent

Common

Cytoplasmic hyaline globules

Absent

Common

Extramedullary hematopoiesis

Often present

Absent

CLINICAL FEATURE

MACROSCOPIC APPEARANCE

MICROSCOPIC APPEARANCE

HCC, hepatocellular carcinoma.

Treatment, Outcome, and Prognostic Factors The most import factor in predicting patient outcomes remains the resectability of the tumor. Timing of hepatic resection varies between different study groups. In the United States, current treatment guidelines recommend initial surgery for all resectable tumors, followed by adjuvant chemotherapy for all except completely resected (stage I) HB with pure fetal histology. Because of the excellent prognosis of these patients, no chemotherapy is recommended (327). Approximately one-half of HBs will be too large or extensive for surgical resection at the time of clinical presentation. Techniques such as preoperative chemotherapy and liver transplantation allow resectability of tumor in up to 80% of these cases and have increased overall survival rates to approximately 70% (320). Stage I tumors (using the Children’s Oncology Group system, Table 37.20) have an almost 100% survival, while stage II show around 80% survival. TABLE 37.20 Staging of Hepatoblastoma According to the Children’s Oncology Group Stage I—Complete resection Stage II—Microscopic residual tumor after resection Stage III—No distant metastases with Unresectable tumor or Gross residual disease after resection or Positive lymph nodes Stage IV—Distant metastases

HEPATOBILIARY RHABDOMYOSARCOMA Hepatobiliary embryonal rhabdomyosarcoma usually occurs in children less than 5 years of age, although cases have been reported in older children and adults (328,329). Pediatric embryonal rhabdomyosarcoma is seen in males and females with equal frequency. Clinical presentations include obstructive jaundice, hepatomegaly, and fever (329). The tumor most commonly arises in the common bile duct and shows secondary involvement of the liver parenchyma. Grossly, the tumor consists of multiple soft, gelatinous, grape-like masses projecting into the bile duct lumen (“sarcoma botryoides”). The tumor often demonstrates a “billowy” appearance due to the folds of affected bile ducts. On microscopic examination, the polypoid projections of tumor are covered with bile duct epithelium and often show ulceration and inflammation. A “cambium” layer is seen immediately under the epithelium (a dense band of tumor cells). The neoplastic cells are spindle, round, irregular, or strap shaped, with eosinophilic cytoplasm and occasional cross-striations. Nuclei are elongated, irregular, with blunt ends and frequent mitoses. The tumor cells are arranged within a myxoid stroma and scattered collagen fibers. Necrosis, hemorrhage, and mixed inflammation may be present. Marked pleomorphism following chemotherapy is common (329). The tumor cells are positive for myoglobin, myosin, muscle-specific actin, and desmin. MyoD1 is positive within the nucleus and remains one of the most specific markers available. Therapeutic options include surgical resection, multidrug chemotherapy, and radiation therapy. In a study of 10 patients treated by the multimodality approach, survival ranged from 3 to 7 years following diagnosis (330). INFLAMMATORY MYOFIBROBLASTIC TUMOR Clinical Features Inflammatory myofibroblastic tumor (IMT) is a tumor composed of neoplastic spindled to epithelioid myofibroblasts admixed with lymphocytes, plasma cells, and eosinophils. Common sites for IMT are lung and the soft tissues of the abdominal cavity, including mesentery, omentum, and retroperitoneum (331). Liver involvement by IMT is relatively rare (300). Historically, the terms IMT and inflammatory pseudotumor were used interchangeably, but with advances in knowledge, IMT (a neoplastic process) is now known to be a different entity than an inflammatory pseudotumor (a benign reactive process). The demographics for IMT vary considerably between studies, but there is a definite peak in children (332) and, based on SEER data, possibly second and third peaks in the 30’s and in persons >50 years of age (331). IMTs have no gender predilection (331) and, when present in the liver, almost always represent spread from other organs. Patients with IMT can present with a wide variety of symptoms, such as recurrent fever and weight loss. IMTs are genetically heterogeneous lesions but in children and young adults contain clonal rearrangements resulting in ALK gene fusions. In such cases, ALK oncoproteins may be identified in the tumor with immunohistochemistry directed against ALK-1. Pathologic Findings and Differential Diagnosis IMTs are solitary in the majority of cases and are well circumscribed, firm, and tan to yellow-white, with a size ranging from 1 cm to more than 20 cm. A solid and whorled quality is seen on cut section; areas of necrosis or degenerative myxoid change may be noted. Microscopically, IMT is characterized by spindled fibroblastic/myofibroblastic spindle cells intermixed with an inflammatory infiltrate of plasma cells, lymphocytes, and eosinophils The spindled stromal cells of IMT demonstrate immunohistochemical and ultrastructural characteristics of myofibroblasts. The cells are immunoreactive for smooth muscle actin, muscle-specific actin, desmin, and vimentin (333). In addition, most IMTs show immunoreactivity for ALK protein. The differential diagnosis includes follicular dendritic cell sarcomas and inflammatory pseudotumors. Follicular dendritic cell sarcomas are often Epstein-Barr virus (EBV) positive and show immunoreactivity for CD21 and CD35 (334). In contrast to IMT, the mean age for inflammatory pseudotumors is around 50 years (332), there is a male predominance, and most cases are isolated to the liver.

METASTATIC TUMOR IN THE LIVER SOLID TUMORS Clinical Features Metastatic tumors are the most common malignant tumors found in the liver. Modes of metastatic spread include hematogenous (most common), lymphatic, or through peritoneal fluid. It is important to note that malignant tumors found in cirrhotic livers are most commonly HCC (335), but metastases still occur to cirrhotic livers, primarily from tumors within the GI tract. Patients with liver metastases can show signs and symptoms referable to the liver, including hepatomegaly, right upper quadrant abdominal pain, anorexia, and weight loss. Biliary obstruction and acute hepatic failure rarely occur. Metastasis from a carcinoid tumor results in the classic carcinoid syndrome, including flushing, diarrhea, and palpitations. Liver enzymes are frequently elevated in cases of metastatic disease. The most common sites of origin for metastatic tumors found in the liver are colon, breast, esophagus, lung, pancreas, and neuroendocrine (336,337). Lymphoma and sarcoma may involve the liver, although less frequently than carcinoma. Treatment modalities for metastatic tumors are dependent on the histologic type. Chemotherapeutic options are generally first-line in patients with metastatic disease. Hepatic arterial embolization and chemoembolization can also be used. Surgical resection of isolated liver metastases is employed with increasing frequency, especially in cases of metastatic colorectal carcinoma. Metastatic welldifferentiated NETs are also treated with arterial embolization, surgical resection, or even hepatic transplantation. Pathologic Findings Grossly, many metastatic tumors present as multiple masses of varying sizes and are present in both liver lobes. The tumor nodules may be quite large and fill the liver. Areas of necrosis within the tumor can cause a cystic appearance on imaging. Metastatic colorectal carcinoma often causes scarring and retraction that can lead to an umbilicated appearance grossly. The tumor nodules often show extensive necrosis following chemotherapy. Squamous cell carcinomas are typically firm, white, and granular; mucinous carcinomas show a soft, glistening cut surface, and melanomas may be brown-black. The characteristic “fish flesh” appearance can be seen in metastatic sarcomas. On histologic examination, the metastatic tumors are typically discrete nodules with histological findings similar to the primary. Rare growth patterns include sinusoidal growth, where tumor cells grow within the sinusoids, a pattern most commonly seen with breast carcinoma, pancreatic adenocarcinoma, and melanoma (75). Metastatic adenocarcinoma can also rarely grow within bile ducts, simulating a primary CC; the most common primary site for this pattern is the colon, but also includes the stomach, pancreas, and breast (338). Metastatic NETs and melanoma may grow in a trabecular pattern, which mimics HCC. The possibility of metastasis should always be entertained when evaluating a core needle biopsy and immunohistochemical markers can be chosen in the context of the clinical situation. On H&E examination, a primary cholangiocarcinoma is indistinguishable from metastatic adenocarcinoma from the extrahepatic biliary tree and pancreas, but most cholangiocarcinomas will be ALB-ISH positive (80%-90%) while most metastatic adenocarcinomas are negative. Of note, metastatic acinar cell carcinoma of the pancreas is positive for ALB-ISH in about 25% of cases (339), but will also be trypsin positive, while cholangiocarcinomas are trypsin negative. Clinical history and imaging are obviously essential in the evaluation of primary sites. The use of immunohistochemical markers may be necessary if the primary cancer is not available for histologic review, if there is no known site or origin, or if the patient has multiple potential primary sites. A panel of markers is more helpful than single immunohistochemical studies and should be individualized for the case based on the tumor morphology, clinical history, and differential diagnosis. LEUKEMIA AND LYMPHOMA

Clinical Features Involvement of the liver by disseminated lymphoma is seen in approximately 5% to 10% of Hodgkin lymphomas and 15% to 40% of non-Hodgkin lymphomas at the time of diagnosis (340). Leukemia commonly involves the liver but is only rarely associated with signs and symptoms of hepatic dysfunction (341) and is only rarely biopsied. Pathologic Findings Large cell lymphomas most often appear as solitary or multiple bulky, well-defined, white-tan masses (as described in section “Primary Hepatic Lymphoma” earlier). Hodgkin lymphoma may manifest as large, bulky masses or as multiple small nodules in a diffuse pattern (“miliary”). Leukemia most commonly causes hepatomegaly in the absence of discrete masses. Hodgkin lymphoma may show variable histologic appearances; the most common pattern consists of portal-based, enlarged, atypical mononuclear cells with prominent nucleoli in a polymorphous inflammatory cell background. Diagnostic Reed-Sternberg cells are rarely seen. Large cell lymphomas may cause bile duct damage and lead to prominent cholestasis and ductopenia. Langerhans cell histiocytosis may involve the liver and manifests as both portal-based and parenchymal nodules with diffuse infiltration of sinusoids. Langerhans cells are characteristically positive for S-100 and CD1a. Generalized mastocytosis has also been associated with hepatic involvement, leading to hepatomegaly and noncirrhotic portal hypertension (342). The tumor cells are most often portal-based but may show sinusoidal infiltration. Mast cells are often difficult to identify on H&E examination; immunohistochemical stains for CD117 (c-kit) or mast cell tryptase are useful for identifying the tumor and extent of disease. Portal fibrosis, bridging fibrosis, and cirrhosis may be seen; in addition, veno-occlusive disease and NRH have been described, consistent with the clinical history of noncirrhotic portal hypertension (343). Extramedullary hematopoiesis is commonly seen in generalized mastocytosis. Myeloid leukemia is seen histologically as a diffusely infiltrative sinusoidal disease. In contrast, lymphoid leukemias tend to be portal tract centered. Hairy cell leukemia involves the liver in a majority of cases. Biopsies are not part of routine clinical management, but on histologic examination, tumor cells can involve both portal tracts and sinusoids (344,345). The tumor cells can form small cavities lined by the tumor cells and containing blood (“angiomatoid” lesions; Fig. 37.38) (346).

FIGURE 37.38 Hairy cell leukemia, angiomatoid lesion. The sinusoids are congested and are focally lined by round to slightly indented lymphoid cells (“angiomatoid” foci). Note the “beading” pattern and separation of the tumor cells in the adjacent sinusoids. Photograph courtesy of Scott Saul, MD, Chester County Hospital, West Chester, PA.

Granulomas may be identified in any of the earlier mentioned diseases, most commonly in hairy cell leukemia and Hodgkin lymphoma. The differential diagnosis for granulomas in the liver disease is extensive, and the possibility of lymphoma or leukemia must always be considered.

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Kim MJ, Lee S, An C. Problematic lesions in cirrhotic liver mimicking hepatocellular carcinoma. Eur Radiol. 2019;29:5101-5110. Torbenson M, Schirmacher P. Liver cancer biopsy—back to the future? Hepatology. 2015;61:431-433. Chen QW, Cheng CS, Chen H, et al. Effectiveness and complications of ultrasound guided fine needle aspiration for primary liver cancer in a Chinese population with serum alpha-fetoprotein levels 20 cores) to rule

out a peripheral zone cancer, and diagnostic transurethral resection to rule out a transition zone malignancy (102). The false-negative rate with core biopsies of the prostate ranges from 12% to 19% (103). These figures vary depending on the type of biopsy performed, degree of palpable abnormality, and number of biopsies performed. Specimens should be considered unsatisfactory when there is only minimal or no prostatic glandular or stromal tissue. Cores of prostatic tissue containing only stroma are not necessarily unsatisfactory specimens and may represent biopsy of a predominantly stromal nodule of hyperplasia. Tumor seeding along the needle track has not been seen with the thin Biopty gun needles, in contrast to prior reports seen with larger perineal needle biopsies. Adenocarcinoma of the prostate also may be diagnosed in the workup for metastatic carcinoma of unknown origin. The importance of identifying these tumors as being of prostatic origin is that even widespread metastatic prostate carcinoma may be hormonally responsive, and treatment may lead to dramatic and sometimes long-term symptomatic relief. Because the bones are a common site of presentation for prostate cancer, IHC stains for prostatic markers should be performed on all cases of metastatic adenocarcinoma to the bone in men without known primaries. Prostate carcinoma also shows a tendency to metastasize to left-sided cervical or other supradiaphragmatic lymph nodes, often as the first manifestation of prostate cancer (104). These cancers often are poorly differentiated and may not be suggestive of prostate carcinoma histologically. Furthermore, patients may have normal rectal examinations and an absence of metastatic bone disease. Because some poorly differentiated prostate adenocarcinomas are negative for PSA, NKX3.1 and P501S (prostein) should also be used in the workup of a poorly differentiated carcinoma with the differential diagnosis of adenocarcinoma of the prostate versus urothelial carcinoma (105,106). Pathologists also should be attuned to prostate carcinoma biopsied as a rectal mass. In some cases, it is the initial manifestation of prostate cancer. In others, it represents prostate cancer recurring several years after diagnosis, such that a new

colonic primary is suspected (107). Endoscopically, the lesion may be indistinguishable from a colonic primary, and in some cases, serum PSA levels may be low. Most cases are poorly differentiated histologically with solid nests, individual cells, microacinar, or cribriform formation. IHC reactivity with prostatic markers is expected. Although typically adenocarcinoma of the prostate is CDX2 negative, uncommonly, they can be diffusely positive (108). Alpha-methylacyl-CoA racemase (AMACR) is not a prostate-specific marker and is positive in urothelial and colonic carcinoma. Metastatic prostate cancer to the lung typically occurs in the setting of advanced prostate cancer with multiple small pulmonary nodules or diffuse lymphatic spread rather than a single large metastatic lung deposit; the first manifestation of prostate cancer would not be expected to be seen on a lung biopsy. GLEASON GRADING OF NEEDLE BIOPSIES The Gleason system is based on the glandular pattern, where cytology is not factored (Figs. 45.14, 45.15, 45.16, 45.17, 45.18, 45.19, 45.20) (109-112). Based on architecture, Gleason’s originally assigned patterns 1 to 5, with 1 being the most differentiated and 5 being undifferentiated. In the original Gleason system, the most common and second most common grades were combined. In 2005, the Gleason system was updated and modified with one change being that, on biopsy, the most common and highest grade patterns on a given core are added to result in the Gleason score (112,113). If a tumor has only one histologic pattern, then for uniformity, both patterns are given the same grade. Although in theory, the Gleason scores range from 2 (1 + 1 = 2), which represents tumors uniformly composed of Gleason pattern 1 tumor, to 10 (5 + 5 = 10), which represents totally undifferentiated tumors, Gleason patterns 1 and 2 are not assigned in current practice (114).

FIGURE 45.14 Schematic diagram of the Gleason grading system.

FIGURE 45.15 Gleason pattern 1 tumor composed of a circumscribed nodule of uniform, single, separate, closely packed glands.

FIGURE 45.16 In Gleason pattern 2, although the tumor is still fairly circumscribed, at the edge of the tumor nodule, there can be minimal extension by neoplastic glands into the surrounding nonneoplastic prostate. The glands in pattern 2 are still single and separate, yet they are more loosely arranged and not quite as uniform as in pattern 1.

FIGURE 45.17 Gleason pattern 3 tumor infiltrates in and among the nonneoplastic prostate, with the glands having marked variation in size and shape. Many of the glands are smaller than those seen in pattern 1 or 2 tumors.

FIGURE 45.18 Gleason pattern 4 cancer with large cribriform glands.

FIGURE 45.19 The Gleason pattern 5 tumor shows no glandular differentiation with either solid masses of cells or individually infiltrating cells.

FIGURE 45.20 Gleason pattern 5 with central comedonecrosis.

A more contemporary grouping of Gleason scores based on differing prognoses is Gleason scores less than or equal to 6; 3 + 4 = 7; 4 + 3 = 7; 8; 9 to 10, which reflects Grade Groups 1-5, respectively (115,116). It is reasonable to assign a full Gleason score even to small foci of cancer on needle biopsy because it has been demonstrated that the grade assigned to these minimal cancers is just as accurate compared with cases with more extensive cancer on biopsy (117). Gleason patterns 1-3 cancer consists of variably sized individual glands that are well-formed. In contrast to Gleason pattern 4, the glands in Gleason pattern 3 are discrete units (118). If one can mentally draw a circle around well-formed individual glands, then it is Gleason pattern 3. One should assign a Gleason score at relatively low power (i.e., 4× or 10× objective). The presence of a few poorly formed glands at high power, which could represent a tangential section off of small well-formed glands, is still consistent with Gleason pattern 3 tumor. There are certain situations that lead to overgrading of Gleason pattern 3 as pattern 4. Crowded glands at low magnification can have the appearance of fused glands, mimicking Gleason pattern 4 cancer (118). Small glands are

acceptable for Gleason pattern 3 as long as they are well-formed and not fused with other glands. The delicate ingrowth of fibrous tissue seen with mucinous fibroplasia (collagenous micronodules) can result in glands appearing to be fused resembling cribriform structures, although the underlying architecture is really that of individual discrete rounded glands invested by loose collagen. When glands surround a nerve (perineural invasion), the glands often develop a more complex papillary, crowded appearance; one should be cautious in diagnosing Gleason pattern 4 based on glands within perineural invasion unless they are overt cribriform glands. Branching glands appear more complex than simple round glands, yet as long as they are not fused or cribriform, branching glands are still consistent with Gleason pattern 3. Gleason pattern 4 is diagnosed in the presence of cribriform glands, fused glands, and ill-defined glands with poorly formed glandular lumina (119,120). There is growing data from many studies that cribriform Gleason pattern 4 is associated with a worse prognosis than poorly formed or fused glands (121-127). Only when there is a cluster of poorly formed glands, where a tangential section of Gleason pattern 3 glands cannot account for the histology, should the focus be graded as Gleason pattern 4 (118). On needle biopsy, cribriform Gleason pattern 4 tumor often manifests as fragments of cribriform carcinoma as there is little supporting stroma in larger cribriform glands. Glomerulations represent an early stage of cribriform pattern 4 cancer and should be assigned Gleason pattern 4 (128). Gleason pattern 5 consists of sheets of tumor, individual cells, and cords of cells. Less commonly, there are nests of cells. Solid nests of cells with vague microacinar or only occasional gland space formation are still consistent with Gleason pattern 5. A relatively uncommon morphology is comedonecrosis with solid nests. Occasionally, one can see necrosis with cribriform masses that by themselves might be cribriform pattern 4; the consensus is that these patterns should be regarded as Gleason pattern 5 (113). Most cases with comedonecrosis represent intraductal carcinoma as opposed to invasive Gleason pattern 5 carcinoma (129). There is a

tendency for pathologists to undergrade Gleason pattern 5 (130,131). One of the most frequent causes of discordant grading is the assessment of tumors that bridge two grades. As shown on Gleason’s schematic diagram (Fig. 45.14), there is a continuum of differentiation between the various Gleason patterns such that the grade assigned at the extremes of a particular Gleason pattern may be somewhat subjective. Interobserver reproducibility studies of Gleason grading have highlighted these problem areas (132,133). The Gleason score on biopsy material has been shown to correlate fairly well with that of the subsequent radical prostatectomy (134). In general, a Gleason score of 6 on biopsy corresponds to a Gleason score less than or equal to 6 in the radical prostatectomy in about 70% of cases. An unavoidable cause of discrepant grading between the biopsy and subsequent prostatectomy specimen is that caused by sampling error by the needle biopsy. The following factors are associated with upgrading from the needle biopsy to the radical prostatectomy: increased cancer extent on biopsy; increased serum PSA levels, smaller prostates; and fewer cores sampling the prostate (135). When different cores have different Gleason scores, pathologists should report the grades of each core separately as long as the cores are submitted in separate containers or the cores are in the same container, yet specified by the urologist as to their location (i.e., by different color inks) (113). If different cores with different grades are present within the same specimen container without a designation as to site, a single grade should be assigned as if they were one long core. High-grade tumor of any quantity on needle biopsy should be included within the Gleason score. Consequently, a needle biopsy with 98% Gleason pattern 3 and 2% Gleason pattern 4 should be graded as Gleason score 3 + 4 = 7. In all specimens, in the setting of high-grade cancer, one should ignore lower-grade patterns if they occupy less than 5% of the area of the tumor (113). For example, tumor composed of 98% Gleason pattern 4 and 2% Gleason pattern 3 should be graded as Gleason score 4 + 4 = 8. One of the major outcomes of the 2014 Consensus Conference was

the recommendation to report percent pattern 4 with Gleason score 7 (Grade Groups 2-3) in both needle biopsies and radical prostatectomy specimens (120,136). The Genitourinary Pathology Society (GUPS) consensus paper on grading prostate cancer gives details as to the rationale of reporting percent pattern 4 and how to record this parameter (137). Over a 2- to 3-year period, the grade of prostate cancer on needle biopsy does not tend to change (138). The Gleason grading system is one of the more powerful prognostic indicators in prostate cancer. Gleason score correlates with all important pathologic parameters seen in the radical prostatectomy specimen, with prognosis after radical prostatectomy and with outcome following radiotherapy (118). The major shift in terms of the likelihood of having adverse findings in the prostatectomy or with failure following prostatectomy or radiotherapy is between a Gleason score of 6 and 7 (Figs. 45.21 and 45.22). The importance of grade is evidenced by the use of various nomograms using preoperative variables such as Gleason score, clinical stage, serum PSA, and, in a more recent study, the extent of cancer on biopsy to predict pathologic stage, post–radical prostatectomy progression, and postradiotherapy failure (94,139-142). Grade on biopsy can influence whether definitive treatment is given (active surveillance vs. surgery or radiation), what type of treatment (surgery vs. radiation), and decisions within a therapy (brachytherapy vs. external beam therapy and whether and for how long to get hormone therapy with radiation; whether to resect the neurovascular bundle[s] at radical prostatectomy; whether to remove lymph nodes at surgery).

FIGURE 45.21 Adenocarcinoma of the prostate, Gleason grade 3 + 3 = 6 on needle biopsy. Note small glands infiltrating in and among larger benign glands.

FIGURE 45.22 Adenocarcinoma, Gleason grade 3 + 4 = 7. Note well-formed glands consistent with Gleason pattern 3 (lower right) compared with fused glands of Gleason pattern 4 (upper left).

EXTENT OF CANCER, PERINEURAL INVASION, AND EXTRAPROSTATIC EXTENSION ON NEEDLE BIOPSY

Multiple techniques of quantifying the amount of cancer found on needle biopsy have been developed and studied, including measurement of (a) the number of positive cores, (b) the percentage of positive cores, (c) total millimeters of cancer among all cores, (d) percentage of each core occupied by cancer, and (e) total percentage of cancer in the entire specimen. There are multiple studies claiming superiority of one technique over the other, with no one method being clearly superior (143). When cancer discontinuously involves a core with intervening benign prostate tissue, studies support an end-to-end measurement be provided (144-147). In order that the report accurately reflects the nature of the cancer, we report such cases as “discontinuously involving X% of the length of the core.” Pathologists should report the number of cores containing cancer as well as one other system quantifying tumor extent per core. There should be precise labeling of the initial biopsies according to sextant site to localize the sites of an initial atypical diagnosis and to direct the location of repeat biopsies, because increased sampling of the initial atypical sextant site and adjacent sextant sites increases the yield of cancer detection on repeat biopsy (148). Although the majority of stage T1c cancers are significant tumors warranting definitive therapy, approximately 25% of these tumors detected by needle biopsy are thought to be “insignificant” tumors. Although limited cancer on the biopsy by itself is not predictive, a combination of relatively low PSA values and minimal findings of cancer on needle biopsy can help predict preoperatively which patients may have insignificant tumors and which may be candidates for conservative therapy (149). Extensive cancer on biopsy cores on systematic prostate biopsy is a powerful predictor of adverse pathologic findings at radical prostatectomy. In about 5% of radical prostatectomy specimens, minute cancer will be found and, in about 3% of cases, no tumor will initially be seen in the entirely submitted specimen. A methodical, limited, targeted approach to identifying cancer can identify cancer in 73% of the cases with no initial cancer. After reviewing the biopsy, our protocol for looking for tumor in the radical prostatectomy with no initial tumor is:

1. Perform immunostains on any suspicious foci. 2. Perform levels on blocks with high-grade prostatic intraepithelial neoplasia (HGPIN). 3. Perform three levels on the sextant and adjacent sextant region of the prostate where the cancer was identified on biopsy. 4. Flip the blocks in these regions and perform three additional levels. In only a rare case when no tumor is found in the prostate is it the result of a switch in needle biopsy specimens. Rather, the explanation is minute cancer sampled on needle biopsy that is not identified despite completed submission of the prostate for analysis (150). The presence of perineural invasion on biopsy is associated with higher risk of biochemical recurrence in prostate cancer patients following radical prostatectomy or radiotherapy (151). There is a stronger linkage of perineural invasion in needle core tissue and response to radiation therapy, compared to surgery (152-156). Perineural invasion should be noted on the biopsy pathology report. However, it is not necessary to report for each part, but its presence or absence can be reported for the entire case. The diagnosis of extraprostatic extension on needle biopsy can be established when tumor is seen admixed with adipose tissue on needle biopsy. Although rarely one can see intraprostatic adipose tissue, the likelihood of cancer involving intraprostatic adipose tissue is so rare that one can assume the adipose tissue in this situation is extraprostatic. Extraprostatic extension on core needle biopsy of the prostate is typically seen with extensive high-grade prostatic adenocarcinoma, such that its usefulness as an isolated prognostic factor is relatively limited (157). DIAGNOSIS OF LIMITED PROSTATE CANCER Misdiagnosis of clinically relevant features on prostate biopsy can be minimized with histologic review of three levels per tissue core (158). One of the most common problems with the evaluation of needle

biopsy material from the prostate is the diagnosis of carcinoma as a result of the limited amount of tumor present (159,160). Commonly, biopsies of adenocarcinoma of the prostate yield several cores of tissue containing benign glands and stroma, with only a few neoplastic glands insinuating themselves within the benign tissue. Evaluating an atypical focus in a needle biopsy of the prostate should be a methodical process. When reviewing needle biopsies, one should develop a mental balance sheet where on one side of the column are features favoring the diagnosis of carcinoma and on the other side of the column are features against the diagnosis of cancer (Table 45.1). A definitive diagnosis can be made if all of the criteria are listed on one side of the column or the other at the end of evaluating a case. The diagnosis of cancer should be based on a constellation of features rather than relying on any one criterion by itself. TABLE 45.1 Features Favoring and against the Diagnosis of Prostate Cancer on Needle Biopsy Favoring Cancer

Against Cancer

Prominent nucleoli Enlarged nuclei Nuclear hyperchromasia Mitotic figures Amphophilic cytoplasm Sharp luminal border Apoptotic bodies Blue-tinged mucinous secretions Pink amorphous secretions Crystalloids Mucinous fibroplasia Perineural invasion Glomerulations

Atrophic features Larger glands with undulations (r/o HGPIN) Small, crowded glands merge in with benign glands (r/o adenosis) Corpora amylacea Inflammation Small atypical glands next to HGPIN (r/o PINATYP)

HGPIN, high-grade prostatic intraepithelial neoplasia; PINATYP, HGPIN with possible tangential sections or outpouchings; r/o, rule out.

The recognition of prostatic cancer in these cases rests on both the architectural abnormalities resulting from the infiltrating neoplastic glands and the cytologic features of the neoplastic epithelium. In order to identify limited amounts of cancer on needle biopsy material, one must first identify the normal nonneoplastic prostate and then look for glands that do not fit in. Although most prostates are relatively similar in their histologic appearance, some contain numerous small foci of crowded glands similar to adenosis. In such a case, the diagnosis of cancer based on a small focus of crowded glands with minimal cytologic atypia should be performed with caution. Other men’s prostate glands are characterized by widespread atrophy; one should, in these cases, hesitate to diagnose cancer if the atypical glands have scant cytoplasm. Architectural patterns suspicious for carcinoma are (a) crowded glands, (b) small glands situated between larger benign glands, and (c) a row of glands going across the core (Figs. 45.23 and 45.24).

FIGURE 45.23 Small focus of prostate carcinoma infiltrating between nonneoplastic glands.

FIGURE 45.24 Adenocarcinoma of the prostate, Gleason grade 3 + 3 = 6. Infiltrative appearance among benign glands is diagnostic of cancer.

In most cases of limited cancer on needle biopsy, there are cytologic differences in the malignant glands when compared with surrounding benign glands (Fig. 45.25). Although the finding of prominent nucleoli in the small glands is reassuring, it is not necessary to diagnose carcinoma. If one does not see nucleoli in the majority of prostate cancers sampled on needle biopsy fixed in formalin, careful attention to microtomy and staining can improve the situation; sections that are too thick or overstained result in hyperchromatic nuclei without visible nucleoli. Often, only significant nuclear enlargement or nuclear hyperchromasia discriminates cancer from the surrounding benign glands. Other nuclear features favoring the diagnosis of cancer are mitotic figures and apoptotic bodies (161). In addition to differentiating nuclear features, neoplastic glands may have amphophilic cytoplasm contrasted with the pale-to-clear cytoplasm of adjacent benign glands (Fig. 45.26). Intraluminal pink, acellular, dense secretions; blue-tinged mucinous secretions; and crystalloids seen in small atypical glands may be additional features that help to differentiate malignant from benign glands, given their greater frequency in malignancy (Figs. 45.27 and 45.28). Prostatic crystalloids are dense, eosinophilic, crystalloid structures that appear in various geometric shapes (162). If the differential diagnosis is between adenosis (see section “Mimickers of

Prostate Cancer”) and low-grade adenocarcinoma (i.e., a lobular collection of crowded glands), the presence of crystalloids is not helpful. If the focus consists of small, atypical glands infiltrating between larger, benign glands, in which adenosis is not in the differential, then the findings of crystalloids can help to establish a malignant diagnosis. Crystalloids also may be seen in scattered benign glands on needle biopsy, where there is no increased likelihood of cancer on repeated biopsy (163).

FIGURE 45.25 Neoplastic glands composed of a single layer of cells with enlarged nuclei, some with prominent nucleoli.

FIGURE 45.26 Small glands of infiltrating adenocarcinoma with amphophilic cytoplasm noted between larger, pale-staining, benign glands.

FIGURE 45.27 Adenocarcinoma of the prostate with blue-tinged mucinous secretions seen on H&E-stained section.

FIGURE 45.28 Adenocarcinoma with small glands with enlarged nuclei, crystalloids, and mitotic figure. Adjacent benign prostate glands contain lipofuscin pigment.

Although nuclear features play a prominent role in the diagnosis of adenocarcinoma of the prostate on needle biopsy material, they may not be as helpful in diagnosing low-grade adenocarcinoma on TURP specimens. Often, low-grade adenocarcinomas of the prostate lack enlarged nuclei and prominent nucleoli, and mitoses are rarely found. Cytoplasmic features are also often not very helpful, because they are often pale-clear, similar to benign glands. The most useful feature in diagnosing low-grade adenocarcinoma on TURP is the recognition of cancer’s architectural growth pattern as seen at relatively low magnification (Fig. 45.29). Benign prostatic glands tend to grow either as circumscribed nodules within BPH or radiate in columns out from the urethra in a linear fashion. In contrast, adenocarcinoma of the prostate grows in a haphazard fashion.

FIGURE 45.29 Adenocarcinoma of the prostate with an infiltrative appearance. Glands infiltrate at right angles to each other.

Some cancers closely resemble benign prostate glands in their architectural pattern or cytologic features and may not be recognized as malignant. Atrophic adenocarcinomas mimic benign atrophy and are distinguished by (a) an infiltrative pattern of growth; (b) the presence of macronucleoli (Fig. 45.30); and (c) the presence of adjacent, nonatrophic cancer (Fig. 45.31) (164,165). Another unusual pattern of adenocarcinoma seen on needle biopsy that may be underdiagnosed involves cancers with voluminous xanthomatousappearing cytoplasm in which nuclei are small and often show no or minimal atypia, termed foamy gland carcinoma; intraluminal pink homogeneous secretions are often present (Fig. 45.32) (166). A third pattern of prostatic adenocarcinoma that may resemble benign glands is called pseudohyperplastic adenocarcinoma (167,168). Pseudohyperplastic prostate cancer is characterized by the presence of larger glands, often with branching and papillary infolding (Fig. 45.33). The recognition of cancer with this pattern is based on the architectural pattern of numerous closely packed glands as well as nuclear features more typical of carcinoma. A

variant of pseudohyperplastic adenocarcinoma is composed of numerous large glands that are almost back-to-back with straight, even luminal borders and abundant cytoplasm (Fig. 45.34). Comparably sized benign glands have papillary infolding, undulations, or ruffling of their luminal border or atrophic cytoplasm. The use of basal cell–specific keratin antibodies or p63 is often essential for the diagnosis of these cancers that mimic benign glands. In rare cases, carcinoma may mimic crushed stroma or inflammation. The identification of this tissue as epithelial and of prostatic origin by the use of prostate-specific markers and keratins may help to establish the diagnosis of prostatic carcinoma.

FIGURE 45.30 Atrophic adenocarcinoma of the prostate with very prominent nucleoli.

FIGURE 45.31 Atrophic adenocarcinoma of the prostate (left) merging with more typical adenocarcinoma of the prostate (right).

FIGURE 45.32 Adenocarcinoma of the prostate with abundant xanthomatous cytoplasm; small nuclei; and pink, homogeneous, intraluminal secretions. Compare cytoplasm with more typical adenocarcinoma (right).

FIGURE 45.33 Pseudohyperplastic adenocarcinoma where cancerous glands are relatively large and have papillary infolding, architecturally resembling benign glands or high-grade prostatic intraepithelial neoplasia (HGPIN). The glands show prominent nucleoli, yet are too crowded to represent HGPIN. Stains for highmolecular-weight cytokeratin were also negative in the entire focus, in support of the diagnosis of cancer.

FIGURE 45.34 Low-grade adenocarcinoma showing large, back-to-back glands in which the luminal surfaces have an even, straight edge without papillary infolding and cells have abundant cytoplasm. Stains for high-molecular-weight cytokeratin were negative in these glands.

There are four features that have not been identified in benign glands to date and that are in and of themselves diagnostic of cancer. These are perineural invasion, mucinous fibroplasia (collagenous micronodules), glomerulations, and mucin extravasation (Figs. 45.35 to 45.36, 45.37) (169). Although perineural indentation by benign prostatic glands has been reported, the glands in these cases appear totally benign and are present at only one edge of the nerve or are intraneural rather than circumferentially involving the perineural space, as can be seen in carcinoma (Fig. 45.38) (170).

FIGURE 45.35 Adenocarcinoma of the prostate with perineural invasion. Occasionally, glands with perineural invasion will show papillary infolding, mimicking a benign gland.

FIGURE 45.36 Collagenous nodules with mucin undergoing organization to delicate, loose fibrosis.

FIGURE 45.37 Glomerulations with adenocarcinoma showing cribriform formation that is not transluminal, resembling glomeruli.

FIGURE 45.38 Perineural indentation by benign prostate gland.

FINDINGS OF ATYPICAL GLANDS SUSPICIOUS FOR CANCER: IMMUNOHISTOCHEMICAL ADJUNCTS IN THE DIAGNOSIS OF

PROSTATE CANCER When certain features are present on needle biopsy that favor the diagnosis of prostate cancer but there are insufficient features for a definitive malignant diagnosis, a report of “atypical, suspicious for carcinoma” may be rendered (Table 45.1). The incidence of atypical glands on needle biopsy is approximately 5% (171). Approximately 50% of cases with an atypical diagnosis have cancer on repeat biopsy (148,171). However, some urologists consider Gleason score 6 (Grade Group 1) cancer following an atypical diagnosis to be insignificant, which would not need therapy. On average, 25% of the cancers detected on repeat biopsy after an atypical diagnosis are higher than Grade Group 1 (172-178). Even in the patients who are found to have Grade Group 1 cancer on repeat biopsy, an advantage of finding the cancer is that these men can be followed on active surveillance with one-third having reclassification to higher-grade cancer on follow-up biopsies. Patients with an initial atypical biopsy should be counseled on the probability of harboring clinically significant and insignificant prostate cancer. A shared decision on whether to do a repeat biopsy taking into consideration the patient’s age, overall health, patient preference, along with potentially factoring in newer serum or urine markers and imaging. If there are numerous atypical glands that are negative for basal cell–specific markers, a diagnosis of carcinoma may be rendered (Fig. 45.39) (5,9,11,12). Because benign prostate glands show some heterogeneity in staining with these antibodies, negative staining for basal cell–specific markers in only a few glands suggestive of cancer is not proof of their malignancy. In cases of prostate cancer mimickers, positive basal cell–specific marker staining may definitively label a focus as benign. Uncommonly, prostate adenocarcinoma tumor cells show cross-reactive staining for highmolecular-weight cytokeratin, yet not in a basal cell distribution. Exceedingly rarely, unequivocal cancer on hematoxylin and eosin (H&E)–stained tissue sections reveals focal basal cell staining (179). Rare carcinomas, often having a distinctive morphology consisting of glands, nests, and cords with atrophic cytoplasm, hyperchromatic

nuclei, and visible nucleoli, have diffuse aberrant p63 positivity but negative staining for high-molecular-weight cytokeratin (180-182).

FIGURE 45.39 High-molecular-weight cytokeratin stain of Figure 45.23 showing lack of basal cells in small glands, consistent with adenocarcinoma.

Another IHC marker that is helpful in diagnosing limited adenocarcinoma on prostate needle biopsy is AMACR. Approximately 80% of small foci of prostate cancer on biopsy are positive for AMACR, where benign glands are usually negative (183). In addition, a high proportion of HGPIN and some foci of adenosis and partial atrophy also are positive for the marker. When the pathologist favors a diagnosis of cancer on H&E stained sections and stains for basal cells are negative, positive staining for AMACR can provide additional confidence to establish a definitive malignant diagnosis (Fig. 45.40) (184-188).

FIGURE 45.40 Adenocarcinoma of the prostate with strong immunoreactivity for racemase. Note adjacent benign glands, which are nonreactive.

MIMICKERS OF PROSTATE CANCER Fully atrophic glands stand out at scanning magnification because of their open lumina lined by cells with crowded nuclei and scant apical and lateral cytoplasm, resulting in a very basophilic appearance to the glands (Fig. 45.41). When there is an increased number of crowded atrophic glands, the term postatrophic hyperplasia (PAH) is used (189,190). Within the center of these small, atrophic glands, there may be seen a dilated gland surrounded by fibrosis. These small glands, despite their atrophic appearance, have an increased proliferation rate (191). In contrast, gland-forming adenocarcinomas at low magnification usually appear pale or amphophilic, with cells showing abundant cytoplasm and basally situated nuclei

(Fig. 45.42). Small glands with partial atrophy may be crowded and have a disorganized infiltrative appearance and be misdiagnosed as carcinoma (192,193). In partial atrophy, the small glands have paleto-clear attenuated cytoplasm with small, somewhat crinkly nuclei without prominent nucleoli (Fig. 45.43). Although the apical cytoplasm in partial atrophy is attenuated, the nuclei in partial atrophy are more spaced apart with more laterally placed cytoplasm, giving rise to a pale appearance at low magnification in contrast to PAH. Basal cell–specific cytokeratin staining may be positive in a patchy fashion or can be negative in a small focus of partial atrophy. AMACR may also be positive, such that the staining pattern of negative basal cells and positive AMACR can be confused with cancer.

FIGURE 45.41 Needle biopsy showing sclerotic atrophy with an infiltrative appearance.

FIGURE 45.42 Benign prostatic atrophy (left) compared with adenocarcinoma with amphophilic cytoplasm (right). Even compared with an amphophilic-appearing carcinoma, atrophy appears more basophilic at low power.

FIGURE 45.43 Needle biopsy showing glands with partial atrophy.

Another potential source of overdiagnosing prostatic adenocarcinoma is the presence of seminal vesicles or histologically similar ejaculatory duct epithelium on needle biopsy. A common finding on needle biopsy of the seminal vesicle is to see at the tip or edge of the core of tissue an irregular row of glandular epithelium that represents the lining of the central dilated seminal vesicle lumen, because the core of tissue fractures at this interface (Fig. 45.44). Surrounding this lumen may be numerous small, glandular diverticula of the seminal vesicle, which may be confused with carcinoma. The presence of prominent lipofuscin granules within seminal vesicle epithelium is an important diagnostic aid, although it should be recognized that normal prostate glands also may demonstrate lipofuscin pigment with refractile red-brown granules corresponding to lysosomes (8). In addition, seminal vesicles characteristically have scattered cells showing prominent nuclear atypia, although the atypia appears degenerative in nature (194) (Fig. 45.45). Seminal vesicle–type epithelium is negative for prostate-specific markers and has a basal cell layer identified with high-molecular-weight cytokeratin and p63 staining.

FIGURE 45.44 Needle biopsy of seminal vesicles showing multiple small glands arranged around a central lumen.

FIGURE 45.45 Scattered markedly atypical nuclei with a degenerative appearance, characteristic of seminal vesicle epithelium. Prominent lipofuscin pigment noted.

One of the greatest difficulties with TUR material, also seen in approximately 1% of needle biopsy specimens, is the distinction of adenocarcinoma from adenosis (Table 45.2) (atypical adenomatous hyperplasia) (Figs. 45.46, 45.47, 45.48, 45.49, 45.50, 45.51) (195198). Adenosis at low magnification is composed of numerous crowded, small, pale-staining glands that resemble a nodule of lowgrade adenocarcinoma of the prostate. Whereas the glands in adenosis have a lobular configuration, the glands in adenocarcinoma are arranged in a haphazard array, with glands often appearing to infiltrate into the stroma at right angles to each other. In adenosis, there is a gradual transition between the small glands with pale-toclear cytoplasm and adjacent, more recognizably benign glands; some of the architectural features seen in the benign glands include branching, larger, more irregular shapes, and papillary infolding. Typically, the nucleoli are unapparent in adenosis, although on occasion, they may be focally prominent (197). Features that do not differentiate adenosis from low-grade adenocarcinoma are (a) backto-back crowded glands; (b) intraluminal crystalloids; (c) medium-

sized nucleoli; (d) scattered, poorly formed glands and single cells; and (e) minimal infiltration at the periphery of the nodule. The use of basal cell markers can help diagnose adenosis, especially when there are some atypical features. Within a nodule of adenosis, there are scattered glands, often very few, with positive staining of basal cells with high-molecular-weight cytokeratin or p63. The staining is typically patchy within a positive gland, sometimes consisting of only a few flattened, immunoreactive basal cells. Adenosis tends to be located centrally within the gland, such that it is most commonly seen in TUR specimens. In these specimens, where there is an entire nodule of adenosis to evaluate, it is highly unlikely for the entire focus to show no immunoreactivity with basal cell–specific cytokeratin. Consequently, the lack of basal cell–specific cytokeratin staining in a nodule of glands with a differential diagnosis of adenosis and low-grade adenocarcinoma is supportive of the diagnosis of adenocarcinoma. There is no evidence that patients with adenosis have an increased risk of adenocarcinoma, either at the time of diagnosis or in the future (199).

FIGURE 45.46 Low magnification of adenosis. Lesion is very lobular with only minimal infiltration at perimeter. Small, crowded glands mimicking cancer merge with more typically benign glands (left).

FIGURE 45.47 Adenosis showing lobular collection of crowded glands. Note benign glands with papillary infolding admixed with small, crowded glands showing similar nuclear and cytoplasmic features.

FIGURE 45.48 Higher magnification of adenosis showing glands with pale-toclear cytoplasm, relatively benign-appearing nuclei, and a focally identifiable basal cell layer.

FIGURE 45.49 High-molecular-weight cytokeratin immunostaining demonstrates a patchy basal cell layer surrounding some of the glands within a nodule of adenosis.

FIGURE 45.50 Crowded glands of adenosis on needle biopsy.

FIGURE 45.51 Adenosis on needle biopsy showing crowded glands with pale-toclear cytoplasm and benign-appearing nuclei, which are similar to adjacent more recognizably benign glands (left). Stains for high-molecular-weight cytokeratin were positive in this case, demonstrating basal cells.

TABLE 45.2 Diagnostic Criteria for Adenosis Adenosis

Adenocarcinoma

Lobular growth

May be infiltrative/haphazard

Small, crowded glands admixed with larger glands

May be pure population of small crowded glands

Huge nucleoli absent

Occasionally huge nucleoli present

Small glands share cytoplasmic and nuclear features

Small glands differ from surrounding benign glands with admixed larger, benign glands

Pale-to-clear cytoplasm

May have amphophilic cytoplasm

Blue-tinged mucinous secretions (rare)

Blue-tinged mucinous secretions (common)

Corpora amylacea (common)

Corpora amylacea (rare)

Occasional glands with basal cells

Basal cells absent

Basal cell–specific antibodies label basal cells in some small glands

Small glands are not immunoreactive with basal cell–specific antibodies

A variant of adenosis that may be confused with intermediate- to high-grade adenocarcinoma of the prostate is sclerosing adenosis of the prostate (200). Sclerosing adenosis consists of a small, relatively localized focus of glands that resembles ordinary adenosis, which merge with cytologically similar cords and individual cells (Fig. 45.52). Nucleoli may be fairly prominent. A thick hyaline basement membrane–like sheath invests some of the glands. Between the glands and individual epithelial cells, there is a cellular spindle cell component, in contrast to prostatic carcinoma, which typically lacks a cellular stromal reaction. Immunohistochemistry with actin and S-100 demonstrates that there is myoepithelial cell differentiation to the basal cells around the glands in sclerosing adenosis and in the spindle cells between the glands, which differs from basal cells in other prostate conditions, which lack myoepithelial cell differentiation (201,202).

FIGURE 45.52 Sclerosing adenosis showing glands of adenosis (left) merging with cytologically similar cords and spindle cells (right).

Lesions that may be misdiagnosed as Gleason scores 2 to 6 and Gleason scores ≥7 adenocarcinoma are listed in Tables 45.3 and 45.4, respectively (Figs. 45.53, 45.54, 45.55) (40,190,192,194196,200,203-217).

FIGURE 45.53 Cowper gland on needle biopsy. Note dimorphic population with duct surrounded by mucinous glands. The lesion is present in skeletal muscle.

FIGURE 45.54 Verumontanum mucosal gland hyperplasia consisting of crowded glands beneath urethra (top). Glands contain distinctive orange-red concretions and corpora amylacea.

FIGURE 45.55 Xanthoma of the prostate.

TABLE 45.3 Benign Mimickers of Gleason Score 2 to 6 Adenocarcinoma Entity

Predominant Mode of Sampling

Atrophy

TURP = Needle

Cowper glands

TURP = Needle

Radiation atypia

TURP = Needle

Adenosis

TURP > Needle

Basal cell hyperplasia

TURP > Needle

Nephrogenic adenoma

TURP >> Needle

Seminal vesicles

Needle > TURP

Verumontanum hyperplasia

Needle > TURP

Mesonephric hyperplasia

TURP

Colonic mucosa

Needle

TURP, transurethral resection of the prostate.

TABLE 45.4 Benign Mimickers of Gleason Score 7 to 10 Adenocarcinoma Entity

Predominant Mode of Sampling

Nonspecific granulomatous prostatitis

TURP = Needle

Paraganglia

TURP = Needle

Clear cell cribriform hyperplasia

TURP > Needle

Sclerosing adenosis

TURP >> Needle

Xanthoma

TURP > Needle

Signet ring cell lymphocytes

TURP

TURP, transurethral resection of the prostate.

PROSTATIC INTRAEPITHELIAL NEOPLASIA Prostatic intraepithelial neoplasia (PIN) consists of architecturally benign prostatic acini or ducts lined by cytologically atypical cells and is subclassified into low-grade PIN (LGPIN) and high-grade PIN (HGPIN) (Figs. 45.56, 45.57, 45.58) (228-220). The distinction between LGPIN and HGPIN is the finding of prominent nucleoli in HGPIN. Diagnostic reports should not comment on LGPIN. First, pathologists cannot reproducibly distinguish between LGPIN and benign prostate tissue. Second, when LGPIN is diagnosed on needle biopsy, these patients are at no greater risk of having carcinoma on repeated biopsy than are men with a benign biopsy finding (171). These authors threshold for diagnosing HGPIN is whether prominent nucleoli can be visualized using the 20X lens (200X magnification) (221). The average incidence of HGPIN reported on needle biopsy is

4% to 5% (171). Abundant evidence demonstrates that HGPIN is a precursor lesion to some carcinomas of the prostate, especially those in the peripheral zone (222-229).

FIGURE 45.56 Low-grade prostatic intraepithelial neoplasia (LGPIN) with slight enlargement of nuclei and stratification, yet no prominent nucleoli.

FIGURE 45.57 Low magnification of HGPIN, showing glands with a normal architectural pattern, yet a basophilic appearance.

FIGURE 45.58 Basophilia of gland with HGPIN resulting from nuclear stratification and high nuclear-to-cytoplasmic ratio with large nuclei and prominent nucleoli. Note in left portion of field within a single gland the abrupt transition between benign-appearing nuclei and PIN nuclei.

Although HGPIN is characterized by nuclear atypia, often, there are accompanying architectural abnormalities. At low magnification, glands are separated by a modest amount of stroma and have a normal overall architectural pattern. These glands resemble benign glands in that they are large, branched, and have papillary and undulating luminal surfaces. At low magnification, glands with HGPIN have a basophilic appearance resulting from a combination of features, including enlarged nuclei, hyperchromasia, overlapping nuclei, amphophilic cytoplasm, and epithelial hyperplasia (Figs. 45.57 and 45.58). The earliest form of HGPIN is characterized by nuclear atypia within epithelium without epithelial hyperplasia (i.e., flat pattern) (230). Tufting HGPIN is the most common pattern, consisting of slight mounds of hyperplastic, cytologically atypical epithelium in a preexisting benign gland. With more pronounced

forms of HGPIN, nuclei project into the lumen as micropapillary PIN, composed of tall epithelial buds lacking fibrovascular cores. Lesions in the past referred to as cribriform HGPIN currently are diagnosed as either Atypical Intraductal Proliferation (AIP) or Intraductal carcinoma of the prostate (IDC-P), depending on whether there is greater than 50% epithelium relative to lumina (Figs. 45.59 and 45.60). In HGPIN, the nuclei toward the center of the gland tend to have a blander cytologic appearance compared to nuclei peripherally located up against the basement membrane. The grade of PIN is assigned based on assessment of the nuclei peripherally located up against the basement membrane (Fig. 45.59). Unusual subtypes of HGPIN include cases with signet ring, small cell neuroendocrine, mucinous, foamy, and inverted nuclear features (231-233).

FIGURE 45.59 Prominent tall papillary tufts within HGPIN. Nuclei appear more benign toward the luminal surface of the papillary projections. Note large nuclei with frequent nucleoli, diagnostic of HGPIN, toward the edge of the gland up against the basement membrane.

FIGURE 45.60 Atypical intraductal proliferation.

DIFFERENTIAL DIAGNOSIS PIN must be differentiated from variants of infiltrating carcinoma as well as several benign entities. Cytologically, HGPIN may be indistinguishable from infiltrating acinar carcinoma. The diagnosis of infiltrating carcinoma is made when the cytologically atypical glands are too small or crowded, in contrast to HGPIN, which occurs when benign glands are replaced by cytologically atypical cells. In contrast to PIN, ductal adenocarcinomas have papillary formations with fibrovascular cores or show prominent cribriform patterns often with extensive comedonecrosis. A very close mimicker of PIN is PIN-like ductal adenocarcinoma (see “Variants of Prostatic Adenocarcinoma”). In some cases, HGPIN is surrounded by a few small atypical glands (PINATYP). It may be difficult to distinguish between outpouchings or tangential sections of HGPIN as opposed to PIN with associated infiltrating carcinoma. As opposed to PIN with associated infiltrating carcinoma. The greater distance of the small atypical glands away from the HGPIN and the greater number of small crowded atypical glands favor the diagnosis of infiltrating carcinoma associated with HGPIN (Figs. 45.61 and 45.62) (234).

Most of these cases will require immunohistochemistry for basal cell markers, where cancer should be diagnosed only when there is a large cluster of entirely negative glands. Even a few small glands with a patchy basal cell layer result in a diagnosis of PINATYP.

FIGURE 45.61 HGPIN (left) with only a few atypical small glands. We cannot distinguish between PIN and adjacent infiltrating carcinoma versus outpouchings or tangential sections off of HGPIN.

FIGURE 45.62 HGPIN (left) with adjacent small atypical glands. The number of small atypical glands and their greater distance away from the HGPIN is diagnostic of infiltrating carcinoma with adjacent HGPIN.

Basal cell hyperplasia may mimic HGPIN as basal cell hyperplasia may show prominent nucleoli along with mitotic activity (207,208) (Table 45.5). TABLE 45.5 Differential Diagnosis: High-Grade Prostatic Intraepithelial Neoplasia and Basal Cell Hyperplasia with Nucleoli Basal Cell Hyperplasia

HGPIN

Often small, crowded glands

Large glands without crowding

Occasional solid nests

Glands with well-formed lumina

Basal cells with atypical nuclei (blue) undermine secretory cells with benign nuclei (red/violet)

No two distinct cell populations

Basal cell nuclei stream parallel to basement membrane

Atypical cells oriented perpendicular to basement membrane

Atypical nuclei positive for basal cell markers

Atypical nuclei negative for basal cell markers; underlying flattened, benign-appearing cells positive

HGPIN, high-grade intraepithelial neoplasia.

It is recommended that men do not need a routine repeat needle biopsy within the first year following the diagnosis of HGPIN on a single core as it is not associated with an increased risk of cancer on rebiopsy relative to men with a benign diagnosis on the initial biopsy (171). In men with two or more cores containing HGPIN, there is a higher risk of cancer on rebiopsy, comparable to men with an atypical focus on biopsy (235,236). The significance of HGPIN on TUR is not clear, with conflicting data as to the risk for subsequent discovery of cancer (237-239). In an elderly patient with HGPIN on TUR, often, no further workup is instituted. In a younger man, a more aggressive workup to rule out a clinically significant tumor is warranted. If HGPIN is found on TUR and the specimen has not been put through entirely, the remainder should be processed to look for infiltrating carcinoma. IDC-P is an atypical glandular lesion that spans the entire lumen of prostatic ducts or acini while the normal architecture of ducts or acini is still maintained. It exceeds in its atypia from HGPIN either by its architectural or cytologic features (Table 45.6). In the vast majority of cases, IDC-P is thought to represent extension of high-grade invasive carcinoma into ducts and acini. IDC-P is almost always associated with adjacent invasive, typically high-grade, adenocarcinoma of the prostate (240-245). Rarely, IDC-P may be identified in radical prostatectomy specimens without adjacent highgrade carcinoma, such that in these cases, it represents a highgrade precursor lesion (245-247). IDC-P also may be rarely identified on biopsy material in the absence of infiltrating carcinoma or only with Gleason score 3 + 3 = 6 cancer (246,248,249). Guo and Epstein’s stringent criteria are highly specific for the diagnosis of IDC-P since they recommend that definitive therapy be performed for this diagnosis even in the absence of invasive carcinoma (248). As a

result of the high specificity, the sensitivity of these criteria for the diagnosis of IDC-P is lower and does not encompass the entire morphologic spectrum of IDC-P. A significant proportion of atypical intraductal proliferations (AIP) may exhibit “low-grade” features that fall short of Guo and Epstein's criteria (245,250-252). In cases that are borderline between IDC-P and HGPIN (i.e., AIP) and in the absence of invasive carcinoma, repeat biopsy is warranted. The differential diagnosis includes cribriform clear cell hyperplasia, which lacks the cytologic atypia seen in IDC-P (Fig. 45.63) (214). Glands within the central zone at the base of the prostate also mimic IDC-P, because they may show Roman bridge and cribriform patterns and are lined by tall, pseudostratified epithelium, yet also lack cytologic atypia (Fig. 45.64) (253). Infiltrating cribriform acinar adenocarcinoma (Gleason pattern 4 or Gleason pattern 5 with comedonecrosis) closely mimics cribriform IDC-P. It is not necessary to perform basal cell immunostains on biopsy and radical prostatectomy to identify IDC-P if the results will not change the overall (highest) grade per case (137). The Genitourinary Pathology Society (GUPS) recommends not to include IDC-P in determining the final Gleason Score/Grade Group on biopsy and/or radical prostatectomy (137).

FIGURE 45.63 Clear cell cribriform hyperplasia showing cribriform glands with clear cytoplasm, benign-appearing nuclei, and a well-defined basal cell layer surrounding many of the glands.

FIGURE 45.64 Central zone with Roman bridge and cribriform formation. Note lack of nuclear atypia.

TABLE 45.6 Intraductal Carcinoma of the Prostate Malignant epithelial cells filling large acini and prostatic ducts, with preservation of basal cells and: Solid or dense cribriform pattern Or Loose cribriform or micropapillary pattern with either: Bizarre nuclear atypia Comedonecrosis

CARCINOMA: CLINICAL STAGES T2 (PALPABLE) AND T1C (NONPALPABLE) CANCER DETECTED ON NEEDLE BIOPSY METHODS OF PROCESSING RADICAL PROSTATECTOMY SPECIMENS In institutions that do not totally embed radical prostatectomy specimens, these authors recommend two methods to sample the prostate, depending on whether gross tumor is visible (254-256). In both methods, routine sections include (a) amputation of the distal (apical) 1 cm of the prostate and serial section of this specimen parallel to the urethra (Fig. 45.65); (b) either a thin shave of the proximal (base) margins or amputation of the base analogous to that described for the apex; (c) base of seminal vesicles at the juncture with the prostate; and (d) right and left vas deferens margins. If there is grossly visible tumor, sections are submitted in which gross tumor is identified (Fig. 45.66). If no visible tumor is seen, the posterior sections are submitted along with one midanterior section from each side. No additional sections are required if this midanterior section shows no or only minimal tumor. If these sections demonstrate sizable tumor, then one should go back to the wet tissue and submit the anterior sections on the ipsilateral side, because it indicates a

large transition zone tumor component where anterior positive margins or anterior extraprostatic extension could be present.

FIGURE 45.65 Handling of the apical margin. (A-C) The distal 1 cm is amputated, inked to designate left versus right, and then serially sectioned parallel to the urethra.

FIGURE 45.66 Gross appearance of adenocarcinoma of the prostate. Note more solid, homogeneous, gray-white appearance to carcinoma (left) as compared with contralateral benign prostate with a spongier appearance.

Whole-mount sectioning of the prostate provides more esthetically pleasing sections for teaching and publications, yet the information obtained by using routine sections is identical; in a small minority of cases, routinely processed thinly sliced sections identify positive margins not identified by using thicker slices of tissue, which is necessary for whole-mount processing. Whole-mount sections facilitate defining the anatomic location of the tumor(s). TUMOR LOCATION Most clinical stage T2 adenocarcinomas of the prostate show predominantly peripherally located tumor in the posterior and posterolateral regions of the prostate (257,258). In a few cases, large transition zone tumors may extend peripherally and be palpated and diagnosed on needle biopsy. Only when a peripheral zone tumor becomes very large does it extend into the transition zone. Stage T1c cancers also are predominantly located peripherally, although 15% have the tumor predominantly within the transition zone (149,259). High-grade transition zone cancers compared to high-grade peripheral zone cancers have higher tumor volumes, increased positive surgical margins, but lower incidence of intraductal carcinoma, seminal vesicle invasion, lymph node involvement, and biochemical failure after radical prostatectomy (260). Bladder neck/anterior margin positivity is more common in transition zone tumors, and positive posterolateral margins are more common in peripheral zone cancers. Multifocal adenocarcinoma of the prostate is present in more than 85% of prostates (257). TUMOR PROGRESSION The term progression is used throughout this section, unless otherwise specified, to indicate an elevated postoperative PSA serum level (>0.2 ng/mL). One-third of men with PSA progression develop metastatic disease at a median interval of 8 years from the time of PSA level elevation (median follow-up: 5 years) (261). EXTRAPROSTATIC EXTENSION AND PERINEURAL INVASION

The prostatic edge is not well defined histologically (262). In some areas, there may appear to be a fibrous or fibromuscular band at the edge of the prostate, although in other areas, normal prostatic glands extend out to the edge of the prostate without any appearance that there is a capsule (Fig. 45.67). In most areas, there appears to be no apparent histologic barrier between the prostatic and periprostatic tissue. Nevertheless, commonly, there is a wall of prostatic carcinoma along the edge of the gland without extension into the adjacent soft tissue, demonstrating that the edge of the prostate physiologically acts as a fairly effective barrier (Fig. 45.68). Because the prostate lacks a discrete capsule, the term extraprostatic extension is recommended to denote non–organconfined disease. Because of the lack of a well-defined prostatic capsule, the term “capsular invasion” should not be used. Assessment for extraprostatic extension is dependent on location in the prostate (263). Peripheral zone carcinomas most frequently exhibit extraprostatic extension posterolaterally or posteriorly. At these sites, direct extension or spread along nerves into periprostatic adipose tissue is an indicator of extraprostatic extension. However, it is not necessary to see tumor in fat to diagnose extraprostatic extension. Posterior, posterolaterally, and laterally if tumor extends beyond the condensed smooth muscle of the prostate into loose connective tissue or into associated dense desmoplastic stromal reaction that is also extraprostatic extension. Extensive tumor may also manifest as a bulge into periprostatic tissue (264). To diagnose extraprostatic extension anteriorly requires identification of tumor invading adipose tissue, as the boundary of the prostate in this region is not well-defined. Similarly, the border of the prostate at the apex is ill-defined where benign prostate glands are admixed with skeletal muscle. The consensus is that tumor in skeletal muscle without benign glands at the inked distal apical perpendicular margin should be reported as organ-confined disease (pT2x) (265). Microscopic invasion into the urinary bladder neck should be considered pT3a disease. In radical prostatectomy specimens, perineural invasion need not be reported as it does not predict recurrence after surgery or survival (266).The degree of

extraprostatic extension varies from only a few glands outside the prostate, which is termed focal extraprostatic extension, to cases with more extensive extraprostatic spread, which is designated as nonfocal extraprostatic extension (Figs. 45.69 and 45.70). The degree of extraprostatic extension correlates with risk of progression after radical prostatectomy (264,267). Other more objective measurements of quantifying the extent of extraprostatic extension have been proposed, but they correlate with the subjective “focal” versus “nonfocal” dichotomy; none have been proven sufficiently superior to recommend as the preferred method (263,268).

FIGURE 45.67 Edge of the prostate toward the base of the gland. Benign glands appear to extend out of the prostate with no well-defined prostatic capsule.

FIGURE 45.68 Adenocarcinoma of the prostate extending to the edge of the prostate but not into periprostatic tissue.

FIGURE 45.69 Focal extraprostatic extension with only a few neoplastic glands situated exterior to the edge of the prostate.

FIGURE 45.70 Nonfocal extraprostatic extension with multiple neoplastic glands exterior to the prostate. Glands show prominent perineural invasion. Margins are still negative, because tumor does not extend to the inked edge of the gland.

SEMINAL VESICLE INVASION Although both seminal vesicle invasion and extraprostatic extension are pathologic stage T3 disease, seminal vesicle invasion is a much more dire prognostic finding, with a 65% 5-year progression rate after surgery (264,269). The most common route of seminal vesicle invasion is through spread of the tumor out of the gland at the base, with growth and extension into periseminal vesicle soft tissue and eventually into the seminal vesicles. Less commonly, there may be direct extension through the ejaculatory ducts into the seminal vesicles or direct extension from the base of the prostate into the wall of the seminal vesicles. Examining the base of the seminal vesicles is the best way to identify seminal vesicle invasion, because this is always the first region to be invaded by carcinoma. Seminal vesicle invasion should be diagnosed when tumor invades the muscular coat of the seminal vesicle (Fig. 45.71).

FIGURE 45.71 Adenocarcinoma of the prostate invading the seminal vesicles.

LYMPH NODE METASTASES The most common site of initial metastatic spread of prostatic carcinoma is the regional pelvic lymph nodes. Progression-free survival at 5 years after radical prostatectomy with positive pelvic lymph nodes has been reported to range from 27% to 74% and from 11% to 64% at 7 and 10 years after surgery, respectively (270,271). Cancer-specific survival at 10 years has ranged from 47% to 86% (270,272-274). Pelvic lymph nodes examined at the time of radical prostatectomy may contain foamy xanthomatous histiocytes. These may be seen as a reaction to a hip prosthesis or in men with nodal metastases who have received preoperative hormonal therapy (275,276). RADICAL PROSTATECTOMY GRADE One should assign a separate Gleason score to each dominant tumor nodule(s) (113). Most often, the dominant nodule is the largest tumor, which is also the tumor associated with the highest stage and highest grade. In the unusual occurrence of a nondominant nodule

that is the highest grade focus within the prostate, the grade of this smaller nodule should also be recorded. Associated small foci of Gleason score 2 to 6 cancers that often coexist with dominant tumor nodules need not be graded or mentioned in the report. GUPS recommends replacing “tertiary grade pattern” with the term “minor tertiary pattern 5,” to be used only in cases with Grade Groups 2 or 3 (Gleason score 3 + 4 = 7 or 4 + 3 = 7) with less than or equal to 5% Gleason pattern 5 in a radical prostatectomy (137). In a dominant tumor nodule with Gleason patterns 3, 4, and 5, and where Gleason pattern 5 is the least amount but exceeds 5% of the cancer, it should be considered as the secondary grade and it should be incorporated into the final Gleason score. As there is no consensus whether a minor tertiary pattern 5 worsens the prognosis to the next highestGrade Group, GUPS currently recommends that the Grade Group is not changed in this setting (137). Minor tertiary pattern 5 should be noted along with the Gleason score, and the Grade Group based on the Gleason score [i.e., Gleason score 3 + 4 = 7 (Grade Group 2) with minor tertiary pattern 5 or Gleason score 4 + 3 = 7 (Grade Group 3) with minor tertiary pattern 5]. The Gleason score at radical prostatectomy correlates well with prognosis. The 5-year postoperative biochemical risk free of disease is 96.6%, 88.1%, 69.7%, 63.7%, and 34.5% for Gleason scores less than or equal to 6; 3 + 4 = 7; 4 + 3 = 7; 8; and 9 to 10, respectively (Fig. 45.72) (115,116). Tumors that are pure Gleason score 6 at surgery have no potential for lymph node metastases (277).

FIGURE 45.72 Correlation of Gleason score of cancer in radical prostatectomy specimen with progression after surgery.

MARGINS OF RESECTION Much of the difficulty in assessing the prostatic margins of resection results from the close relation of the prostate to surrounding vital structures, such as the urogenital diaphragm, pelvic sidewall, rectum, and bladder neck (264,278). Consequently, on pathologic examination of radical prostatectomy specimens, there is often only a scant amount of periprostatic tissue. The incidence of positive margins varies according to extent of the cancer and the expertise of the urologist. The mean rate of positive margins in robotic-assisted radical prostatectomy series published between 2008 and 2011 was 15%, ranging from 6.5% to 32% (279). The apex is one of the most frequent sites of a positive margin (280). The value of frozen sections of the distal margin at the time of radical prostatectomy is questionable because often the urologist has taken as much tissue as possible at the site. Removal of additional tissue risks injuring the urogenital diaphragm and increasing the incidence of postoperative incontinence. Also, frozen section at the apex has low sensitivity with many false negatives due

to sampling error (281). When tumor is at the inked margin at the apex, the pathologic stage is pT2x as long as the tumor is organconfined elsewhere. This staging uncertainty reflects that at the apex of the prostate, the boundaries of the prostate are especially vague, where it cannot be determined whether a cancer with a positive margin in this site is intraprostatic or extraprostatic. The proximal margin in radical prostatectomy specimens consists of the bladder neck, which is composed of thick muscle bundles. Positive proximal margins usually correlate with extensive tumor, although the site of positive margin lacks independent prognostic significance (282). Tumor extending to the anterior margin with uncommon exception occurs in the setting of extraprostatic extension, because the surgeon transects this region as far anteriorly away from the prostate as possible. Posterior, posterolateral, and lateral sites account for a sizable proportion of positive margins. They parallel the location of most stage T2 and T1c carcinomas. In assessing these margins of resection, it is critical to first determine whether the tumor is organconfined or has extended out of the prostate. When the tumor is organ-confined, the cleavage plane that the urologist dissects is usually between the prostate and the uninvolved periprostatic adipose tissue, such that in these cases, the outer edge of the prostate has a smooth, rounded contour at low magnification. The prostate is often covered only by a film-like connective tissue layer (Fig. 45.73) that may be unapparent or easily disrupted during intraoperative or postoperative handling of the prostate, leading to the picture seen in Figure 45.74. If the tumor extends just to the inked surface of the prostate (which has a smooth, rounded edge at low magnification), the margins are considered negative for tumor, because the tumor is confined to the prostate and has been removed in its entirety. When tumor extends out of the prostate, the contour of the prostate at scanning magnification has an irregular surface resulting from the associated fibrous response in which extraprostatic tumor adheres to the gland (Fig. 45.70). In these cases with extraprostatic extension, the adequacy of resection depends on whether the tumor extends to the inked margin

(Figs. 45.70 and 45.75). However, even in these cases, if there is only a scant amount of benign soft tissue separating the tumor from the ink, the margin should be considered negative. The preponderance of studies shows that the distance from tumor to ink does not affect the risk of recurrence (283-287). The importance of distinguishing among posterior, posterolateral, and lateral positive margins in a pathology report is that one of the few sites the urologist may modify the surgical procedure is in the posterolateral regions bilaterally in the regions of the neurovascular bundle (Fig. 45.76). The neurovascular bundles run along this aspect of the gland and have been shown to be a major contributor to potency. The surgeon, based on either preoperative or intraoperative factors, can decide whether to spare the neurovascular bundles (i.e., leave them within the patient for potency), at which point the pathologist will find only a scant amount of periprostatic soft tissue in the posterolateral regions of the gland. Alternatively, the surgeon can sacrifice (resect) the entirety or portions of the neurovascular bundle, resulting in an additional approximately 5 mm of soft tissue in this region. In contrast, laterally and posteriorly, the surgeon cannot take additional tissue because of the presence of pelvic sidewall and rectum. In general, most authorities recognize that judicious preservation of one or both of the neurovascular bundles can be performed without compromising removal of cancer. Even when margins are thought to be positive, additional tissue removed from that site does not always show tumor (288,289). A few institutions have reported on performing frozen sections in the area of the neurovascular bundles to determine whether they should be resected, but this is not commonly practiced (288,289).

FIGURE 45.73 Organ-confined adenocarcinoma of the prostate. Edge of the prostate has a smooth, rounded border. Flimsy periprostatic soft tissue may be easily disrupted.

FIGURE 45.74 Adenocarcinoma extending close to the inked edge of the gland, which has a smooth, rounded surface. This margin is negative for tumor and is associated with a low risk of progression.

FIGURE 45.75 Adenocarcinoma of the prostate, which has extended out of the prostate where the surgeon transected tumor, resulting in a shaggy, irregular surface of the gland. The tumor in this region extends to the inked margin and therefore is positive for tumor.

FIGURE 45.76 Whole-mount cross section of prostate showing posterolateral unilaterally resected neurovascular bundle (lower left) and contralateral region posterolaterally where neurovascular bundle has been spared (lower right)

In only approximately 50% of patients with positive margins is there evidence of progression as measured by elevated postoperative serum PSA levels (264). Because of the uncertainty as to whether positive margins always translate into residual tumor left within the patient, many authorities monitor patients who have positive margins until progression is demonstrated before administering adjuvant therapy. Factors that can aid in the clinician’s decision whether to administer adjuvant radiation therapy are the extent of positive margins and the grade of the tumor at the margins (290-295). Positive margins also may arise as a result of the surgical transection of intraprostatic tumor (intraprostatic incision). The reported incidence of intraprostatic incision ranges from 1.3% to as high as 71% (264). This variation may reflect surgeon’s experience as well as difficulties in pathologists recognizing extraprostatic extension. If extraprostatic tumor associated with a fibrous stromal response at a margin is misdiagnosed as organ-confined, it will be misclassified as a positive margin due to intraprostatic incision. The other relatively frequent site of intraprostatic incision is in the regions of the neurovascular bundles, where the urologist tries to preserve the bundle for potency, yet cuts into the prostate. Only if both tumor and benign glands are transected in the same area and are present at the inked margin should one diagnose a positive margin as a result of intraprostatic incision. Intraprostatic incision is associated with an increased risk of postoperative progression equivalent to that associated with focal extraprostatic extension and a positive margin (294). Gleason grade, presence and degree of extraprostatic extension, and margins of resection are all independent predictors of post– radical prostatectomy progression. TUMOR VOLUME Tumor volume in the radical prostatectomy specimen correlates well with pathologic stage and Gleason grade in clinical stage T2 cancers (296,297). Multiple methods of measuring tumor volume have been

proposed (264). Most large series do not demonstrate an independent association with outcome, but other studies support recording tumor volume as a prognostic maker (298-302). Despite the preponderance of evidence showing a lack of independent prognostic significance of tumor size, it has been recommended that an estimate of tumor size be reported for all radical prostatectomy cases, with the weak argument being that tumor volume is given in other organ systems (303,304). It is further recommended that this measure should be a quantitative figure (302). Because tumor volume is not an independent predictor and will in general not affect subsequent therapy, it is these authors’ recommendation that if one feels obliged to report radical prostatectomy tumor volume, the simplest and fastest method should be employed, such as a rough estimate of the overall percentage of the prostate involved by tumor or the maximum diameter of tumor. Although, in general, larger tumors are high grade and small tumors low grade, exceptions occur. There is a tendency to hypothesize that tumors begin as lowgrade tumors and, on reaching a certain size, dedifferentiate into higher grade lesions, accounting for the relation between size and grade. Alternatively, high-grade tumors may be high grade at their inception, yet because of their rapid growth are detected at an advanced size (305). Similarly, low-grade tumors may evolve so slowly that they tend to be detected at lower volumes. VASCULAR INVASION Vascular invasion is uncommonly identified in radical prostatectomy specimens, seen in 7% of tumors smaller than 4 cc (most tumors seen today at radical prostatectomy are 90%) (379-383). However, we have seen rare cases of prostate adenocarcinoma that diffusely

expressed GATA3 (106,377). Less sensitive markers include uroplakin and thrombomodulin (49%-69% sensitivity) and p63 and high-molecular-weight cytokeratin (60%-70% sensitivity) (106). INTRADUCTAL UROTHELIAL CARCINOMA INVOLVING THE PROSTATE Intraductal urothelial carcinoma is seen when urothelial carcinoma extends down into the prostatic ducts and acini in a patient with carcinoma in situ (CIS) of the prostatic urethra and bladder. If identified on TURP or transurethral biopsy, patients usually are recommended for radical cystoprostatectomy, because topical chemotherapy for superficial bladder carcinomas appears to act by direct contact with neoplastic epithelium and does not effectively treat prostatic involvement by urothelial carcinoma. Intraductal urothelial carcinoma ranges from subtle pagetoid spread within the prostatic epithelium to filling and expanding ducts, often with central comedonecrosis (Fig. 45.90). Once resected by cystoprostatectomy, the noninvasive involvement of the prostate by urothelial carcinoma does not adversely affect survival; the prognosis is determined by the stage of the bladder tumor (373,374,384,385). When intraductal urothelial carcinoma is seen on needle biopsy, the prognosis is still poor, reflecting unsampled invasive tumor (375).

FIGURE 45.90 Intraductal urothelial carcinoma with rounded, malignant urothelial nests. Associated stromal invasion characterized by cords and single cells of infiltrating urothelial carcinoma.

In prostates with intraductal urothelial carcinoma and prostatic stromal invasion, the associated bladder tumors tend to be highstage, in which case the already poor prognosis is not affected by the prostatic involvement (373). However, intraductal and infiltrating prostatic urothelial carcinoma also may be associated with low-stage bladder tumors, with a deleterious effect on prognosis (384,385). Urothelial carcinoma involving the prostate is substaged into whether invasive carcinoma invades only the suburethral tissue from the overlying prostatic urethra (pT1) or the prostatic stroma from invasive carcinoma arising from CIS involving prostate acini (pT2) (386,387). It is not clear how to report the staging of both the bladder urothelial carcinoma and prostatic involvement in cystoprostatectomy specimens. These authors report the findings in both the bladder and prostate, assigning separate stages for each organ. Alternatively, one can just report the bladder or prostatic stage, depending on which is higher, and then describe the extent of disease in the other organ. Extensive sampling of the periurethral area in cystoprostatectomy specimens performed for urothelial carcinoma is

necessary to identify and to evaluate the prostate for urothelial carcinoma. With intraductal urothelial carcinoma of the prostate, nests of urothelial carcinoma have the contours and distribution of prostatic ducts and acini, and the stroma lacks a desmoplastic response. Infiltrating urothelial carcinoma is characterized by small cords, nests, or individual cells often with retraction artifact, eliciting a desmoplastic and inflammatory stromal response. PRIMARY UROTHELIAL CARCINOMA Primary urothelial carcinoma of the prostate without bladder involvement is rare (388-392). It is characterized by intraductal growth, almost always accompanied by stromal infiltration. Primary urothelial carcinomas of the prostate tend to infiltrate the bladder neck and surrounding soft tissue such that more than 50% of patients present with tumors extending out of the prostate. In approximately 20% of cases, patients present with distant metastases, bone and liver being the most common sites. In contrast to adenocarcinoma of the prostate, bone metastases usually are osteolytic.

MESENCHYMAL TUMORS Benign soft tissue tumors of the prostate are rare, the most common being leiomyoma. The difficulty in diagnosing a leiomyoma of the prostate is that smooth muscle predominant stromal nodules are often found in prostates with hyperplasia. These stromal nodules lack the well-organized fascicles of a leiomyoma and do not have the other degenerative features commonly seen in leiomyomas, such as hyalinization, necrosis, and calcification. Large, single, symptomatic leiomyomas of the prostate are rare (393). Sarcomas and related proliferative lesions of specialized prostatic stroma are rare. Lesions are classified into stromal tumors of uncertain malignant potential (STUMPs) and prostatic stromal

sarcoma based on the degree of stromal cellularity, presence of mitotic figures, necrosis, and stromal overgrowth (Fig. 45.91) (394,395). There are several different patterns of STUMP: (a) hypercellular stroma with scattered atypical, but degenerativeappearing cells admixed with benign prostatic glands; (b) hypercellular bland stroma with eosinophilic cytoplasm admixed with benign glands; (c) phyllodes pattern; and (d) myxoid stroma (Figs. 45.92 and 45.93). Most recently, a fifth round cell pattern was reported, which is the most subtle, consisting of mildly increased stromal cellularity with rounded nuclei and more eosinophilic cytoplasm (396). Cases can exhibit a mixture of the above patterns. STUMPs are often associated with a wide range and extent of associated epithelial proliferations (397). Although most cases of prostatic STUMP do not behave in an aggressive fashion, occasional cases have recurred rapidly after resection. Cases with mixed STUMP and sarcoma exist, and a few STUMPS have progressed to stromal sarcoma. Although many STUMPs may behave in an indolent fashion, their unpredictability in a minority of cases and the lack of correlation between different histologic patterns of STUMPs and sarcomatous dedifferentiation warrant close follow-up and consideration of definitive resection in younger individuals. STUMPS and stromal sarcomas typically express CD34. They are also reactive for progesterone receptors and, less commonly, estrogen receptors, similar to normal prostatic stroma (398,399). Stromal sarcomas have the potential for metastatic behavior. Small, incidentally discovered prostatic lesions with the structure of fibroadenomas also have been described (400).

FIGURE 45.91 (A,B) Stromal sarcoma of the prostate.

FIGURE 45.92 Stromal proliferation of uncertain malignant potential with appearance of benign phyllodes tumor.

FIGURE 45.93 Stromal proliferation of uncertain malignant potential consisting of scattered atypical, yet degenerative-appearing stromal cells between benign glands.

Sarcomas (excluding those of specialized prostatic stroma) account for 0.1% to 0.2% of all malignant prostatic tumors. Rhabdomyosarcoma is the most frequent mesenchymal tumor within the prostate and is seen almost exclusively in childhood. The distinction of a bladder versus a prostate primary is often not possible such that bladder/prostate rhabdomyosarcomas are evaluated in the literature as a single disease entity (401). The majority of patients present with localized disease, with approximately 15% presenting with metastases. Approximately 10% are alveolar subtype, with the rest embryonal rhabdomyosarcoma. Prostate/bladder rhabdomyosarcomas can be subdivided into low-, intermediate-, and unfavorable-risk prognostic groups (402). Low risk is embryonal subtype either completely resected or with microscopic residual disease. Intermediate risk is either: (1) embryonal with gross residual disease; (2) embryonal in patients less than 10 years old with metastases; or (3) alveolar subtype with no metastases. Unfavorable risk is alveolar with metastases or embryonal in patients greater than 10 years old with metastases. The 5-year survival is

90%, 65% to 75%, 40% to 55% for low-, intermediate-, and unfavorable-risk groups, respectively. Leiomyosarcomas are the most common sarcomas involving the prostate in adults (403,404). The majority of patients are between 40 and 70 years old, although in some series, up to 20% of leiomyosarcomas occurred in young adults or children. After either local excision or resection, the clinical course tends to be characterized by multiple recurrences. Metastases, when present, usually are found in the lung and liver. The average survival is between 3 and 4 years. A spindle cell lesion arising most commonly in the bladder but also in the prostate has been described by a variety of terms in the literature, including pseudosarcomatous fibromyxoid tumor, myofibroblastoma, nodular fasciitis of bladder, pseudosarcomatous myofibroblastic proliferation, inflammatory pseudotumor, and, most recently, inflammatory myofibroblastic tumor (IMT) (405,406). Other rare mesenchymal lesions of the prostate are hemangioma (407), chondroma (408), cartilaginous metaplasia (409), malignant peripheral nerve sheath tumor (410), schwannoma (411), chondrosarcoma (412), synovial sarcoma (413,414), granular cell tumor (415), angiosarcoma (416), neurofibroma (417), solitary fibrous tumor (418), and postradiation sarcomas (419). The one case reported of an osteosarcoma most likely represents a sarcomatoid carcinoma (420). Gastrointestinal stromal tumor (GIST) lesions may clinically present as primary prostatic processes on imaging studies and clinical exam, arising from the rectum or perirectal space with compression of the prostate, where they are sampled on prostatic needle biopsy (421).

BASAL CELL HYPERPLASIA AND CARCINOMA A spectrum of basaloid lesions exists, ranging from hyperplasia to carcinoma. Basal cell lesions are preferentially located in the transition zone and are seen on TURP, although occasionally, they may be found on needle biopsy. Basal cell hyperplasia is

characterized by collections of small, solid nests or tubules of uniform cells similar to those of the normal prostatic basal cells (206209) (Fig. 45.94). Basal cell hyperplasia may reveal focal cribriform and more commonly pseudocribriform glands. Pseudocribriform hyperplasia consists of back-to-back small round glands of basal cell hyperplasia rather than a solid nest of cells with punched-out lumina that characterize true cribriform glands (209). Basal cell hyperplasia is one of the few prostatic entities that contain well-formed lamellar calcifications; carcinomas rarely contain calcifications; when present, they usually consist of fine, calcified grains within central necrosis in high-grade cancers. Another unique feature seen within the cells of basal cell hyperplasia is the presence of intracytoplasmic eosinophilic globules (209). Basal cell hyperplasia may reveal prominent nucleoli, rare mitotic figures, nuclear enlargement, individual cell necrosis, necrotic intraluminal secretions, and bluetinged mucinous secretions (Fig. 45.95) (207,208). Basal cell hyperplasia is distinguished from acinar adenocarcinoma by the multilayering of its nuclei, solid nests, and atrophic cytoplasm. The distinction of cytologically atypical basal cell hyperplasia from HGPIN is outlined in Table 45.5. There is no known association between basal cell hyperplasia showing prominent nucleoli and either acinar adenocarcinoma or basal cell carcinoma. Basal cell hyperplasia may be admixed with ordinary BPH, be present as distinct nodules, or have a diffusely infiltrative appearance. When a well-formed, distinct nodule of basaloid nests is formed, the term basal cell adenoma sometimes is used, although others prefer to consider these lesions more pronounced examples of basal cell hyperplasia. Basal cell hyperplasia can be differentiated from prostatic adenocarcinoma by its immunoreactivity for high-molecular-weight cytokeratin and p63.

FIGURE 45.94 Basal cell hyperplasia characterized by glandular structures with multiple cell layers.

FIGURE 45.95 Basal cell hyperplasia with prominent nucleoli and mitotic figures.

Basal cell carcinomas may have multiple unique patterns not seen with basal cell hyperplasia: (a) large basaloid nests with peripheral

palisading and necrosis, (b) adenoid cystic carcinoma–like, (c) anastomosing basaloid nests and tubules centrally lined by eosinophilic cells, and (d) variably small- to medium-sized nests with irregular shapes (422-425). To diagnose basal cell carcinoma in cases resembling basal cell hyperplasia requires either a desmoplastic stromal response, perineural invasion, necrosis, or widespread infiltration into surrounding tissue (Fig. 45.96). A high Ki67 proliferative index and overexpression of bcl-2 also favor basal cell carcinoma over hyperplasia (425). Basal cell carcinomas of the prostate appear do have relatively low malignant potential, with metastases typically seen in cases composed of solid basaloid nests with necrosis.

FIGURE 45.96 Basaloid carcinoma of the prostate with desmoplastic stromal reaction.

PROSTATIC URETHRAL POLYPS Urethral polyps are benign usually single, polypoid lesions found within the prostatic urethra in and around the verumontanum (426,427). Lesions have been described from adolescence to old age. The surface of urethral polyps often appears papillary, with the

broad papillae lined by urothelium, prostatic epithelial cells, or a combination of both (Fig. 45.97). The cores of the papillary projections contain prostatic stroma and prostatic glands.

FIGURE 45.97 Prostatic urethral polyp lined by urothelium and prostatic epithelium, with its core containing benign prostatic glands.

Other benign polypoid lesions found in the prostatic urethra that should be distinguished from urethral polyps include nephrogenic adenomas; papillary pseudotumors of the prostatic urethra composed of large, edematous, urethral mucosal folds, and fibroepithelial polyps (210,428).

HEMATOLOGIC MALIGNANCIES Hematologic malignancies can involve the prostate or pelvic lymph nodes in 1.2% of radical prostatectomy specimens, as well as on biopsy or TURP (429,430). Primary prostatic lymphoma without lymph node involvement appears to be much less common than secondary infiltration of the prostate (431). Most reported lymphomas have been of the large cell and small-cleaved-cell types

with a diffuse pattern. The entire spectrum of malignant lymphomas seen at other sites may become manifest in the prostate (160). Malignant lymphoma involving the prostate is associated with a poor prognosis regardless of patient age, stage of presentation, or treatment regimen. The poor prognosis of prostatic lymphoma is related to the generalized disease that eventually results rather than to the prostatic involvement. The one exception to the poor prognosis associated with prostatic lymphoma is small lymphocytic lymphoma/chronic lymphocytic leukemia, where the prognosis is not affected by the prostatic involvement. The most common form of leukemic involvement of the prostate is that of chronic lymphocytic leukemia, although other types also have been described in the prostate (432,433). Most patients have known leukemia or have the diagnosis established at the time of workup for urinary symptoms. It is often unclear whether the prostatic leukemic infiltrate in chronic lymphocytic leukemia is an incidental finding in patients with BPH or is the cause of their obstructive symptoms.

MISCELLANEOUS MALIGNANT PROSTATIC TUMORS Sarcomatoid carcinoma (carcinosarcoma) is a rare type of prostatic cancer, where both the malignant epithelial and spindle cell components are clonally related (434). The vast majority of patients have a known treatment history of acinar adenocarcinoma treated by external beam radiation, brachytherapy, and/or hormone therapy (mean interval 6.8 years) (435). The epithelial component is either moderately or poorly differentiated adenocarcinoma, usually acinar but occasionally ductal, small cell, adenosquamous, or basal cell carcinoma. The spindle cell component commonly is undifferentiated or shows a wide range of specific mesenchymal differentiation. Typically, the spindle component is negative for prostate markers, although it may react with antibodies to keratin (436). Sarcomatoid carcinomas have a dismal prognosis with a relatively better

prognosis for the minority of tumors that are clinically not invading the bladder and not metastatic at diagnosis (435,437). Excluding hematopoietic neoplasms, even at autopsy, the prostate is rarely involved by metastatic tumor. Metastases from malignant melanoma and carcinoma of the lung predominate (438). Other malignant tumors of the prostate include reports of a malignant mixed tumor resembling that seen in the salivary gland (439), endodermal sinus tumor (yolk sac tumor) (440), seminoma (441), teratoma (442), malignant mixed germ cell tumor (443), rhabdoid tumor (444), papillary cystadenocarcinoma (445), tubulocystic clear cell adenocarcinoma as seen in the female genital tract (446), renaltype clear cell carcinoma (447), ectomesenchymoma with rhabdomyosarcoma and ganglioneuroma (448), peripheral neuroectodermal tumor (PNET) (449), malignant perivascular epithelioid cell tumor (PECOMA) (450), and Wilms tumor (451). Prostate adenocarcinomas have also been described with lymphoepithelioma-like (452), pleomorphic giant cell (476,453) oncocytic features (454), and a plasmacytoid variant (455). Rare case reports of malignant melanoma of primary prostatic origin have been reported (49,50,456).

DIAGNOSTIC MOLECULAR BIOMARKERS IN PROSTATE CANCER The section below focuses on tissue-based molecular tests for risk stratification that are currently in use clinically or likely to be in use in the near future. COMMERCIAL GENOMIC ASSAYS Several commercial genomic tests seek to provide individualized risk stratification based on an analysis of the prostate cancer on needle biopsy core to aid in clinical decision-making and treatment selection (457-464). The most common RNA-based prognostic tests for tumor tissue that have been used to aid in treatment selection (i.e.,

definitive treatment vs. active surveillance) are OncotypeDX GPS, Prolaris, and Decipher. An additional test, Confirm MDx, predicts risk of subsequent cancer diagnosis using a negative prostate biopsy. Whether any of these tests are being utilized, and if so which one, varies depending on the treating clinician and/or institution. PTEN LOSS Outside of commercial genomic assays, one marker that has been validated to have prognostic utility in clinical practice in the active surveillance setting is immunohistochemistry for PTEN. PTEN is the most commonly deleted tumor suppressor gene in primary prostate cancer and its loss is associated with poor clinical outcomes (465469). Additional studies may elucidate a potential role for PTEN in selecting patients for active surveillance for some men with Gleason score 3 + 4 = 7 disease (Grade Group 2) (470,471). DNA DAMAGE REPAIR GENES Alterations in homologous recombination DNA repair pathway, which includes mutations in the BRCA2, BRCA1 and ATM genes, are found in up to 20% of metastatic castration-resistant prostate cancer. Furthermore, in 10% of such patients, the homologous recombination DNA repair pathway alterations are germline in nature (472). The dramatic enrichment for these alterations in metastatic compared to primary prostate cancer supports their association with the development of aggressive disease (473-475). Moreover, in aggressive histologic subsets of primary prostate cancer (including ductal, cribriform, tumors with intraductal prostate cancer and Grade Group 5 tumors) the prevalence of homologous recombination DNA repair pathway mutations may approach that seen in metastatic disease (476,477). Defects in genes involved in the mismatch repair (MMR) pathway are seen in up to 10% of castration-resistant prostate cancer cases, compared to less than 3% of primary tumors (478). MMR mutations are also enriched among primary tumors with aggressive histology, including ductal and Grade Group 5 tumors (476). Compared to homologous recombination DNA repair pathway

alterations, fewer of the MMR mutations in prostate cancer are germline, though prostate cancer is enriched among patients with Lynch Syndrome (479). The above-mentioned findings in DNA repair genes have important implications for genetic counseling and therapeutic decision-making in prostate cancer patients. Currently, the NCCN guidelines recommend germline testing in all patients with metastatic disease and high-risk clinically localized prostate cancer (Grade Group 4 or 5 tumors) (480). Given the promising treatment response with poly-ADP ribosylase (PARP) inhibitors in prostate cancers with homologous recombination DNA repair pathway alterations and the recent FDA approval of pembrolizumab treatment for advanced tumors with MMR defects, metastatic prostate cancers should undergo genomic sequencing to detect somatic mutations in DNA repair genes(478). TMPRSS2-ERG GENE FUSIONS Fusions between the androgen-regulated transmembrane protease serine 2 gene (TMPRSS2) and the ERG gene is present in approximately 40% to 50% of prostate adenocarcinomas (481). This gene fusion is highly specific for prostate cancer, with the exception that 16% to 20% of HGPIN also show the gene fusion. TMPRSS2ERG gene fusions monoclonal anti-ERG antibodies are available that correlate well with fusion-positive cancer (481-484). The major limitation of ERG as a diagnostic test is its low sensitivity, such that a negative stain does not exclude prostate carcinoma. Another weakness of this marker is that in 16% to 28% of cancers, there is heterogeneous or weak ERG expression, further contributing to false-negative staining on biopsy (485-489). We have not adopted ERG immunostaining in our routine workup of atypical foci.

SEMINAL VESICLES Seminal vesicle cysts may be congenital or they may be acquired as a result of obstruction resulting from inflammation (490). Both types

of seminal vesicle cysts are usually unilateral and unilocular and histologically resemble dilated seminal vesicles lined by either flattened or papillary epithelium. Seminal vesicle cysts are usually found in men in their 20s. Not infrequently, ipsilateral renal agenesis is identified (491); in some cases, ectopic ureters may enter the cysts (492). Seminal vesicle cystadenomas are found incidentally in elderly men (493). The lesions are multilocular and are lined by a single layer of columnar or cuboidal epithelium. The cyst walls contain a nondescript fibromuscular stroma, which may be inflamed. Primary carcinoma of the seminal vesicles exhibit a wide range of microscopic patterns. Microscopically, tumors have nonspecific histologic features with papillary, trabecular, and glandular patterns and varying degrees of differentiation (494-497). Rarely, tumors may be undifferentiated or may produce abundant extracellular mucin (colloid carcinoma). The diagnosis requires exclusion of secondary involvement by carcinomas of the prostate, bladder, or rectum based on clinical information and immunohistochemistry. Normal seminal vesicles and seminal vesicle adenocarcinomas are positive for carcinoembryonic antigen (CEA), CK7, and PAX8 while negative for PSA, prostatic acid phosphatase, and CK20. Most patients present with metastases, and in 95% of patients, the survival is less than 3 years. Benign mesenchymal tumors of the seminal vesicle include leiomyoma, solitary fibrous tumor, and schwannoma (498). Sarcomas of the seminal vesicle are even more rare than carcinomas. As with carcinomas, many of the published cases are poorly documented in terms of both the origin of tumor and the histologic subtype. Well-documented cases of angiosarcoma, rhabdomyosarcoma, and leiomyosarcoma of the seminal vesicles have been reported (498-500). Mixed epithelial–stromal tumors are composed of both neoplastic epithelial and stromal elements. They range in biologic behavior from benign, such as adenofibroma and adenomyoma, to low-grade and high-grade malignant mixed epithelial–stromal tumors, with the

grade determined based on stromal cellularity, atypia, mitoses, and necrosis (501,502). Rare primary germ cell tumors of the seminal vesicle have been reported, such as seminoma and choriocarcinoma (503,504). These probably arise from germ cells entrapped there during fetal development. Other rare tumors reported in the seminal vesicle include an adnexal tumor of probable Wolffian origin, squamous cell carcinoma, small cell carcinoma, PNET (498), and paraganglioma (505). Benign ectopic prostatic tissue can be seen in the seminal vesicle (506), and we have seen a primary adenocarcinoma of the prostate arising in the seminal vesicle.

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cases with clinical follow-up. BJU Int. 2010;106(3):330-333. Brat DJ, Wills ML, Lecksell KL, Epstein JI. How often are diagnostic features missed with less extensive histologic sampling of prostate needle biopsy specimens? Am J Surg Pathol. 1999;23(3):257-262. Epstein JI. Diagnostic criteria of limited adenocarcinoma of the prostate on needle biopsy. Hum Pathol. 1995;26(2):223-229. Epstein JI, Netto GJ. Biopsy Interpretation of the Prostate. 4th ed. Lippincott Williams Wilkins; 2008. Aydin H, Zhou M, Herawi M, Epstein JI. Number and location of nucleoli and presence of apoptotic bodies in diagnostically challenging cases of prostate adenocarcinoma on needle biopsy. Hum Pathol. 2005;36(11):1172-1177. Ro JY, Grignon DJ, Troncoso P, Ayala AG. Intraluminal crystalloids in wholeorgan sections of prostate. Prostate. 1988;13(3):233-239. Henneberry JM, Kahane H, Humphrey PA, Keetch DW, Epstein JI. The significance of intraluminal crystalloids in benign prostatic glands on needle biopsy. Am J Surg Pathol. 1997;21(6):725-728. Cina SJ, Epstein JI. Adenocarcinoma of the prostate with atrophic features. Am J Surg Pathol. 1997;21(3):289-295. Egan AJ, Lopez-Beltran A, Bostwick DG. Prostatic adenocarcinoma with atrophic features: malignancy mimicking a benign process. Am J Surg Pathol. 1997;21(8):931-935. Nelson RS, Epstein JI. Prostatic carcinoma with abundant xanthomatous cytoplasm. foamy gland carcinoma. Am J Surg Pathol. 1996;20(4):419-426. Levi AW, Epstein JI. Pseudohyperplastic prostatic adenocarcinoma on needle biopsy and simple prostatectomy. Am J Surg Pathol. 2000;24(8):1039-1046. Humphrey PA, Kaleem Z, Swanson PE, Vollmer RT. Pseudohyperplastic prostatic adenocarcinoma. Am J Surg Pathol. 1998;22(10):1239-1246. Baisden BL, Kahane H, Epstein JI. Perineural invasion, mucinous fibroplasia, and glomerulations: diagnostic features of limited cancer on prostate needle biopsy. Am J Surg Pathol. 1999;23(8):918-924. Ali TZ, Epstein JI. Perineural involvement by benign prostatic glands on needle biopsy. Am J Surg Pathol. 2005;29(9):1159-1163. Epstein JI, Herawi M. Prostate needle biopsies containing prostatic intraepithelial neoplasia or atypical foci suspicious for carcinoma: implications for patient care. J Urol. 2006;175(3 Pt 1):820-834. Dorin RP, Wiener S, Harris CD, Wagner JR. Prostate atypia: does repeat biopsy detect clinically significant prostate cancer? Prostate. 2015;75(7):673678. Leone A, Gershman B, Rotker K, et al. Atypical small acinar proliferation (ASAP): is a repeat biopsy necessary ASAP? A multi-institutional review. Prostate Cancer Prostatic Dis. 2016;19(1):68-71.

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275. Albores-Saavedra J, Vuitch F, Delgado R, Wiley E, Hagler H. Sinus histiocytosis of pelvic lymph nodes after hip replacement. A histiocytic proliferation induced by cobalt-chromium and titanium. Am J Surg Pathol. 1994;18(1):83-90. 276. Schned AR, Gormley EA. Florid xanthomatous pelvic lymph node reaction to metastatic prostatic adenocarcinoma. A sequela of preoperative androgen deprivation therapy. Arch Pathol Lab Med. 1996;120(1):96-100. 277. Ross HM, Kryvenko ON, Cowan JE, Simko JP, Wheeler TM, Epstein JI. Do adenocarcinomas of the prostate with Gleason score (GS) 2 cm and ≤5 cm N3: Lymph node mass >5 cm MX: Unknown status of distant metastases M0: No distant metastases M1a: Nonregional nodal or lung metastases M1b: Distant metastasis other than nonregional nodal or lung SX: No marker studies available S0: All marker studies normal aLDH

levels expressed as elevations above upper limit of normal (N). AFP, α-fetoprotein; hCG, human chorionic gonadotropin; LDH, lactate dehydrogenase; TNM, tumor-node-metastasis LDHa

hCG (mIU/mL)

AFP (ng/mL)

S1:

10,000

GERM CELL TUMORS CLASSIFICATION The overwhelming majority of primary testicular tumors are of germ cell origin. The classification in Table 47.1 represents the 2016 WHO classification (9) and is fundamentally different from the previous WHO classification in that it is based on pathogenesis rather than histomorphology (10). There appears to be widespread uniform acceptance of the new WHO classification, which was endorsed by the International Society of Urological Pathologists (11,12). An overarching attempt at classification of all germ cell tumors from all sites has been attempted by Oosterhuis and Looijenga (Table 47.3) (13). The subdivision of germ cell tumors in this system provides a morphomolecular root of the classification of all testicular germ cell tumors. TABLE 47.3 Types of Germ Cell Tumor Type

Age and Sex

Site

Classification

Genetics

I

Both sexes, usually in infants and children younger than 6 years; only rarely in adults. Wide age range only when occurring in the ovary

Testis, ovary, mediastinum, sacrum, retroperitoneum, and other midline structures

Prepubertal-type teratoma, prepubertal-type yolk sac tumor, and mixed prepubertal-type teratoma and yolk sac tumor

Teratomas diploid, yolk sac tumors, aneuploid yolk sac. No gain of 12p

II

Largely in males, postpubertal, but also in disorders of sex development, Klinefelter and Down syndrome

Testis, ovary, dysgenetic gonads, mediastinum, and other midline structures

All tumors derived from GCNIS

Usually hyperdiploid, with gain of 12p

III

Exclusively male, usually older

Testis

Spermatocytic tumor

Gain of chromosome 9

IV

Female only, reproductive age

Ovary

Ovarian “dermoid cysts” also known as benign cystic teratomas

Usually diploid

V

Female only, reproductive age

Placenta/uterus

Gestational trophoblastic neoplasia

Diploid (90% XX or 10% XY)

Modified by permission from Nature: Oosterhuis JW, Looijenga LHJ. Human germ cell tumours from a developmental perspective. Nat Rev Cancer. 2019;19(9):522-537. GCNIS, germ cell neoplasia in situ.

The radical changes behind the 2016 WHO classification for germ cell tumors are centered on a number of issues (11). First, the long-standing global differences in nomenclature of germ cell neoplasia in situ (GCNIS; previously termed “carcinoma in situ,” “intratubular germ cell neoplasia, unclassified,” and “testicular intraepithelial neoplasia”) (14). This newly modified term has had widespread acceptance by uniting European and American practices, reducing confusion, and also being more terminologically correct: the lesion is not a carcinoma nor is there doubt about its nature (14). Second, and more importantly, it takes into account differences in pathogenesis, molecular signature, histomorphology, and outcome. It was recognized that germ cell tumors in the testis could be split into a larger category comprising those associated with gain of 12p (often as an isochromosome) and/or GCNIS, and a smaller group of tumors that were neither associated with GCNIS nor with i(12)p. This latter, somewhat heterogeneous, group includes mostly pediatric germ cell tumors and spermatocytic tumor. The aim of the classification is not merely for sterile erudition: it has the aim of avoiding both over or undertreatment by misclassification and misunderstandings of biologic potential.

HISTOGENESIS The histogenetic relationship among the various morphologic types of germ cell tumors has been a matter of continued interest and controversy. Recent evidence from several studies supports that the initial event in most adult testicular germ cell tumors is an in utero mutational event or transcriptional modification in intratubular germ cells that causes them to retain immature characteristics in postnatal life (15). There is support that defective development of Sertoli cells, likely induced in some cases by exposure to estrogenic substances during development, is permissive of delayed germ cell maturation (16). Additional events and exposure to pubertal hormones lead to the development of an intratubular malignant germ cell that shares many of the features of seminoma cells. Continued proliferation of such cells within the seminiferous tubules yields the lesion that is termed germ cell neoplasia in situ (GCNIS). These cells eventually develop into an invasive tumor, and a critical event in the development of invasion appears to be the acquisition of additional copies of genes on the short arm of chromosome 12, often in the form of an isochromosome, i(12p), that inhibits apoptosis and permits extratubular growth of invasive tumor cells that do not rely on Sertoli cells for their survival (17). The direct invasive derivative

of GCNIS appears to be seminoma, given the remarkable similarity of these two entities, not only morphologically but also by immunohistochemistry (18-23), ultrastructure (24,25), ploidy analysis (26), lectin-binding patterns (27), number of nucleolar-organizer regions (28), and genetic analysis (29). This supports that seminoma is a common precursor to many other forms of invasive germ cell tumor, a concept further supported by reports showing foci of nonseminomatous elements at the periphery of nodules of seminoma (30,31), the frequent presence of nonseminomatous elements in autopsy studies of patients who died of metastatic germ cell tumor subsequent to an orchiectomy showing pure seminoma (32,33), ultrastructural evidence of epithelial differentiation in some seminomas (34), the greater DNA content of seminoma compared to nonseminomatous tumors (supporting evolution from seminoma secondary to gene loss), and studies showing similar patterns of loss of heterozygosity in the seminomatous and nonseminomatous components of mixed germ cell tumors (29). However, other forms of intratubular germ cell neoplasia are also recognized, and these may also be precursor lesions. As this is still not proven, these lesions are still termed “intratubular” rather than “in situ.” Intratubular embryonal carcinoma has been identified by morphology (35) and immunochemistry for CD30 (36) in a significant percentage of nonseminomas and may be more common than previously recognized; however, this is disputed as CD30 may also stain immature Sertoli cells (37). Intratubular embryonal carcinoma is always associated with GCNIS, suggesting that it may be derived from it, and provides an alternative path for the development of nonseminomatous tumors. Intratubular trophoblast has also been identified in association with seminomas that have a component of trophoblastic giant cells (38). A further lesion, intratubular seminoma, where the seminiferous tubules are packed and expanded with seminoma-like cells, probably represents an end stage of GCNIS and may be associated with all germ cell tumor types (39). A current model of germ cell tumor histogenesis, therefore, recognizes the pivotal role of GCNIS in the development of the various germ cell tumor types, which may be by several alternative pathways. However, primacy of the pathway whereby most tumors are derived through seminoma is assumed (Fig. 47.3). It is important to understand, however, that this model does not apply to the germ cell tumors not derived from GCNIS: pediatric germ cell tumors, spermatocytic tumor, and prepubertal-type teratomas; their pathogenesis is still unknown.

FIGURE 47.3 Diagram of testicular germ cell tumor histogenesis. Note the derivation of the pediatric tumors, spermatocytic tumor, and dermoid and epidermoid cyst, independent of germ cell neoplasia in situ. Also note the key role of seminoma in giving rise to other types of tumors. Benign lesions are shown in green; low-grade tumors are in yellow; in situ malignancies are in orange; and invasive malignancies are in red.

EPIDEMIOLOGY Germ cell tumors of the testis have characteristic associations. The GCNIS-derived tumors occur primarily in young men, most commonly from 15 to 45 years of age. The GCNIS-unrelated tumors occur with a much smaller peak in childhood and with spermatocytic tumors in the elderly. The incidence of testicular germ cell tumors increased progressively in the 20th century (Fig. 47.4), as verified by independent studies in several different countries (40-48), with the exception of cohorts born during World War II in Nazi-occupied countries (49,50). Some authorities consider this increase to be of “epidemic” proportions. This alarming increase in incidence, however, only applies to GCNIS-related cases, with the rate in prepubertal patients remaining relatively constant throughout this same interval (Fig. 47.4) (51). The recent annual incidences are on the order of 10 per 100,000 male population in Denmark and Switzerland, the countries with the highest rates of testicular germ cell tumors; the annual incidence in the US white population and in the United Kingdom is approximately 6 per 100,000 male population. Racial variations in the incidence of testicular cancer are quite apparent; the nonwhite populations in the United States have a substantially lower incidence than does the white population. In fact, the only nonwhite population with a comparably high incidence is the Maoris of New Zealand (52,53). Furthermore, testicular germ cells tumors occur more commonly among professional workers and those of higher socioeconomic class than among laborers and those of lower socioeconomic class (54-57). Isolated reports have implicated exposures to certain agents or association with particular occupations as being of potential etiologic importance (45,54,58-61), but no consistent pattern has emerged. Exposure to estrogenic compounds in utero is implicated in some studies (62-64), as is a familial history of breast cancer (65), early birth order (66), certain human leukocyte antigen (HLA) haplotypes (67-70), Marfan syndrome (71), Down syndrome (71-73), Klinefelter syndrome (74), and the dysplastic nevus syndrome (75). It has been suggested that a hyperestrogenic state in utero leads to defective Sertoli cells, which, in turn, contribute to the process of malignant transformation of germ cells. Hence, testicular germ cell tumors are associated with testicular maldevelopment, the so-called “testicular dysgenesis syndrome” (76-79). No linkage of testicular cancer with smoking, alcohol consumption, radiation exposure, or prior vasectomy is established (52,80-82).

FIGURE 47.4 The incidence of testicular cancer in postpubertal patients has undergone a progressive increase over many decades, whereas the incidence in prepubertal patients has remained the same. Reprinted from Xu Q, Pearce MS, Parker L. Incidence and survival for testicular germ cell tumor in young males: a report from the Northern Region Young Person’s Malignant Disease Registry, United Kingdom. Urol Oncol. 2007;25(1):32-37. Copyright © 2007 Elsevier. With permission.

There are several well-defined positive associations with testicular germ cell tumors: cryptorchidism, a prior testicular germ tumor, a familial history of testicular germ cell tumors, certain disorders of sex

development (intersex syndromes), and oligospermic infertility. Cryptorchidism remains the most common risk factor for testicular germ cell tumors. In many series, approximately 10% of the cases are associated with past (corrected) or present cryptorchidism (83-89). Overall, cryptorchid patients have approximately a four-fold elevated risk of testicular germ cell tumors (90-92). Because of this elevated risk, it has been suggested that cryptorchid patients have diagnostic testicular biopsies to evaluate for the presence of the precursor lesion, GCNIS, which is present in 2% to 4% of the cases (93-95). Follow-up studies of patients with GCNIS on biopsy have verified a high rate of development of invasive testicular germ cell tumors (50% at 5 years (96)) but only very rare cases in patients with negative biopsies (93,97). The normally placed testis opposite a cryptorchid one is also at increased risk, but to a lesser degree (55,92,98). Two to 5% of patients with a testicular germ cell tumor develop another germ cell tumor in the contralateral testis (99-104). There is an especially increased risk if the residual testis is atrophic or cryptorchid (105-109); there is a family history of testicular cancer (110,111), or if the first tumor occurred at a young age (109). Intervals of more than a decade may occur between such metachronous testicular cancers (112), although some may present synchronously, and 50% of second tumors occur within 3 to 5 years (99,106). Again, biopsy of the remaining testis appears to be an effective method of identifying patients at risk for a second primary testicular germ cell tumor (97,108,113). Some advocate biopsy of the opposite testis at the initial orchiectomy (114). In one study, there was a remarkably high (>90%) frequency of C-KIT gene mutations in bilateral tumors compared to their virtual absence (1%) in unilateral cases (115), suggesting their utility in prospectively identifying patients at risk for a contralateral tumor. Unfortunately, other studies have failed to verify this result (116-119). There is good evidence for a familial basis to some testicular germ cell tumors (120-122), with an approximately 2% frequency of testicular cancer in the first-degree male relatives of patients with testicular germ cell tumors compared to 0.4% in a control population (123). Brothers are at highest risk (10 times); sons at intermediate risk (six times); and fathers at lowest risk (four times) (124). Patients with testicular cancer and a familial history are also at increased risk for bilateral tumors, with bilateral involvement occurring in 8% to 14% of such cases (110,120,124). However, specific genes associated with the increased risk of testis cancer are not known (125), although a deletion on the Y chromosome has been shown to be associated with some cases of familial germ cell malignancy (126). Linkage studies implicating genes at Xq27 (127,128) have not been consistent (125). It is also clear that patients with certain disorders of sex development (formerly known as “intersex syndromes”) are at increased risk for germ cell tumors. Patients with gonadal dysgenesis who carry a Y chromosome develop germ cell tumors at an increased rate, most often in association with a preexisting gonadoblastoma (129,130). Approximately 30% of such patients develop gonadoblastoma that may then give rise to the various subtypes of invasive germ cell tumor. For certain subsets of this condition, notably Frasier and Denys-Drash syndromes, the risk may be even higher, although the data are limited (126). Interestingly, a common thread for many of these disorders is mutation of the SOX9 gene or genes in the upstream signaling pathway for SOX9, most notably WT1. Patients with the androgeninsensitivity (testicular feminization) syndrome are also at increased risk, with 5% to 10% developing germ cell tumors (131-133), which is undoubtedly a low estimate because of prophylactic gonadectomy at an early age in many patients. One study reported a 22% frequency of malignant tumors in androgeninsensitivity patients beyond 30 years of age (134), and a consensus statement estimated the risk at 50% for those with partial androgen-insensitivity syndrome and a non–scrotal testis (126). The patients with the partial rather than complete form of the androgen-insensitivity syndrome are those at risk (131133). Most germ cell tumors in the androgen-insensitivity syndrome occur after the full development of female secondary sexual characteristics (131,135), but occasional cases are reported in younger patients (136), making the timing of prophylactic gonadectomy somewhat controversial. This appears to be a safe strategy for those with the complete form of the syndrome (133). Gonadal biopsy may successfully identify GCNIS in at-risk patients with gonadal dysgenesis and the androgen-insensitivity syndrome (137,138), even during childhood (139,140), unlike biopsies in children who have risk factors other than intersex conditions. The independent linkage of testicular germ cell tumors with oligospermic infertility remains less well established than with the other four factors. There is no question that male infertility patients have an elevated risk of testis cancer, with GCNIS being identified in approximately 1% (24,95,141). The

association of cryptorchidism, testicular atrophy, and gonadal dysgenesis with infertility, however, complicates this analysis and casts doubt that infertility is an independent risk factor (142).

GERM CELL NEOPLASIA IN SITU This term replaces the nomenclature “intratubular germ cell neoplasia, unclassified (IGCNU)” and the lesion that was originally described by Skakkebaek as “carcinoma in situ” of the testis. When patients with GCNIS were prospectively followed, invasive germ cell tumors developed in 50% by 5 years of follow-up (143). As additional support of its precursor role, GCNIS occurs at increased frequency in patients known to be at increased risk for testicular germ cell tumors. Thus, GCNIS is identified in 2% to 4% of cryptorchid patients (93-95), in approximately 5% of the contralateral testes of patients with a prior testicular germ cell tumor (105,108,144-146), in 0.4% to 1% of patients with infertility (24,95,105,141), and at high rates in selected cases of gonadal dysgenesis and the androgen-insensitivity syndrome (137,147). GCNIS is observed in the vast majority cases of invasive germ cell tumors of the testis in adults if sufficient residual seminiferous tubules are present (148-150). It is not seen in the tumors now described as “not associated with GCNIS”: spermatocytic tumor (96) and prepubertal-type teratoma and/or yolk sac tumor in adults (to be elucidated later). Most cases of supposed GCNIS in pediatric cases represent a reactive phenomenon (151), delayed germ cell maturation as described in other conditions (Fig. 47.5) (152) or malformed spermatogonia of uncertain significance (Fig. 47.6) (156).

FIGURE 47.5 A pediatric testis with delayed germ cell maturation. A centrally located germ cell (arrow) has the features of a primordial germ cell or gonocyte. Retention of fetal germ cell markers, as seen in germ cell neoplasia in situ, may occur in such cases.

FIGURE 47.6 A pediatric testis with atypical, variably enlarged, frequently multinucleated spermatogonia. These findings do not represent germ cell neoplasia in situ.

In postpubertal patients, GCNIS most commonly appears as germ cells with enlarged, hyperchromatic nuclei and clear cytoplasm (often having retraction artifact in formalin-fixed material) aligned along the basal portion of the seminiferous tubules, known as the “spermatogonial niche” (Fig. 47.7). Nucleoli are conspicuous, and mitotic figures are frequent. The Sertoli cells are often displaced toward the lumen, and spermatogenesis in the affected segment of tubule is almost always absent (Fig. 47.7), although it may appear normal in adjacent tubules. The affected tubules usually have thickened peritubular basement membranes. As GCNIS progresses, the Sertoli cells may be replaced, and a pattern resembling the so-called “intratubular seminoma” may develop. Pagetoid spread of GCNIS into the rete testis (Fig. 47.8) is common (153). The very rare cases of supposed GCNIS described in children reported that the neoplastic cells were not basally located but dispersed at various levels, with the more typical pattern evolving as the patient aged (96,154). More recent studies suggest this reflects germ cells with delayed maturation, (Fig. 47.5) which, in an unknown proportion of cases, may progress to true GCNIS.

FIGURE 47.7 Germ cell neoplasia in situ consisting of cells with enlarged, polygonal nuclei with prominent nucleoli and clear cytoplasm along the basal aspect of a seminiferous tubules lacking spermatogenesis. Sertoli cells are displaced toward the lumen.

FIGURE 47.8 Germ cell neoplasia in situ cells have spread in a pagetoid manner into the rete testis, with an overlying layer of rete epithelium.

The great majority of GCNIS cells are periodic acid-Schiff (PAS) positive and diastase sensitive (150) (Fig. 47.9), but similar positivity may also be identified in nonneoplastic spermatogonia and Sertoli cells (155). A more specific method is immunostaining with antibodies directed against placental alkaline phosphatase (PLAP), which highlight GCNIS in more than 95% of the cases (20,155-158). Unlike PAS stains, PLAP positivity does not occur in nonneoplastic spermatogonia, although rare cases show very focal PLAP positivity in spermatocytes (157). PLAP staining is usually membrane accentuated (Fig. 47.10). Antibodies M2A, 43-9F, TRA-1-60, and D2-40 (podoplanin) and those with specificities against glutathione S-transferase π, angiotensin-converting enzyme, OCT3/4, NANOG, and the C-KIT protooncogene (CD117) protein have also successfully identified GCNIS with very high sensitivities (18,23,158-165). We currently consider OCT3/4 the marker of choice for the detection of GCNIS because of its remarkably high sensitivity and specificity; it shows easily appreciated nuclear reactivity (Fig. 47.11). In addition, we have seen a number of cases where nonneoplastic germ cells were CD117 reactive, perhaps due to increasingly sensitive immunostaining methods that have diminished the specificity of this antibody in its identification of GCNIS. Several ultrastructural studies of GCNIS have identified features similar to those of seminoma (24,166-169); another study noted a similar distribution of nucleolar-organizing regions in GCNIS and seminoma (28).

FIGURE 47.9 Strong periodic acid-Schiff positivity in germ cell neoplasia in situ.

FIGURE 47.10 Placental alkaline phosphatase positivity in germ cell neoplasia in situ with usual membranous pattern.

FIGURE 47.11 OCT3/4 produces strong nuclear reactivity in germ cell neoplasia in situ, whereas the tubules with spermatogenesis are negative.

Testicular biopsies detect GCNIS in at-risk patients with a high sensitivity, and the recommended approach is to take one or two 3-mm biopsies per testis (170). A negative result on adequate biopsies is good evidence of little increased risk of testicular cancer; among almost 2000 patients who had negative biopsies opposite a germ cell tumor, only 0.3% developed a second tumor on follow-up (108). It remains controversial regarding who should be evaluated, although some suggest screening biopsies in patients with a history of cryptorchidism, prior testicular cancer, and somatosexual ambiguity in the presence of a Y chromosome. In patients with these risk factors, there is a much greater probability of a positive biopsy for GCNIS in the presence of testicular atrophy (114). GCNIS is usually treated by orchiectomy or radiation. The chemotherapy that is given to patients with metastatic germ cell tumors may eradicate GCNIS in the remaining testis, but this is not a consistently effective form of treatment (114,171-174). Other forms of intratubular neoplasia include, most commonly, intratubular embryonal carcinoma and intratubular seminoma. Both are usually encountered in testes with already established invasive tumors. With intratubular embryonal carcinoma, the tubules are often expanded by tumor cells with associated central necrosis and calcification. The pleomorphic cells are similar to those of embryonal carcinoma and may be highlighted by immunochemistry for CD30, which also may highlight foci not obvious on routine staining. Intratubular seminoma represents filling and distention of seminiferous tubules by cells morphologically identical to GCNIS (Fig. 47.12). Intratubular syncytiotrophoblastic cells occur adjacent to seminoma in approximately 15% of cases (38), and intratubular spermatocytic tumor is virtually universal adjacent to invasive spermatocytic tumors. Intratubular yolk sac tumor and teratoma are very rare.

FIGURE 47.12 Intratubular seminoma fills several tubules.

GERM CELL TUMORS DERIVED FROM GERM CELL NEOPLASIA IN SITU SEMINOMA “Pure seminoma” (including cases with scattered trophoblastic elements) represents approximately 50% of all testicular germ cell tumors (175-177) and occurs at an average age of 40 years (175), which is 5 to 10 years older than patients with nonseminomatous germ cell tumors (30,175). Between 80% and 90% of patients with seminoma have initial symptoms of testicular swelling or other palpable abnormalities (178,179), although up to 11% may have normal-sized or atrophic testes (180). Tumors with a predominant or exclusive pattern of intertubular growth often do not present as masses but as metastatic tumor or are discovered incidentally (181). Testicular pain occurs in 10% to 20% of cases (178,179). Presenting symptoms secondary to metastases, most commonly lumbar back pain as a result of retroperitoneal spread, occur in 1% to 3% of cases (178,179). Only rare patients develop gynecomastia as a result of elevations of human chorionic gonadotropin (hCG) secondary to admixed trophoblastic elements. Paraneoplastic exophthalmos (182,183), hypercalcemia (184), limbic encephalopathy (185), polycythemia (186), and hemolytic anemia (187) are rare. Serum hCG levels are mildly to moderately elevated in approximately 10% of patients with clinical stage I seminoma and in approximately 25% with metastatic involvement (188), although testicular vein hCG is elevated in a much higher proportion of cases (189). Elevated serum hCG correlates with trophoblast cells in the tumor. α-Fetoprotein (AFP) is not produced by seminoma cells, and an elevated serum AFP in a patient with an apparently pure seminoma is generally indicative of unsampled nonseminomatous elements, although mild AFP elevation may reflect liver disease, including metastatic hepatic involvement (190); in one study, minimal AFP elevations in patients with apparently pure seminomas were not associated with a different behavior (191). Serum PLAP levels are also elevated in approximately 50% of patients with seminoma (192).

The cut surface of the tumor is cream colored to tan to pink and lobulated to multinodular (Fig. 47.13), with a fleshy quality and a tendency to bulge above the surrounding parenchyma. Punctate foci of hemorrhage may correspond to foci of syncytiotrophoblastic cells (193). Well-defined foci of yellow necrosis may be present, but extensive hemorrhage and necrosis are unusual. Tumors with predominantly interstitial growth may resemble small scars or even be grossly in apparent (181). Extension through the tunica albuginea or into the epididymis occurs in less than 10% of cases (194).

FIGURE 47.13 A seminoma has a light tan, lobulated cut surface with punctate foci of hemorrhage corresponding to syncytiotrophoblast cells. Note small focus of necrosis at bottom. Photograph courtesy of S. F. Cramer, MD, Rochester General Hospital, Rochester, NY.

On microscopic examination, seminomas often have a diffuse, sheetlike pattern (Fig. 47.14) or sometimes a lobulated arrangement (Fig. 47.15). There often is an intertubular or cordlike pattern at the periphery of the tumor as neoplastic cells surround but do not destroy seminiferous tubules. Rarely, intertubular growth is predominant (Fig. 47.16), and these tumors may easily be overlooked, although the associated lymphocytes are helpful in their recognition. Branching, fibrous septa often course through seminomas (Fig. 47.15). Edema may cause a microcystic or cribriform arrangement (Fig. 47.17). This pattern may mimic the microcystic pattern of yolk sac tumor, but distinction can usually be made based on the typical seminomatous appearance of other areas; the usually more irregular nature of the cystic spaces, with the frequent presence of intracystic, exfoliated seminoma cells and edema fluid; and the retained cytomorphology of the tumor cells (195). Rarely, a tubular (or pseudotubular) pattern may occur, usually as a focal finding (196,197) (Fig. 47.18). Extensive zones of scarring (“tumor regression”) may occur, with obliteration of major portions of the tumor.

FIGURE 47.14 A seminoma has a sheetlike arrangement of cells with some scattered, perivascular lymphocytes.

FIGURE 47.15 A seminoma has a lobulated appearance created by aggregates of tumor cells separated by fibrous septa containing lymphocytes.

FIGURE 47.16 Small clusters of seminoma cells grow between the tubules and provoke a lymphocytic reaction.

FIGURE 47.17 There are irregularly shaped microcysts in this seminoma.

FIGURE 47.18 A seminoma forms solid tubular structures, a pattern that must be distinguished from Sertoli cell tumor.

Seminoma cells characteristically have clear to lightly eosinophilic cytoplasm and central nuclei, often with slightly “squared” edges, and one or two large central nucleoli (Fig. 47.19). The cells are closely apposed, with well-defined cytoplasmic borders. An abundant amount of cytoplasm causes the nuclei to be well spaced and nonoverlapping (Fig. 47.19). In poorly fixed specimens, however, cytoplasmic autolysis occurs, obscuring the cell borders and causing apparent nuclear overlapping, thus creating confusion with solid patterns of embryonal carcinoma (see section “Embryonal Carcinoma”). Occasionally, the cells have denser, amphophilic to basophilic cytoplasm, imparting a plasmacytoid appearance (Fig. 47.20). These cases often have somewhat more crowded and pleomorphic nuclei and are more apt to be misinterpreted as embryonal carcinoma. We have seen rare cases where emptyappearing cytoplasmic vacuoles imparted a signet-ring appearance to some of the tumor cells (Fig. 47.21) (198).

FIGURE 47.19 Seminoma cells have pale to clear cytoplasm with well-defined cytoplasmic membranes and polygonal nuclei that often have flattened edges. Nucleoli are prominent.

FIGURE 47.20 A seminoma has increased nuclear pleomorphism and a plasmacytoid appearance.

FIGURE 47.21 A seminoma with signet-ring cell change caused by prominent cytoplasmic vacuoles that compress the nuclei.

Lymphocytic infiltrates occur in virtually all seminomas. They are often most intense within and adjacent the fibrous trabeculae but also intermingle with the tumor cells (Fig. 47.22). Germinal center formation can develop in some cases, but most of the lymphoid elements are T cells (199-204) that promote granuloma formation. A granulomatous reaction occurs in up to 50% of the cases (194) and varies from scattered clusters of epithelioid histiocytes to well-defined granulomas with characteristic Langhans giant cells. In rare cases, an extensive granulomatous reaction can obliterate almost all of the underlying seminoma, thus causing a misdiagnosis of granulomatous orchitis (see Chapter 46). It is, therefore, crucial to examine closely any testis with an extensive granulomatous reaction for residual seminoma cells or GCNIS before accepting a case such as granulomatous orchitis. PLAP, OCT3/4, and podoplanin immunostains can prove useful in identifying residual seminoma cells in this circumstance.

FIGURE 47.22 Fibrovascular septa in a seminoma contain a lymphocytic infiltrate.

In up to 20% of seminomas, trophoblast cells are identified in a scattered pattern (205,206). Sometimes, these cells have a distinctly syncytiotrophoblastic appearance with multinucleation and/or intracytoplasmic lacunae (Fig. 47.23). In other cases, they appear as large mononucleated cells. They are often located adjacent to capillaries and may be associated with microfoci of hemorrhage. Although small, nodular aggregates of trophoblast cells may be seen, confluent growth is lacking, which, in conjunction with the absence of a mononucleated trophoblast component, permits distinction from choriocarcinoma.

FIGURE 47.23 Syncytiotrophoblast cells with lacunae containing red blood cells are scattered in a seminoma.

Most seminomas contain glycogen, and PAS stains are usually positive. PLAP immunostains are positive, usually with a membrane-accentuated pattern, in 85% to 98% of cases (22,155,207,208). Reactivity for CD117 and podoplanin (also known as M2A and D2-40) occurs in most (209-214); whereas epithelial membrane antigen (EMA) and CD30 are typically negative (Table 47.4). OCT3/4 is a transcription factor that is positive in seminoma (Fig. 47.24), embryonal carcinoma, and GCNIS (215,216). We consider OCT3/4 the single best marker for seminoma because of its great sensitivity and high specificity. SOX2 negativity and SOX17 positivity in seminomas contrast with the reverse pattern seen in embryonal carcinomas (217-221). Seminomas may also contain cytokeratin (CK) 7, CK8, CK18, and, less commonly, CK4, CK17, and CK19 (222,223), but only rarely in a diffuse cytoplasmic pattern. Intermingled trophoblast cells are positive for CK8, CK18, and CK19 as well as hCG (22,205,206,222,224,225). Positivity of seminomas with antibodies directed against CK8 and CK18 (e.g., CAM 5.2) may, therefore, be seen (usually in isolated cells but rarely more diffusely (22,226,227)

in up to 73% of cases (222). Most routinely processed seminomas, however, are negative or weakly and focally reactive with broad-spectrum CK antibodies.

FIGURE 47.24 There is strong nuclear and weaker cytoplasmic reactivity for OCT3/4 in a seminoma.

TABLE 47.4 Typical Immunohistochemical Reactions in Germ Cell Tumors and Metastatic Carcinoma Germ Cell Neoplasia In Situ

Usual Seminoma

Spermatocytic Tumor

Embryonal Carcinoma

Yolk Sac Tumor

Choriocarcinoma

Teratoma

Metast Carcin

PLAP

+

+

−

±

±

+

−

±

CD117

+

+

±

−

−

−

−

±

D2-40

+a

+a

?

±b

−

−

±

−

AFP

−

−

−

±

+

−

±

±

βhCG

−

−

−

−

−

+

−

±

CD30

−

−

−

+

−

−

−

±

OCT3/4

+

+

−

+

−

−

−

−

NANOG

+

+

−

+

−

−

−

−

AE1/AE3

−

±

−

+

+

+

+

+

CK7

±

±

?

+

−

+

+

±

EMA

−

−

−

−

−

+

+

+

Glypican 3

−

−

?

−

+

+

±

±

SALL4

+

+

+

+

+

±

±

±

SOX2

−

−

−

+

−

−

±c

?

SOX17

+

+

−

−

±

−

±

?

GATA3

−

−

?

−

+

+

+

±

Inhibin

−

−

?

−

−

+

−

±

aDiffuse

membranous; bFocal membranous or cytoplasmic staining; cEmbryonic-type neuroectodermal elements show variable positivity +, usually positive; −, usually negative; ±, variable staining; AFP; α-fetoprotein, βhCG, β human chorionic gonadotropin; CK, cytokeratin; EMA, epithelial membrane antigen; PLAP, placental alkaline phosphatase.

On electron microscopic study, seminomas are primitive cells, often having large aggregates of glycogen and simple cellular organelles that are polarized in the cytoplasm (34,228,229). Cell membranes are closely apposed, but junctions are usually primitive and few. The nuclei have intricate nucleoli but an evenly dispersed euchromatin. They may show epithelial differentiation, including welldefined desmosomes and surface microvilli, despite a typical light microscopic morphology (34). Seminomas have a DNA index of l.66 (230), which is significantly higher than in nonseminomatous tumors (26,230). In addition, isochromosome (12p) or other chromosome 12 anomalies are identified on karyotypic analysis (231-233). Differential Diagnosis Solid patterns of embryonal carcinoma may be confused with seminoma. Poorly defined cell borders, nuclear overlapping, and nuclear irregularity and pleomorphism are features of embryonal carcinoma that differ from most well-fixed seminomas. Furthermore, embryonal carcinomas lack the regular fibrous septa of seminoma. CK and CD30 stains (both positive in embryonal carcinoma) may also prove useful in this differential diagnosis (Table 47.4). CD117, podoplanin (D2-40 antibody), and SOX17 may also be useful because they are positive in seminoma, but not in embryonal carcinoma (234-236). Some seminomas have more pleomorphic foci than others; we require, however, definite evidence of epithelial differentiation in the form of papillae or glands and supportive immunoreactivity in the pleomorphic zones before recognizing embryonal carcinomatous differentiation. Solid patterns of yolk sac tumor may also resemble seminoma, but these are usually associated with more characteristic yolk sac tumor patterns (237). If only a small biopsy is available, solid pattern yolk sac tumor often shows suggestions of true microcyst formation, unlike seminoma, and may show intercellular basement membrane deposits or cytoplasmic hyaline globules (238). In difficult cases, strong positivity for AE1/AE3 CK, Glypican 3, and AFP and negativity for OCT3/4 support solid yolk sac tumor over seminoma (237). CD117 is not helpful in this differential diagnosis as it stains ~60% of solid yolk sac tumors (237). Glypican 3 is more sensitive than AFP in this regard, with the caveat that the staining intensity in solid yolk sac tumor is lower than that of the more classic patterns (such as the reticular/microcystic pattern) (237,239,240). Lymphomas involving the testis may be confused with seminoma. Although primary testicular lymphomas do occur, most cases of testicular involvement represent spread from an extratesticular site. Bilateral involvement in an older patient is a clinical feature in favor of lymphoma over seminoma (241245). Paratesticular involvement on gross examination is more common in lymphoma (245). Lymphomas often have an extensive intertubular pattern with relative tubular preservation (242,243,246) and usually lack the clear cytoplasm and well-defined cell borders of seminoma. The nuclei of lymphomas are frequently irregular and less uniform than those of seminoma. The great majority of seminomas are associated with GCNIS, but lymphomas are not. PLAP and OCT3/4 immunostains are positive in seminomas but almost always negative in lymphomas (215,247), although there are reports of rare large B-cell lymphomas that react for OCT3/4 (248). On the other hand, lymphoid markers show the opposite pattern (22). The distinction of seminoma from SCT is discussed in the section “Sertoli Cell Tumor.” Treatment and Prognosis Seminomas are extremely sensitive to both radiation and chemotherapy, and these modalities usually represent the primary forms of treatment following orchiectomy, although clinical stage I patients are now increasingly managed by surveillance (249-252). Some patients with clinical stage I seminoma may receive radiation directed at the ipsilateral inguinal and iliac lymph nodes as well as to the abdominal paraaortic and paracaval nodes, although some studies suggest that early-stage patients may not need pelvic radiation in the absence of prior inguinal surgery or scrotal involvement (253-255). Cure rates exceeding 95% can be expected for these patients. When recurrence occurs, it is almost always outside

the radiated field. Adjuvant carboplatin is now used in many centers as an alternative to radiotherapy with comparable results, although, with less long-term outcome data, (258) relapses are amenable to further lines of chemotherapy (256). In patients with metastatic seminoma to the retroperitoneum, those with less bulky disease are treated with radiation with cure rates of 90% to 96% (257-259). With bulky retroperitoneal seminoma or supradiaphragmatic involvement, cisplatin-based chemotherapy is the preferred treatment. Survival for these patients when so treated is approximately 80% (260,261). The most important prognostic factor in seminoma is tumor stage (which includes tumor bulk/burden). There is controversy concerning the significance of elevated hCG levels in patients with seminoma; there is some evidence that patients with more than a moderate hCG elevation have a poorer overall survival (262-264), although this may be a surrogate for tumor bulk (189,265). The presence of a lymphoid infiltrate and prominent granulomatous reaction may confer a somewhat improved prognosis (266), but this may be of borderline significance (267,268). As seminoma has almost a uniformly favorable prognosis, the possibility of defining pathologic characteristics to define poor-risk disease is challenging: thousands of cases are needed to adequately power any future study. The recognition of a histologically defined “anaplastic seminoma” with a worse prognosis remains controversial. It is clear that the original criteria (269), based on the mitotic rate of seminomas, are not effective in recognizing an aggressively behaving subset of seminomas (270,271). Often, tumors classified as “anaplastic seminomas” are either poorly fixed seminomas, with obscuring of the diagnostic features, or solid pattern embryonal carcinomas. Tickoo et al (272) described “seminoma with atypia” based on nuclear pleomorphism and crowding, dense cytoplasm, and few lymphocytes. Such tumors were more likely to present with advanced-stage disease and to express CD30 and lose CD117 reactivity. However, most would regard the CD30 positivity as representing early transformation to embryonal carcinoma. EMBRYONAL CARCINOMA Embryonal carcinoma is a germ cell neoplasm composed of primitive epithelial cells mostly arranged in solid, papillary, and glandular configurations. As a pure neoplasm, it is relatively uncommon, representing approximately 10% of germ cell tumors (175,176); however, an embryonal carcinoma component is found in most (87%) of the nonseminomatous neoplasms (175). Many cases of embryonal carcinoma with yolk sac tumor were previously classified as pure embryonal carcinoma. Embryonal carcinomas tend to occur in patients approximately 10 years younger (average age, 31 years) (175) than those with seminomas. Most patients present with a testicular mass, which may be associated with pain. Unlike seminoma, which is limited to the testis at presentation in 70% of the cases, 66% of patients with a tumor composed predominantly of embryonal carcinoma have metastases at diagnosis (273), and presenting symptoms may, therefore, be related to metastatic disease, including back pain, dyspnea, cough, hemoptysis, hematemesis, and neurologic symptoms. Gynecomastia may occur in a minority of patients and generally is a reflection of admixed, hCG-producing trophoblastic elements. Sometimes, widespread metastases are present in the face of a clinically occult primary tumor (274). Patients with pure embryonal carcinomas usually do not have serum AFP elevation (275), although rare embryonal carcinoma cells may stain positively for AFP (22,276). The high rate of positivity for AFP reported by some (277) reflects the frequent and often intimate association of embryonal carcinoma with a yolk sac tumor component. Elevated serum levels of lactate dehydrogenase (LDH) occur in approximately 60% of patients with advanced-stage disease (278), and elevation of PLAP may also be seen (192). On gross examination, embryonal carcinomas are usually pale-gray, poorly demarcated tumors associated with hemorrhage and necrosis (Fig. 47.25), with an average diameter of 2 to 3 cm (266). Extratesticular spread is present in approximately 20% of cases (194).

FIGURE 47.25 An embryonal carcinoma has a hemorrhagic and necrotic appearance.

On microscopic examination, there are typically nodules of large cells with prominent zones of eosinophilic, coagulative necrosis. Within these nodules, several patterns may occur. The tumor may be arranged in papillae that may have stromal cores (Fig. 47.26) or consist purely of epithelium, with intervening slitlike spaces. Cross sections of papillae with prominent vessels result in a “pseudoendodermal sinus” pattern (Fig. 47.27) (279). Glandular patterns are common, with cuboidal to columnar tumor cells arranged around luminal spaces (Fig. 47.28). Solid patterns have a sheetlike proliferation of tumor cells (Fig. 47.29). Intermingled cells with a dark, smudged appearance are common and characteristic (Fig. 47.29) but are nonspecific. This feature has been termed an “appliqué” appearance because the degenerate cells appear to be “applied” to the periphery of tumor nodules. Occasionally, such cells can be difficult to distinguish from trophoblastic elements, causing concern for choriocarcinoma. Immunostaining for hCG is useful in resolving this problem. Additional patterns include anastomosing, sieve like, pseudopapillary, and blastocyst like, but these are uncommon (307). Two patterns with a prominent embryonal carcinoma component include the polyembryoma-like and diffuse embryoma. In the former, embryonal carcinoma is arranged in intimate association with yolk sac tumor to create variably formed simulations of early embryos (see section “Mixed Germ Cell Tumors”). In the latter, also termed a “double-layered pattern” of embryonal carcinoma, ribbons of columnar embryonal carcinoma cells are accompanied by a parallel layer of smaller, flattened cells (279) (Fig. 47.30). The AFP positivity and CD30 negativity of the smaller cell component, as well as its appearance, indicate that this pattern represents a mixed germ cell tumor with a yolk sac tumor component. Thus, both polyembryoma and diffuse embryoma should be regarded as mixed germ cell tumor.

FIGURE 47.26 An embryonal carcinoma forms papillae with fibrous cores.

FIGURE 47.27 Transverse cuts through the papillae of an embryonal carcinoma may produce structures resembling the endodermal sinus-like formations seen in yolk sac tumor.

FIGURE 47.28 An embryonal carcinoma forms glands lined by columnar cells.

FIGURE 47.29 A solid pattern of embryonal carcinoma has admixed “smudged” cells, especially at periphery.

FIGURE 47.30 (A) A pattern referred to as either the “double-layered” pattern of embryonal carcinoma or “diffuse embryoma.” It is a mixed germ cell tumor (embryonal carcinoma with a parallel layer of flat to cuboidal cells of yolk sac tumor), as supported by (B) the CD30 negativity of the smaller cell population.

On high-power examination, embryonal carcinoma cells, in routine paraffin sections, are generally polygonal with amphophilic to lightly basophilic cytoplasm and ill-defined cytoplasmic borders (Fig. 47.31). The nuclei are large and often irregularly shaped, with vesicular chromatin interspersed with clumps of heterochromatin, and have one or more centrally located, large nucleoli. Often, the routine histologic appearance is that of nuclear crowding such that nuclei appear to overlap, although, in plastic sections, this finding is seen to be an effect of the section thickness. The mitotic rate is generally very brisk.

FIGURE 47.31 An embryonal carcinoma has crowded, vesicular nuclei; prominent nucleoli; and poorly defined cell borders.

It is not uncommon, however, for some embryonal carcinomas to have foci reminiscent of seminoma where the cells have distinct cytoplasmic membranes and pale or clear cytoplasm (Fig. 47.32). The nuclei, however, have the more irregular and pleomorphic appearance that is expected. When wellformed glands are present, the cells may be cuboidal or columnar, sometimes with subnuclear and/or supranuclear clear vacuoles reminiscent of secretory endometrium (Fig. 47.33).

FIGURE 47.32 An embryonal carcinoma with cells having clear cytoplasm and well-defined cytoplasmic membranes, creating a “seminoma-like” appearance.

FIGURE 47.33 An embryonal carcinoma with subnuclear vacuoles (secretory-type change).

It is common to identify intratubular embryonal carcinoma adjacent to an invasive lesion. Such intratubular foci often show extensive necrosis and may ultimately undergo total necrosis and calcify (Fig. 47.34). If there is similar regression of the invasive neoplasm, the presence of such coarse, irregular intratubular calcifications (“hematoxylin-staining bodies”) within expansive tubular profiles provides good evidence of a regressed germ cell tumor, specifically embryonal carcinoma (280,281).

FIGURE 47.34 Intratubular embryonal carcinoma showing prominent comedonecrosis. Note the relatively uniform tubular diameters and nonbranching pattern.

A source of controversy is the extent that an embryonal carcinoma may have a stromal component. There is a tradition of permitting a primitive neoplastic mesenchymal component to be associated with the typical epithelial cells of embryonal carcinoma (Fig. 47.35) and yet retain classification of such tumors as pure embryonal carcinoma (194,282), rather than embryonal carcinoma with a teratomatous component. The rationale for this approach is that such a finding represents a reiteration of primitive embryonic development. There is also some evidence that embryonal carcinomas with and without a primitive mesenchymal component have a similar biology based on the experience of the British Testicular Tumour Panel (178), although these data antedate current treatments. We recommend classifying neoplastic stroma associated with embryonal carcinoma as teratoma.

FIGURE 47.35 An undifferentiated, neoplastic stroma is associated with embryonal carcinoma.

With the advent of “surveillance-only” protocols in patients with nonseminomatous tumors, it is important to assess an embryonal carcinoma (and other tumor types) for vascular invasion and extratesticular extension. Artifactual implants of embryonal carcinoma occur easily during tissue cutting because of its extremely cellular and friable nature (3). Intravascular implants consist of loosely cohesive cells that lie randomly in the luminal space. They are often associated with implants that are “buttered” on the surface of the testis and spermatic cord (3). True vascular invasion consists of cohesive groups of cells that conform to the shape of the vessel (Fig. 47.36) or are adherent to its wall by thrombotic material. Vascular invasion is usually easiest to appreciate at the periphery of the main tumor. Intratubular neoplasm may mimic vascular invasion; the identification of residual Sertoli cells in such cases, as well as staining for endothelial markers, can be of assistance. Prominent comedonecrosis supports intratubular rather than intravascular tumor, as does a nonbranching arrangement of rounded tumor nests of relatively uniform diameter (Fig. 47.34).

FIGURE 47.36 An embryonal carcinoma with vascular invasion conforms to the shape of the vessel lumen.

Immunohistochemical stains may assist in the diagnosis of embryonal carcinoma. From 0% to 33% of morphologically typical embryonal carcinomas stain positively for AFP (22,225,276,283,284), with a higher frequency of positivity in the embryonal carcinoma component of a mixed germ cell tumor (276,283), probably indicating its capacity for transformation to yolk sac tumor (Table 47.4). Furthermore, 8% of embryonal carcinoma is positive for Glypican 3 (239). Patchy PLAP reactivity occurs in 86% to 97% of cases (22,155,285), and CK is positive in 95% to 100% (22,286), although EMA positivity occurs in only 2% (22). The presence of CKs other than CK8 and CK18 in embryonal carcinomas contrasts with the limited reactivity (mainly confined to CK8 and CK18 in scattered cells) of

most seminomas (222,227,287). OCT3/4 is uniformly positive in tumor nuclei, but not helpful in distinction from seminoma (215,216). CD30 positivity (Fig. 47.37) occurs in 84% of cases and is uncommonly seen in other types of germ cell tumor (288-290) (Table 47.4). CD30 positivity is sometimes lost in embryonal carcinomas after chemotherapy (291), in which case the use of SOX2 (positive) and SOX17 (negative) may be helpful (217-221). Although embryonal carcinomas are not difficult to recognize in the testis, an embryonal carcinoma presenting in a metastatic site may be extremely difficult to distinguish from an undifferentiated carcinoma of nongerminal type. The presence of OCT3/4, PLAP, and CD30 positivity with EMA negativity in such cases can be of significant diagnostic assistance. Other stains that may be positive in some embryonal carcinomas include α1-antitrypsin, Leu7, vimentin, LDH, ferritin, and human placental lactogen (22,276,292,293).

FIGURE 47.37 An immunostain for CD30 highlighting the cytoplasmic membranes of an embryonal carcinoma.

On ultrastructural examination, embryonal carcinomas appear as poorly differentiated adenocarcinomas, forming small glandular spaces with long tight junctional complexes and a peripheral basal lamina (34,294). The Golgi complex is usually prominent, and the nucleus is typically quite irregular in shape with a large, complex nucleolus and cytoplasmic inclusions. Cytogenetic and molecular analysis of embryonal carcinomas has identified the i(12p) marker chromosome or other 12p alterations, which can be diagnostically useful when embryonal carcinoma presents in a metastatic site without a known testicular lesion (232,295). Differential Diagnosis Embryonal carcinomas must be distinguished from several other types of tumor. It is most important to distinguish embryonal carcinoma from seminoma because of different treatment options offered to patients with seminomatous and nonseminomatous tumors. This important differential diagnosis has been discussed in the section “Seminoma.” Solid pattern yolk sac tumors may be confused with embryonal carcinomas; however, other patterns of yolk sac tumor frequently permit this distinction. In small specimens, solid pattern yolk sac tumor can usually be distinguished from embryonal carcinoma by virtue of the smaller size of its cells and nuclei and its less pleomorphic nature. The presence of hyaline globules or basement membrane deposits favors yolk sac tumor (238), as does AFP or Glypican 3 positivity and CD30, OCT3/4, or SOX2 negativity (Table 47.4). Often, tumors composed predominantly of embryonal carcinoma have foci of contiguous yolk sac tumor composed of smaller, vacuolated tumor cells, frequently in a myxoid background. The differential with “anaplastic” spermatocytic tumor is discussed in the section “Spermatocytic Tumor.” Large cell lymphomas may be confused with embryonal carcinomas but usually occur in older patients with extratesticular disease. Lymphomas often have an interstitial pattern; always lack GCNIS; and are negative for PLAP, OCT3/4 (with rare exceptions), and CK (247) and usually positive for leukocyte

common antigen (LCA) and other lymphoid markers (22,245). CD30 (and rarely OCT3/4) reactivity, however, may occur in both types of tumor. The most difficult diagnosis is the distinction of embryonal carcinoma in an extragonadal site from a metastatic undifferentiated carcinoma. The helpful immunostaining patterns for this problem have been discussed earlier, with OCT3/4 and SALL4 being particularly valuable. CD30 staining has been shown to be less frequently positive in postchemotherapy embryonal carcinomas (291), an important caveat to keep in mind. Mucin stains may be helpful as well because embryonal carcinomas lack cytoplasmic mucin, which may be identified in poorly differentiated carcinomas of somatic origin. The electron microscopic demonstration of the long tight junctions of embryonal carcinoma may also be helpful in this context (294). Treatment and Prognosis Nonseminomatous germ cell tumors, including embryonal carcinoma, are now effectively managed in most cases by surgery and/or chemotherapy. The form of treatment following radical orchiectomy is stage dependent. For clinical stage I disease, there is controversy regarding the best approach; one school of thought advocates retroperitoneal lymph node dissection (RPLND) of the nerve-sparing type in all such patients (296), whereas others advocate careful follow-up without additional treatment unless relapse develops (“surveillance” management). The overall survival for these two approaches is comparable (~98%), although the identification of certain features in the orchiectomy specimen (notably vascular invasion, large tumor size, and a high proportion of embryonal carcinoma) may be considered relative contraindications to surveillance management because of increased risk of relapse. Therefore, it is important for the examining pathologist to carefully examine the orchiectomy specimen for lymphovascular invasion and to estimate the percentage composition of the various components as well as to provide a measurement of the maximum tumor dimension. For patients with limited retroperitoneal metastases, treatment is RPLND and either close follow-up or postoperative adjuvant chemotherapy. Survival rates exceeding 95% are expected (297). In more advanced-stage disease, the usual treatment is initial cisplatin-based chemotherapy followed by resection of residual masses, if any exist. This group of patients has an overall survival of 70% to 80% (260,298), with a key prognostic finding being the nature of any residual tumor, whether teratomatous, which carries a good prognosis, or nonteratomatous germ cell tumor, which carries a less favorable prognosis. Pathologic findings in the resected residual tissue often determine the need for additional therapy (299). Assessment of completeness of excision and volume of residual malignant elements, in conjunction with accurate classification, is particularly important (300,301). For patients with disseminated disease, the prognosis appears to depend on factors, including tumor bulk (261,298,302-304), levels of serum markers (8,261,302-305), and proliferative index (305). Second- and third-line salvage regimens are used after failure of first-line chemotherapy with some success (306-308). POSTPUBERTAL-TYPE YOLK SAC TUMOR Teilum’s studies (309-311) provided the evidence that the neoplasm we now recognize as yolk sac tumor is of germ cell origin with differentiation toward the yolk sac and allantoic membranes. In postpubertal patients, it is much more commonly a component of a mixed germ cell tumor. By definition in the 2016 WHO classification, GCNIS is commonly present in the seminiferous tubules of postpubertal-type yolk sac tumor, but not in prepubertal-type yolk sac tumor (21,312). In Talerman’s study (313), there was a 44% frequency of a yolk sac tumor component in prospectively examined, nonseminomatous germ cell tumors. Most patients present with a mass but in a small proportion of cases, there are symptoms of metastatic disease or gynecomastia. Yolk sac tumor remains the most commonly overlooked component of nonseminomatous germ cell tumors (275,314). From 95% to 100% of patients with tumors containing yolk sac, tumor elements have elevated levels of serum AFP (315,316). This provides both an important diagnostic aid and a means of monitoring therapy and detecting recurrences. On gross examination, yolk sac tumor may appear as a nonencapsulated, gray to tan to yellow nodule, often with cystic change or a myxoid quality (Fig. 47.38); myxoid appearance is also commonly

seen in prepubertal-type cases (Fig. 47.39). Hemorrhagic and necrotic areas are common.

FIGURE 47.38 The cut surface of an adult yolk sac tumor showing areas of hemorrhage and cystic change.

FIGURE 47.39 A pediatric yolk sac tumor appears as a solid, yellow, myxoid nodule.

Yolk sac tumor has numerous patterns that produce an array of appearances. Fortunately, these patterns are usually admixed, thus facilitating their recognition. The following classification of yolk sac tumor patterns represents a modification of Talerman’s work (317); the 11 patterns are endodermal sinus (also designated perivascular or festoon), reticular (microcystic, honeycomb), macrocystic, papillary, solid, glandular-alveolar, myxomatous, sarcomatoid, polyvesicular vitelline, hepatoid, and parietal. The reticular pattern of yolk sac tumor is the most common one (238,318); solid and macrocystic patterns are also relatively frequent (238,279), whereas hepatoid, polyvesicular vitelline, and endodermal sinus structures are relatively infrequent (238,279). The endodermal sinus pattern has a central vessel in a core of mesenchyme that is mantled by a cuboidal to columnar layer of malignant cells and that is recessed into a cystic space (Fig. 47.40). These structures are referred to as “glomeruloid” or Schiller-Duval bodies. In addition, this pattern has fibrous cores of tissue draped (or “festooned”) by tumor cells that alternate with “labyrinthine” spaces (Fig. 47.41).

FIGURE 47.40 An endodermal sinus pattern of yolk sac tumor showing extensive basement membrane deposits (parietal differentiation).

FIGURE 47.41 Festoons of epithelium drape fibrovascular cores with intervening “labyrinthine” spaces, which are findings characteristic of the endodermal sinus pattern.

The reticular pattern is most common and consists of a network of tumor cells arranged in cords. Often, the cells have prominent cytoplasmic vacuolation, resulting in a microcystic or honeycomb appearance (Fig. 47.42). Sometimes, the cells, because of the extensive vacuolation, resemble lipoblasts or signet-ring cells. Often, tumor cords are dispersed in a prominent myxoid background that blends gradually into a myxomatous pattern. Larger cysts may develop in some yolk sac tumors, usually in association with a reticular (microcystic) pattern to produce a macrocystic pattern. It is likely that they derive from coalescence of microcysts.

FIGURE 47.42 Many cells resemble lipoblasts in a microcystic (reticular) yolk sac tumor.

Some yolk sac tumors form cystic spaces into which papillary processes project (Fig. 47.43). These papillae may contain fibrous cores or simply represent piled-up epithelium. Typically, the cells have relatively scant cytoplasm, with high nuclear-to-cytoplasmic ratios and some hobnail configurations. Intracystic, detached clusters of cells are frequent.

FIGURE 47.43 Small papillae, many with “hobnail” cells, are present in a yolk sac tumor.

The solid pattern has a sheetlike configuration of cells that may resemble seminoma, although there is usually a focal microcystic tendency, and the fibrous septation and prominent lymphocytes of seminoma are lacking (Fig. 47.44). Often, the cells have clear cytoplasm and well-defined borders, similar to seminoma, but there is association with other yolk sac tumor patterns. They are more pleomorphic than those of seminoma. Occasionally, the cells are small and primitive, resembling blastema (Fig. 47.45). CK, Glypican 3, and OCT3/4 stains (see Table 47.4) are useful in separating these two possibilities.

FIGURE 47.44 A solid pattern yolk sac tumor showing focal microcysts and lacks lymphoid infiltrates and fibrous septa.

FIGURE 47.45 Solid foci in a yolk sac tumor have a blastema-like quality; microcystic foci are also present.

Distinct glands may occur in yolk sac tumor, often developing from cystic, alveolar-like spaces lined by flattened epithelium. Thirty-four percent of yolk sac tumors had such glandular differentiation in one study (238). The glands have a primitive appearance, often with enteric features, including an apical brush border (Fig. 47.46). They may have extensive subnuclear vacuolation, similar to early secretoryphase endometrial glands. An anastomosing or branching arrangement is common, and rarely, a predominantly or purely glandular pattern occurs, although this is frequent in late recurrences (319). Differentiation of purely glandular yolk sac tumors from teratomas can be problematic; staining for AFP and Glypican 3 may assist. The branching, anastomosing pattern is typical of yolk sac tumor, and these glands lack a circumferential smooth muscle component, unlike many teratomatous glands (320).

FIGURE 47.46 Small glands are dispersed in a fibromyxoid stroma in a yolk sac tumor.

Some yolk sac tumors are extensively myxoid, with trabeculae, thin cords, and individual cells in a mucoid stroma having a high content of hyaluronic acid (Fig. 47.47). Individuals stellate to spindle cells trail into the myxoid and vascular stroma in apparent transition from the epithelium but retain CK reactivity (321). Teilum (322) considered this pattern a reiteration of the extraembryonic mesenchyme of the developing embryo. Occasionally, the spindle cells may undergo stromal differentiation, forming skeletal muscle (Fig. 47.48) and cartilage (321) and blurring the distinction between yolk sac tumor and teratoma. We believe that when such elements are intimately associated with yolk sac tumor, they should be classified as yolk sac tumor with, for example, rhabdomyoblastic differentiation. Some of the sarcomatous tumors observed in metastases following chemotherapy may derive from this mesenchymal component of yolk sac tumor (321,323).

FIGURE 47.47 A myxomatous pattern yolk sac tumor has thin cords of cells in an extensively mucoid stroma.

FIGURE 47.48 There is rhabdomyoblastic differentiation in a myxomatous pattern yolk sac tumor.

It also appears likely that the cellular, sarcomatoid foci that can be identified in occasional yolk sac tumors derive from the proliferation of the spindle and stellate cells originally associated with the myxomatous pattern. The transition of cells with a more epithelial phenotype into stellate and spindled cells represents a form of epithelial-mesenchymal transition (Fig. 47.49). The term “sarcomatoid pattern” is reserved for those cases where spindle cell areas are prominent and not readily viewed within the spectrum of myxoid pattern. This pattern tends to be especially prominent in postchemotherapy resections where it may be confused with a sarcoma. While sarcomatoid yolk sac tumor is much more often found in metastases following chemotherapy, it may also occur de novo in the untreated testis. The morphology is variable, but often multinodular with frequent abrupt transition from myxoid to collagenous areas and from hypocellular to hypercellular foci. The neoplastic cells have spindled and, in most cases, also epithelioid profiles. When the stroma is extensively myxoid, tumor cells may be arranged in a ringlike manner around central acellular, myxoid zones (Fig. 47.50). Sarcomatoid foci retain reactivity for low-molecular-weight CKs and also GPC3 (typically only focally), supporting their derivation from epithelial yolk sac tumor elements (Fig. 47.49). They are not associated with serum AFP elevation and are negative for AFP (324).

FIGURE 47.49 (A) Sarcomatoid pattern yolk sac tumor with spindled and stellate cells demonstrating moderate cytologic atypia in a fibrotic background. (B) The tumor cells are positive for Glypican 3.

The polyvesicular vitelline pattern is relatively unusual, occurring in 8% of cases in one series (238). It consists of vesicles, often with central constrictions, lined by flattened to cuboidal to columnar cells (Fig. 47.51). A transition in epithelial height may occur at the point of the constriction. The columnar cells resemble primitive enteric epithelium similar to that lining the embryonic allantois. Apical and basal vacuoles may occur. Teilum (325) felt that this pattern was analogous to the embryonic subdivision of the primary yolk sac into the secondary yolk sac. It has always been a minor component in testicular yolk sac tumors, in our experience, unlike in the ovary.

FIGURE 47.50 Sarcomatoid yolk sac tumor forming tumor ringlets (center).

FIGURE 47.51 Irregularly shaped and constricted vesicles, lined by flattened and intestinal-type columnar epithelium, are prominent in a polyvesicular vitelline pattern yolk sac tumor.

Some yolk sac tumors show hepatic differentiation, with clusters of polygonal cells with eosinophilic cytoplasm; round, vesicular nuclei; and prominent nucleoli (Fig. 47.52). These hepatoid cells may be arranged in sheetlike, trabecular, or nested patterns. Hepatoid differentiation is generally focal in approximately 20% of yolk sac tumors (238,326), but, in rare cases, may be predominant (327), although this would appear to be more common in ovarian tumors (328). Hepatoid foci are intensely positive for AFP and may contain hyaline globules. We have only rarely identified bile.

FIGURE 47.52 There are nests of polygonal hepatoid cells with abundant eosinophilic cytoplasm in a yolk sac tumor. A syncytiotrophoblast cell is also present.

A parietal pattern consists of confluent deposits of extracellular basement membrane between neoplastic cells. The term originates from the parietal layer of the embryonic yolk sac, which synthesizes a thick basement membrane (Reichert membrane). It is common to find scattered foci of parietal differentiation in yolk sac tumors (92% of cases (238)) where it can aid in diagnosis. It is seen in several patterns, including reticular, endodermal sinus, and solid, and consists of focal, wispy to bandlike deposits of eosinophilic matrix (Fig. 47.40). It is rare, however, to have a predominance of extracellular basement membrane in a primary tumor such that the associated pattern is obliterated; when this occurs, it is designated a “parietal pattern” (Fig. 47.53). Parietal patterns occur with greater frequency after chemotherapy and as late recurrences (319,329).

FIGURE 47.53 A parietal yolk sac tumor showing extensive deposits of basement membrane that efface most of the underlying microcystic pattern.

Hyaline globules commonly occur in yolk sac tumor and can aid in its recognition. These are round, eosinophilic, PAS-positive, diastase-resistant globules of variable size ranging from 1 to 50 µm or more in diameter (Fig. 47.54). They also occur in other neoplasms but are uncommon in seminoma and embryonal carcinoma (238). They generally do not stain for AFP. Such globules should not be confused with the more irregularly shaped, bandlike deposits of basement membrane that are the hallmark of parietal differentiation.

FIGURE 47.54 Numerous eosinophilic hyaline globules are present in a microcystic and solid yolk sac tumor.

AFP can be identified in the cytoplasm of most yolk sac tumors, with frequencies ranging from 55% to 100% (22,224,226,276); the staining is often patchy (Fig. 47.55 and Table 47.4). Glypican 3 is a newer marker for yolk sac tumor with a greater sensitivity (Fig. 47.56), although it may also be seen in syncytiotrophoblastic cells as well as occasional teratomas and embryonal carcinomas (239,240). Hepatoid foci are generally intensely AFP positive and are also usually reactive for HepPar1 and Glypican 3. The enteric glandular structures of yolk sac tumor may stain positively for CEA (22,238,293). α1-Antitrypsin can be identified in approximately 50% of cases (22,224). Pankeratins (e.g., AE1/3) are diffusely positive in virtually all yolk sac tumors (22,330), and vimentin can be demonstrated in the stromal component (330). PLAP is not as likely to be reactive in yolk sac tumor as in most other types of germ cell tumor, with 39% to 85% being positive (22,155,285). EMA is usually negative (22), as is CK7, similar to ovarian examples (331) (Table 47.4).

FIGURE 47.55 There is the characteristic patchy distribution of α-fetoprotein positivity in a yolk sac tumor.

FIGURE 47.56 Strong and diffuse positivity for Glypican 3 in a yolk sac tumor.

By electron microscopy, yolk sac tumors show epithelial cells with tight junctional complexes and apical microvilli. Extracellular basal laminar deposits may be prominent, with similar-appearing material, often with a central lucent zone, within dilated cisternae of endoplasmic reticulum (34). Glycogen is commonly seen, and the nuclei are irregular in shape with a “wandering” nucleolonema. Differential Diagnosis It is important to distinguish solid patterns of yolk sac tumor from seminoma, a differential diagnosis addressed in the section “Seminoma.” Embryonal carcinomas are distinguished based on the absence of the distinctive patterns of yolk sac tumor and their larger, more pleomorphic nuclei. Hyaline globules and deposits of basement membrane are rarely seen in embryonal carcinoma (238), whereas most express CD30, unlike yolk sac tumor (288,290). There are rare cases that appear to be transitional morphologies between embryonal carcinoma and yolk sac tumor, and examination of other sections usually shows both tumor types. Glandular patterns of yolk sac tumor lack the circumferential muscle seen in the glandular components of many teratomas (320). Glandular patterns of yolk sac tumor may be seen as late relapses in the retroperitoneum and are mistaken for adenocarcinomatous transformation, with distinction facilitated by AFP and Glypican 3 positivity and EMA and CK7 negativity of glandular yolk sac tumor (332). Invasion of the rete testis by germ cell tumor elements can cause a hyperplastic epithelial reaction with intracytoplasmic hyaline globules that may be misinterpreted as a yolk sac tumor component (333)

(Fig. 47.57). This intrarete hyperplasia is differentiated from yolk sac tumor based on the characteristic branching pattern, as seen at low magnification, and the bland cytologic features.

FIGURE 47.57 Hyperplasia of the rete testis with hyaline globules simulates yolk sac tumor. Embryonal carcinoma is adjacent.

Treatment and Prognosis The treatment of adult patients with yolk sac tumor is similar to that of other nonseminomatous germ cell tumors (see section “Embryonal Carcinoma”). The presence of a yolk sac tumor component in a testicular primary may be associated with a higher frequency of low-stage disease (334,335). Differentiation from prepubertal-type yolk sac is based primarily on the age of the patient and presence of GCNIS in the background seminiferous tubules; genetic studies are unnecessary. A yolk sac tumor component in a metastatic lesion may impart a worse prognosis (336), despite the opposite impact when in the testis (335). This finding probably reflects a greater chemoresistance of yolk sac tumor than other germ cell tumor elements, a conclusion supported by autopsy studies documenting a much greater frequency of residual yolk sac tumor in patients dying in the chemotherapeutic era compared to those dying before effective chemotherapy (337). Chemoresistant spindle cell tumors of variable morphology are often sarcomatoid yolk sac tumor or derived from yolk sac tumor elements and may be the source of ultimately fatal sarcomatoid tumors (323,324).

TROPHOBLASTIC TUMORS CHORIOCARCINOMA Pure testicular choriocarcinomas are rare (0.3% of testicular tumors (194)), but a component of choriocarcinoma is identified in 16% of mixed germ cell tumors on careful examination (175), typically as microfoci. Patients with pure tumors are usually in the second or third decade and often present with metastatic symptoms rather than a testicular mass; some may have no palpable testicular abnormalities on clinical examination. No cases are described in prepubertal children. Frequent presenting symptoms are hemoptysis, lumbar back pain, gastrointestinal bleeding, and neurologic abnormalities. There is often marked elevation of serum hCG levels and secondary endocrine abnormalities, including gynecomastia (which may also be a presenting complaint) and thyrotoxicosis (because of the thyroidstimulating hormone–like activity of hCG) (338). On gross examination, choriocarcinomas are often small, and the testis may appear normal or atrophic from its external aspect. On cut surface, a hemorrhagic and centrally necrotic nodule is generally identified, often with gray tissue at its periphery (Fig. 47.58).

FIGURE 47.58 A nodule of choriocarcinoma showing the usual hemorrhagic and necrotic features.

Choriocarcinomas consist of a proliferation of malignant trophoblastic cells. In its classic form, a central area of hemorrhage and necrosis is surrounded by an admixture of two distinct populations of cells—mononuclear cells with generally clear or pale cytoplasm and mild-to-moderate nuclear pleomorphism (cytotrophoblast and intermediate trophoblast cells) and multinucleated cells, often with intracytoplasmic lacunae containing erythrocytes, that have abundant amphophilic cytoplasm (syncytiotrophoblastic cells). Smudged nuclear chromatin is common in the syncytiotrophoblastic cells. In the better differentiated cases, the syncytiotrophoblastic cells “cap” the mononucleated trophoblast population (Fig. 47.59), similar to the normal arrangement of trophoblast on placental villi. More commonly, intermingling of these elements is random.

FIGURE 47.59 A classic pattern of choriocarcinoma showing clusters of lightly staining, mononucleated trophoblast cells capped by multinucleated syncytiotrophoblast cells in a hemorrhagic and cystic background.

In some cases, the syncytiotrophoblastic cells are scant, occurring as scattered cells among sheets of mononucleated trophoblast cells (Fig. 47.60) (339). This “monophasic” pattern is one that we have more commonly seen following chemotherapy. Not only may the syncytiotrophoblastic cells be minor in amount, but they may also have a shrunken and degenerate appearance with smudged nuclei. In this circumstance, they are mainly recognizable by their more deeply staining cytoplasm. It can be difficult to

distinguish such cases from embryonal carcinoma with cellular degeneration (“appliqué” pattern). Because syncytiotrophoblastic cells are largely responsible for the production of hCG, immunostains for hCG usually show only focal to rare reactivity in the monophasic pattern. On the other hand, GATA3 is diffusely expressed, and inhibin tends to show greater reactivity than hCG.

FIGURE 47.60 A “monophasic” choriocarcinoma is composed of pleomorphic, mononucleated trophoblast cells in a fibrinoid background without distinct syncytiotrophoblast cells.

There are rare cases in which the separation of the trophoblast elements into two separate populations becomes indistinct, but there is a proliferation of a spectrum of malignant trophoblast cells, from small to large; this pattern, in our experience, is also more common in postchemotherapy specimens. Because of the extensive hemorrhagic necrosis of most choriocarcinomas, diagnostic foci of viable cells may require many sections. Such foci are usually peripheral, and an additional clue to the diagnosis is GCNIS in the adjacent seminiferous tubules. Pyknotic syncytiotrophoblastic cells may be identified within the necrotic zone to encourage continued searching for diagnostic foci. Choriocarcinomas, like physiologic trophoblast, have a marked tendency for angioinvasion that correlates with their clinical aggressiveness. Stains for hCG are valuable in helping to establish a diagnosis of choriocarcinoma and are positive in essentially all cases, mainly in the syncytiotrophoblastic cells but also in scattered large mononuclear cells that may be transitional forms between cytotrophoblast and syncytiotrophoblastic cells (22,224,340). Strong nuclear GATA3 staining occurs in both mononucleated trophoblasts and syncytiotrophoblasts (341) and was found to be sensitive (78%) in the larger experience with gestational choriocarcinomas (342). Glypican 3 is positive in choriocarcinomas, but its reactivity is less intense compared to that of yolk sac tumor (239). Syncytiotrophoblastic cells are also positive for pregnancyspecific β1-glycoprotein (340) and inhibin-α (343-345). Mononucleated trophoblast cells are usually, but not always, hCG negative and SALL4 positive (346). Those having intermediate trophoblast differentiation may stain positively for human placental lactogen and HLA-G (347). PLAP is positive in approximately 50% of choriocarcinomas (22), and CEA is positive in 25% of choriocarcinomas (22,348). Choriocarcinomas contain CK7, CK8, CK18, and CK19 (349). Unlike several forms of germ cell tumor, EMA is positive in a significant number of choriocarcinomas (46%), mainly in the syncytiotrophoblasts (22) (Table 47.4). Differential Diagnosis It is important to distinguish choriocarcinoma from hemorrhagic testicular necrosis as a result of torsion, trauma, vascular lesions, or clotting abnormalities. Clinically, testicular infarction usually produces painful testicular enlargement, whereas patients with choriocarcinomas often have small, nonpainful testes. On cross section, infarction usually has a diffuse hemorrhagic picture, whereas a nodular area of

hemorrhagic necrosis is typical of choriocarcinoma. Coagulative necrosis characterizes infarction, but residual tissue structures cannot be identified in the central hemorrhagic and necrotic areas of choriocarcinomas. The presence of GCNIS and hCG immunostains may be of additional help. It is also important to distinguish choriocarcinomas from other types of germ cell tumors that contain trophoblast cells. This distinction is based on the scattered nature and usually minor amounts of the trophoblastic elements, lack of a mononucleated trophoblast component, and the absence of hemorrhagic necrosis in the latter. Embryonal carcinoma with cellular degeneration may be distinguished from choriocarcinoma by hCG stains. Monophasic choriocarcinoma should be distinguished from solid pattern yolk sac tumor and seminoma with syncytiotrophoblastic cells. The expression of trophoblastic hormones in the mononucleated cells is helpful in this regard, as are the AFP reactivity of yolk sac tumor and the OCT3/4 positivity of seminoma. The placental site trophoblastic tumor (PSTT) (see following section) is distinguished from choriocarcinoma based on the predominant to exclusive presence of intermediate trophoblast cells, as supported by diffuse reactivity for human placental lactogen and the absence of or minor reactivity for hCG. Treatment and Prognosis Choriocarcinomas disseminate rapidly, often via hematogenous routes. Brain involvement occurs with disproportionate frequency (32). It is treated similarly to other nonseminomatous tumors, but a significant choriocarcinomatous component in a testicular germ cell tumor probably worsens the prognosis based on several studies in which multivariate analysis correlated a poor outcome with high hCG titers and nonpulmonary visceral metastases (350-352). In some studies, direct identification of choriocarcinoma or “trophoblastic” elements correlated with a poorer prognosis (350,351,353,354). A “choriocarcinoma” syndrome has been identified in which patients have hemorrhagic visceral metastases and a high mortality despite aggressive therapy (350). Regardless, cures of metastatic choriocarcinoma do occur (350,355). Choriocarcinomas as a component of mixed germ cell tumors may behave less aggressively than do pure choriocarcinomas (266,356-358). PLACENTAL SITE TROPHOBLASTIC TUMOR Rare cases of testis tumors resemble the PSTT of the uterus. One tumor occurred in a 16-month-old infant and consisted of an interstitial proliferation of intermediate trophoblast cells (Fig. 47.61). The patient was well on follow-up, 8 years after orchiectomy (339). Small foci of similar appearance may be seen in some typical choriocarcinomas (340) and with teratoma (359). On immunochemistry, they are typically positive for GATA3 and human placental lactogen and negative for p63. Knowledge on behavior is very limited; the two reported cases did not recur during long follow-up after orchiectomy (339,359).

FIGURE 47.61 A placental site trophoblastic tumor showing interstitial growth of intermediate trophoblasts admixed with Leydig cells.

EPITHELIOID TROPHOBLASTIC TUMOR

Epithelioid trophoblastic tumor (ETT) has been seen in both untreated primary tumors and metastases after chemotherapy (360,361). It consists of squamoid-looking cells, which are in fact intermediate trophoblast with highly eosinophilic cytoplasm (Fig. 47.62). Hyaline globules are seen frequently, which contain apoptotic nuclear debris. On immunochemistry, they are typically positive for GATA3, p63, and cyclin E and usually negative or only focally positive for human placental lactogen. Data on the behavior of ETT are limited; most patients were alive and well after tumor resection, with one rare case of death due to liver metastasis (361).

FIGURE 47.62 Epithelioid trophoblastic tumor with densely eosinophilic cytoplasm, prominent cytoplasmic membranes, nuclear pleomorphism, and intracytoplasmic hyaline globules.

CYSTIC TROPHOBLASTIC TUMOR This lesion is characteristically seen after chemotherapy and, therefore, usually in a metastatic site (362), but it also occurs de novo in the testis (363). It consists of a cyst lined by mostly mononucleated trophoblast cells that usually have irregular, pleomorphic nuclei but inconspicuous mitotic activity (Fig. 47.63). Hemorrhage and tumor necrosis are absent. Stains for hCG are usually only focally positive. In metastases, it appears to behave similarly to teratoma and, therefore, does not require adjuvant therapy (362).

FIGURE 47.63 Cystic trophoblastic tumor consisting of a cyst lined by squamoid, occasionally vacuolated, mononucleated trophoblast cells admixed with fibrinoid material.

POSTPUBERTAL-TYPE TERATOMA

In adult patients, postpubertal-type teratoma usually occurs as one component of a mixed germ cell tumor, with more than 50% of all mixed germ cell tumors having a teratomatous component (176,364); pure cases in adults are uncommon. The adult patients have the typical features of a testicular germ cell tumor (i.e., they are generally young, present with a testicular mass, but may have symptoms secondary to metastatic involvement and may have serum marker elevations depending on the nature of the associated components). One puzzling aspect about the biology of testicular teratomas in adults is the occasional development of nonteratomatous metastases in patients with apparently pure teratomas (358). This phenomenon is probably due to the usual development of the adult form of teratoma from a nonteratomatous precursor element (for instance, embryonal carcinoma) that can metastasize as such but also transform to teratoma in the testis (365). Transformation to teratoma in the metastatic site as well explains cases reported as “mature teratoma metastasizing as mature teratoma” (366-369). Ninety percent of adult patients with pure teratoma also have GCNIS (370) and harbor the same common abnormalities seen in other malignant germ cell tumors. Therefore, it is important to recognize that postpubertal-type teratoma of the testis always has malignant potential. Serum marker studies of patients with pure testicular teratomas are generally negative, although the immunohistochemical detection of AFP in teratomatous glands indicates a potential for abnormal AFP values (206,224,293). On gross examination, teratomas are often multinodular, and their cut surface varies from multicystic (Fig. 47.64) to solid. Nodules of translucent, white cartilage may be seen. The cysts may be filled with clear or mucoid fluid or keratinous material. Hair is not identified in postpubertal-type teratomas. Areas of immature tissue may have a fleshy, encephaloid character.

FIGURE 47.64 A pure teratoma with multiple cysts. Photograph courtesy of D. J. Gersell, MD, St. Louis, MO.

The characterization of testicular teratomas as “mature” or “immature” had been removed from the WHO classification (10). This is because immaturity in the epithelial or stromal components has been shown to have no prognostic significance. On microscopic examination, pure teratomas, by definition, are composed of tissues resembling somatic tissues (Fig. 47.65). In postpubertal patients, there is frequent cytologic atypia of such tissues that reflects their malignant potential and development from GCNIS. This is supported by the demonstration of aneuploidy (371,372). Common findings include nodules of atypical cartilage (Fig. 47.66), glands lined by gastrointestinal or respiratory epithelium with muscular cuffs, squamous islands, transitional epithelium, neuroglia, pigmented retinal epithelium, and fibrous stroma. Bone, liver, pancreas, thyroid, prostate, meninges, kidney, and choroid plexus are uncommon. Sometimes, the glands are architecturally complex (Fig. 47.67). A granulomatous reaction to extravasated keratin is common.

FIGURE 47.65 A teratoma consists of intestinal-type glands, fat, and stroma.

FIGURE 47.66 The cartilage in a testicular teratoma from an adult patient showing cytologic atypia.

FIGURE 47.67 The glands are architecturally complex in a teratoma.

Islands of immature neuroectoderm (Fig. 47.68), as well as primitive tubules, similar to those seen in nephroblastoma and often with accompanying blastema (Fig. 47.69), may also occur, as may embryonic skeletal muscle and nonspecific cellular stroma. There is no point, however, in grading the degree of immaturity in the adult tumors, unlike in ovarian cases, because they derive from invasive malignant germ cell tumors and may, therefore, have associated metastases even if completely mature.

Immunohistochemical staining of teratomas yields the expected results for the specific elements examined. Thus, neural markers are identified in neural tissues (373), chromogranin in neuroendocrine cells (343), CKs in epithelia, vimentin in stromal tissues, and desmin in muscle (330). AFP may be seen in enteric- or respiratory-type glandular structures as well as in liver (206,224,293). CEA, α1-antitrypsin, and ferritin occur in teratomatous epithelium in approximately 50% of cases (293), and PLAP is also positive in glandular teratomatous structures in a minority of cases (155,207,285). p53 protein may be identified in teratomatous epithelium (374). SALL4 reactivity occurs in some teratomas, particularly in enteric-type glands and immature neuroectodermal tissue (346).

FIGURE 47.68 There are focal immature tissues in a teratoma, with small collections of embryonic-appearing neuroectoderm.

FIGURE 47.69 Collections of primitive-appearing tubules and blastema-like cells are seen in a teratoma.

Treatment and Prognosis Pure postpubertal teratomas in adults are uncommon with most examples associated with other germ cell elements, which determine the prognosis. If pure, they may nonetheless have associated metastases of teratomatous or nonteratomatous germ cell tumors for reasons already discussed. At two referral centers for treatment, the proportion of pure postpubertal teratomas with metastases was slightly more than 40% (375,376), a high figure that likely reflects referral bias. In the Armed Forces Institute of Pathology series, 21% metastasized (377). It is not uncommon for the testes in these cases to exhibit parenchymal scarring, suggestive of regression of other nonteratomatous germ cell tumor elements. SOMATIC-TYPE MALIGNANCY ARISING IN TERATOMA

It is important to recognize the development of independently evolving neoplasms from overgrowth of neuroectoderm, nephroblastoma-like tissues, rhabdomyoblastic cells, or other types of primitive cells in any teratoma. However, these assessments are challenging. A guideline for such overgrowth is size in excess of the majority of a low-power field (×4 objective, 5 mm in diameter); overgrowth of neuroectoderm to this extent results in a PNET (see next paragraph; Fig. 47.70) (378) and similarly for a nephroblastoma-like tumor (Fig. 47.71) (379) and embryonal rhabdomyosarcoma. Alternatively, malignant but “mature” stromal tissues can also overgrow teratomas, giving rise, for example, to “teratoma with leiomyosarcoma.” Secondary carcinomas in teratoma are appreciable when there is evidence of stromal invasion by malignant-appearing epithelium (Fig. 47.72). It should be noted that in some studies, no adverse prognostic significance was associated with secondary teratomatous malignancies when identified in the testis (380), although their presence in metastatic sites and especially in the mediastinum was ominous (378,380-383). Grading of the sarcomatous tumors into lowand high-grade groups, using the criteria of the French grading system for sarcomas, may be prognostically useful.

FIGURE 47.70 Overgrowth of primitive neuroectoderm in a teratoma results in a primitive neuroectodermal tumor (new terminology “embryonic-type neuroectodermal tumor”).

FIGURE 47.71 An area in a teratoma has the features of an epithelial-predominant nephroblastoma.

FIGURE 47.72 An adenocarcinoma in a teratoma showing invasive growth.

Occasionally, it is presumed that a single neoplastic element completely overgrows the initial teratoma, leading to a monodermal teratoma. PNETs of the testis are also considered monodermal teratomas consisting of small cells arranged in diffuse sheets, tubules, rosettes, and pseudorosettes (Fig. 47.70) (384) They will undergo a name change (“embryonic-type neuroectodermal tumors [ENTs]”) to align with the modifications made in the WHO CNS classification and to avoid confusion with Ewing sarcoma. ENTs represent overgrowth of primitive neural elements of teratoma and, hence, are usually the components of germ cell tumors with teratomatous and other elements (378,385). Rarely, they represent a pure testicular tumor (384,386,387). On gross examination, the pure cases are fleshy, gray white, and partially necrotic. Microscopically, small cells with hyperchromatic nuclei are arranged in sheets, neural-type tubules, and rosettes. Neuron-specific enolase, synaptophysin, chromogranin, and CD99 are positive in varying proportions of cases (378). Neurosecretory granules may be present on ultrastructural examination (387). The absence of chromosome 22 rearrangements on fluorescence in situ hybridization (FISH) study supports that they are usually not tumors related to Ewing sarcoma (388). Instead, they most resemble “embryonal” tumors as seen in the central nervous system (CNS) of children. Distinction from teratoma with neuroectodermal elements is based on the overgrowth of embryonic-type neuroectoderm. Differential diagnostic considerations for ENT are discussed below. Small cell carcinoma metastatic to the testis typically occurs in older patients, does not form the tubular structures characteristic of ENT, has more impressive CK reactivity, and lacks associated GCNIS. Nephroblastoma-like tumors have better defined tubular structures and usually blastema and stroma (379); the neural markers are negative and WT1 positive. Clinical stage I patients with a component of ENT have a higher rate of relapse than comparable patients lacking such a component (389). Metastatic cases of ENT have a very poor outcome (378,389).

MIXED GERM CELL TUMORS Although the various types of germ cell tumors discussed in the previous sections may occur as pure tumors, it is much more common, except for seminoma, to see combinations of different neoplastic types. From 69% to 91% of the nonseminomatous testicular germ cell tumors are of mixed types (175,364). In diagnosing such cases, the recommended term is mixed germ cell tumor, followed by a parenthetical listing of the components with an estimate of their relative proportions in percentages. On gross examination, mixed germ cell tumors are characteristically variegated, reflecting their different components, with foci of hemorrhage and necrosis (Fig. 47.73). Microscopically, the appearance is identical to the various pure neoplasms. There is a tendency for many pathologists to overlook foci of yolk sac tumor (275) that characteristically develop in close association with an embryonal carcinoma component. The “double-layered” pattern of embryonal carcinoma (279) (Fig. 47.30) should be regarded as an example of a mixed germ cell tumor composed of embryonal carcinoma and yolk sac tumor.

There is evidence that tumors composed of embryonal carcinoma and teratoma behave in a less aggressive manner than those consisting of pure embryonal carcinoma (390), and yolk sac tumor elements may also favorably alter the behavior of mixed germ cell tumors (335).

FIGURE 47.73 A mixed germ cell tumor has the typical variegated appearance, with areas of hemorrhage, necrosis, and cystic change.

POLYEMBRYOMA AND DIFFUSE EMBRYOMA Both polyembryoma and diffuse embryoma are distinctive forms of mixed germ cell tumors. Their behavior is typical of nonseminomatous germ cell tumors. Polyembryoma consists of small, scattered, embryo-like bodies having a central core of embryonal carcinoma cells (resembling the embryonic plate), an associated amniotic-like cavity, and a yolk sac tumor component resembling the embryonic yolk sac (391-393) (Fig. 47.74). Syncytiotrophoblasts are often associated with the amniotic-like and yolk sac–like components, and intestinal, hepatic, and squamous differentiation may occur (391). Thus, these tumors are considered to have components of embryonal carcinoma and yolk sac tumor and, sometimes, teratomatous and syncytiotrophoblastic components. In diffuse embryoma, there is a diffuse admixture of approximately equal amounts of embryonal carcinoma and yolk sac tumor elements (Fig. 47.75) (394,395). These components retain their expected immunoreactivities.

FIGURE 47.74 An “embryoid body” in a polyembryoma has an amniotic-like cavity, core of embryonal carcinoma, and yolk sac tumor, resembling the embryonic yolk sac.

FIGURE 47.75 A “diffuse embryoma” consists of admixed embryonal carcinoma and yolk sac tumor in approximately equal amounts.

REGRESSED GERM CELL TUMORS Some patients with metastatic germ cell tumors are noted, either at autopsy or orchiectomy, to have testicular scars that may be associated with GCNIS, intratubular coarse calcifications, or teratomatous elements. These patients are felt to have had regression of most or all of their primary neoplasm (280). Approximately 10% of male patients dying of metastatic germ cell tumors have such “burnt-out” primary tumors (396), and it is especially likely to occur in patients with metastatic choriocarcinoma (397) but may be associated with other germ cell tumor types as well. It is likely that most patients with isolated retroperitoneal germ cell tumors have metastatic retroperitoneal disease with tumor regression in the testis (398). On microscopic examination of the testis, there are fibrotic scars with prominent numbers of small blood vessels that may also contain lymphoplasmacytic infiltrates, hemosiderin-laden macrophages, “ghost” tubules, and intratubular coarse calcifications (Fig. 47.76) (281). The latter probably correspond to complete comedo-type necrosis of intratubular embryonal carcinoma with dystrophic calcification. GCNIS may be identified in residual seminiferous tubules in approximately half of the cases (281,398). The testis peripheral to the scar invariably shows atrophy with impaired spermatogenesis (281), reflecting the usual background on which testicular germ cell tumors develop. Many cases of “pure teratoma” were likely originally mixed germ cell tumors with regression of the nonteratomatous elements (399).

FIGURE 47.76 A “burnt-out” germ cell tumor consists of a scar containing coarse intratubular calcifications.

Immunochemistry may be helpful in defining whether a necrotic or mostly necrotic tumor is a seminoma or nonseminoma (400). A CK, OCT3/4, CD117, and CD30 panel can show differential patterns of expression even in severely necrotic tumors, although only distinct reactivity in the appropriate cellular location ought to be considered positive.

POSTCHEMOTHERAPY RESECTIONS Patients with metastatic testicular germ cell tumors commonly undergo postchemotherapy resection of residual masses, and the interpretation of these specimens can pose problems, even to experienced pathologists (299). The diagnosis of residual, nonteratomatous germ cell tumor in these cases is often considered an indication for additional chemotherapy, whereas teratoma, necrosis, and fibrotic or reparative reactions are not (401,402). Some studies have suggested that patients with very small volumes of nonteratoma on RPLND may escape further salvage therapy; thus, quantification of the residual tumor may be important (300,301). Necrotic foci are often surrounded by a prominent infiltrate of foamy macrophages, sometimes containing hemosiderin, and an active fibroblastic proliferation (Fig. 47.77). The cytoplasmic clarity of the macrophages may cause a misinterpretation as seminoma, but the nuclei, although sometimes “active appearing” with small- to moderate-sized nucleoli, lack malignant features. In the central portion of the necrosis, pyknotic nuclei in the “ghost-like” outlines of tumor cells should not be considered evidence of persistent germ cell tumor.

FIGURE 47.77 A postchemotherapy specimen showing the typical coagulative necrosis surrounded by a prominent foamy macrophage reaction with cholesterol clefts.

Fibrotic foci often contain widely scattered, spindle-shaped to epithelioid-appearing cells with nuclear atypia. CK stains may highlight these cells, confirming they have an epithelial phenotype and supporting the belief they often derive from the “mesenchymal” component of yolk sac tumor and, indeed, such stroma has the same genetic profile of the associated germ cell tumor elements (403). Others may represent persistent trophoblastic cells. As long as the atypical cells are widely scattered as individual or small clusters and show no or only rare mitotic figures, we do not further qualify our diagnosis of “fibrosis” in this context. A greater degree of cellularity or proliferative activity may merit comment, but it is unlikely to alter the future therapy (i.e., close follow-up rather than additional chemotherapy). Occasionally, repeated recurrences show progressively increased cellularity and atypia of “fibrotic” foci until a clear-cut “sarcoma” is recognizable (323). Some of these “sarcomas” appear, however, to represent sarcomatoid differentiation of yolk sac tumor (see section “Postpubertal-Type Yolk Sac Tumor”) (324). Persistent teratoma often shows significant cytologic atypia in both mesenchymal and epithelial components. In the absence of stromal invasion or overgrowth, this atypia has no significant impact on

prognosis and is not considered an indication for additional chemotherapy (404). The development of carcinoma or sarcoma, manifest by invasion or overgrowth, is associated with an aggressive course, but the treatment primarily remains surgical excision (381,404). Most of the examples of ENT and nephroblastoma-like tumor in patients with testicular germ cell tumors are seen in postchemotherapy resections of residual masses (378,379). Although most forms of germ cell tumor in postchemotherapy resections have a similar morphology as in untreated cases, immunochemistry for CD30 is often negative in embryonal carcinomas after chemotherapy (291). Also, we have noted a tendency for some choriocarcinomas to lack a well-defined biphasic pattern and mainly consist of mononucleated trophoblast cells (monophasic choriocarcinoma), as was noted in treated gestational choriocarcinoma (405). Tumors with a monophasic appearance are regarded as persistent choriocarcinoma, and most patients, as a result, receive additional chemotherapy. Marked cystic transformation of choriocarcinoma, with a lining epithelium resembling atypical squamoid cells (cystic trophoblastic tumor), may also occur but should not be considered a reason for additional chemotherapy as these behave similarly to teratomas (362). Persistent (or late recurrent) yolk sac tumor may have more prominent parietal, hepatoid, and glandular features than in untreated patients and may be mistaken for adenocarcinoma in the latter circumstance, although SALL4, AFP, and/or GPC3 positivity and negativity for EMA and CK7 are helpful in recognizing glandular yolk sac tumor. OCT3/4 is useful in identification of seminoma or embryonal carcinoma, but only nuclear positivity should be regarded because cytoplasmic positivity may represent other nonneoplastic tissue, such as paraganglia (463).

GERM CELL TUMORS UNRELATED TO GERM CELL NEOPLASIA IN SITU This group of tumors, new to the 2016 WHO classification, forms a group of two unrelated types of germ cell tumor. They are classified by the Looijenga/Oosterhuis model (Table 47.3) as type I and type III germ cell neoplasia and are brought together in the 2016 classification for ease, rather than any relationship between the mainly pediatric tumors and spermatocytic tumor. PREPUBERTAL-TYPE TERATOMA In prepubertal patients, they almost always are pure neoplasms and represent approximately 15% of testicular germ cell tumors (the others being yolk sac tumors) (406,407). The mean age at diagnosis is 20 months, and occurrence beyond 4 years is unusual (406). Most patients present with a testicular mass identified by a parent. The biology of prepubertal-type teratomas is significantly different from those associated with GCNIS. Karyotypic and molecular studies of pediatric prepubertal testicular teratomas have yielded normal results (408,409). It has been recognized that the specific features and natural history of prepubertal teratomas can be seen in adult patients (410,411), leading to the term prepubertal type. In adults, this is a diagnosis of exclusion as it has clinical consequences entirely different from postpubertal-type teratomas. In postpubertal patients, diagnosis should be limited to pure teratomas with no cytologic atypia, no associated GCNIS, and no evidence of regressive or atrophic changes in the background parenchyma. Diagnosis can also be supported by a lack of isochromosome 12p. These cases have a typical organoid arrangement of tissues, particularly of respiratory type, and otherwise have similar features to the dermoid-type teratomas. These cases have had a benign outcome. It has been suggested that they may represent a prepubertal teratoma that had not been identified in childhood and persisted into adult life (410,412). Although immaturity is disregarded in the classification of postpubertal-type testicular teratomas, it has potential value in the prepubertal group, mainly because they are closely related to teratomas of the ovary and sacrococcygeal region. Even though grading has yet to be shown to have prognostic value, we recommend using the same method for assessing the quantity of immature neuroectodermal tissue in ovarian and sacrococcygeal teratomas (413,414). Epidermoid and Dermoid Cyst

Dermoid cyst and epidermoid cyst should be considered a specialized form of prepubertal-type testicular teratoma. They are both benign lesions. Epidermoid cyst, by definition, is a squamous epithelial-lined cyst, typically filled with keratin; it represents approximately 1% of testicular masses (415,416). Epidermoid cysts are most common from the second to fourth decades and present as palpable masses (415). They are usually approximately 2 cm in diameter (417) and contain the characteristic yellow-white, “cheesy” debris, often having a laminated appearance typical of keratin (Fig. 47.78). On microscopic examination, a fibrous wall is lined by keratinizing squamous epithelium with a granular cell layer but no skin appendages (Fig. 47.79). Areas of rupture may allow keratin to escape and result in a granulomatous and fibrous reaction. There is no associated GCNIS (370,418), and spermatogenesis, except for the effects of compression atrophy, is generally intact. They are benign lesions (415,416) but must be distinguished from postpubertal teratoma because the absence of GCNIS and “other” teratomatous elements is key in this regard. Epidermoid cyst may be managed by conservative local excision if the diagnosis can be assured, typically by biopsies of the surrounding testis to exclude GCNIS (418).

FIGURE 47.78 An epidermoid cyst contains laminated, white keratinous material. Photograph courtesy of S. F. Cramer, MD, Rochester General Hospital, Rochester, NY.

FIGURE 47.79 An epidermoid cyst is lined by keratinizing squamous epithelium and contains central masses of keratin; the adjacent seminiferous tubules lack intratubular germ cell neoplasia, unclassified type.

Dermoid cyst was initially described in patients in the typical age range for germ cell tumors (419). It is now classified as a subtype of prepubertal teratoma, with differentiation purely to skin appendage structures. On gross examination, there is a cyst filled with keratin and, in some cases, hair. On microscopic examination, pilosebaceous units are oriented in a skin-like arrangement to a squamous epithelial surface (Fig. 47.80). It also lacks GCNIS and cytologic atypia and is associated with normal

spermatogenesis and the absence of chromosome 12p abnormalities (410). Many examples have an associated lipogranulomatous reaction. Parenchymal scarring suggestive of partial regression is absent.

FIGURE 47.80 A dermoid cyst is lined by keratinizing, squamous epithelium with numerous pilosebaceous units but also contains a dilated gland with goblet cells.

Differential Diagnosis It is vital to distinguish postpubertal-type teratomas from prepubertal-type teratomas in adults (194,410,415,416,419-421). In children, this is hardly an issue. The absence of GCNIS and elements other than a squamous epithelial-lined cyst serves to distinguish epidermoid cyst from mature teratoma. Although dermoid cyst may have other teratomatous elements, the organoid arrangement of pilosebaceous units to an epidermal surface, the absence of GCNIS, lack of cytologic atypia, normal surrounding parenchyma, and the frequently associated lipogranulomatous reaction allow distinction from teratoma. As mentioned earlier, they lack i(12p) or other forms of 12p alterations (410). Treatment and Prognosis Prepubertal-type teratomas, including immature ones, are generally benign with exceedingly rare case reports of malignant behavior (422,423). Both tumors were in pediatric patients (3- and 20-month old) and immature, then later metastasized to a retroperitoneal lymph node. Orchiectomy is curative (358,424). Assessment of molecular abnormalities would be vital in any such case, especially in adult patients, given its rarity in this population. WELL-DIFFERENTIATED NEUROENDOCRINE TUMOR (MONODERMAL TERATOMA) Testicular well-differentiated neuroendocrine tumors (“carcinoid tumors”) are considered to be monodermal teratomas because, although most are pure neoplasms, approximately 20% are associated with teratomatous elements (415-418). It has been suggested they are associated more specifically with prepubertal-type teratoma and thus are discussed here (411). They are rare and occur over an age range from the second to ninth decades, with a mean age of 46 years (425-429). Most patients present with a mass, sometimes of several years in duration (428). A clinical carcinoid syndrome is infrequent, occurring in approximately 10% of cases, but serotonin metabolites are detected in blood in a higher proportion (428). On gross examination, they are solid, yellow to tan, well-circumscribed nodules (Fig. 47.81); cysts may be identified if other teratomatous structures are present. Microscopically, they generally appear similar to mid-gut well-differentiated neuroendocrine tumors, having islands of cells that form small acini (Fig. 47.82). The nuclei are round with granular chromatin, and there is granular, eosinophilic cytoplasm. Often, discrete pink granules can be seen in the cytoplasm, especially at the periphery of nests and glands. Other teratomatous elements may be identified, most frequently mucinous and glandular epithelium. The lack of GCNIS in the few pure neoplasms that we have seen, nor have others (430), with one possible exception (431) again suggests alignment with the non–GCNIS-related monodermal

teratomas. One study reported i(12p) in four cases, at odds with this observation (432). Serotonin, neuron-specific enolase, chromogranin, synaptophysin, and CK may be identified immunohistochemically (428,433,434). Polymorphous neurosecretory granules are seen by electron microscopy (426,428,433).

FIGURE 47.81 A yellow cut surface and well-circumscribed margins are typical gross features of testicular carcinoid. Photograph courtesy of D. J. Gersell, MD, St. Louis, MO.

FIGURE 47.82 There are solid islands of cells with small acini in a testicular well-differentiated neuroendocrine tumor.

It is important to distinguish primary testicular well- differentiated neuroendocrine tumors from metastatic tumors. The presence of teratomatous elements supports a primary lesion; metastases are frequently bilateral and multifocal and show lymphatic/vascular invasion, and the patients have evidence of extratesticular tumor. The general prognosis of primary tumors is good; two of 12 patients in the series of Berdjis and Mostofi (425) developed metastatic disease, which may occur many years following orchiectomy (435). The largest series of Wang et al showed that 20 typical carcinoids had no recurrences, although recurrence was noted in a case of atypical carcinoid. A clinical carcinoid syndrome correlates with malignant behavior, as does large tumor size (mean size = 7.3 cm for malignant cases) (428). Orchiectomy with follow-up is the usual therapy. PREPUBERTAL-TYPE YOLK SAC TUMOR Prepubertal-type yolk sac tumor is the most common form of testicular tumor in prepubertal children in whom it occurs as a pure neoplasm at a median age of 14 to 20 months, with an age ranging from 3 months to 8 years (436-439). It may rarely be found in postpubertal patients, but the greatest number of cases occurs between the ages of 1 and 2 years (438). Childhood yolk sac tumor is not associated with

cryptorchidism and occurs with roughly equivalent frequencies in African American and white populations (440). Eighty percent of children presenting with yolk sac tumor have clinical stage I disease (436,437). Prepubertal-type yolk sac tumors are more likely to be AFP negative (276); however, AFP is usually raised, but it is important not to overinterpret high AFP levels in young children because physiologic AFP “elevations” may occur (441). On macroscopy, prepubertal-type yolk sac tumors are usually more homogeneous than postpubertal forms, often appearing as a solid, yellow/tan myxoid nodule (Fig. 47.39). On microscopy, prepubertal yolk sac tumors are very similar morphologically to postpubertal tumors, though they tend to be more limited in morphology and lack GCNIS and regressive features in the surrounding parenchyma. These tumors lack gain of 12p (409). Although prognosis is excellent, necrosis and large size are associated with more aggressive behavior. In one study, pathologic tumor stage was pT1 in 28%, pT2 in 66%, and pT3 in 6%, with the presence of lymphovascular invasion contributing to the high frequency of pT2 disease (438). Differential Diagnosis Pediatric yolk sac tumors may be confused with juvenile granulosa cell tumors (442,443,444) because both share a solid and cystic pattern, show cytologic atypia, and may have high mitotic rates. Yolk sac tumor typically occurs in older children (>1-year-old) than the neonates and very young infants (5 cm); in those with infiltrative margins; in the presence of necrosis, significant nuclear atypia, and a mitotic rate of more than three to five mitoses per 10 high-power fields; and with vascular invasion, atypical mitotic figures, DNA aneuploidy, MIB-1 staining in excess of 5%, and increased expression of p53 protein (497,532). For patients with malignant Leydig cell tumors, RPLND is often performed. Radiation and chemotherapy are usually not effective. Recurrences may develop after several years, although the mean survival for patients with malignant Leydig cell tumors is 4 years (530). SERTOLI CELL TUMOR SCT represents only approximately 1% of testicular tumors in children (533) and adults. Three histologic types are described: SCT-NOS, LCCSCT, and intratubular large cell hyalinizing Sertoli cell neoplasia (534). Sclerosing SCT was removed from the 2016 WHO classification and is now considered a variant of SCT-NOS. Sertoli Cell Tumor, Not Otherwise Specified The age range is 9 to 80 years old, with a mean in the fifth decade (535,536); most patients present with a testicular mass (499,536), although occasional tumors may produce clinical estrogenic manifestations such as gynecomastia and impotence, which may, therefore, be the presenting features (498,536). Gynecomastia in a child in the absence of virilism is an important diagnostic clue to a possible SCT and contrasts with the situation in childhood Leydig cell tumor where gynecomastia does not occur in the absence of virilism (499). A rare case of bilateral SCTs-NOS in a patient with familial adenomatous polyposis (Peutz-Jeghers syndrome) suggests an association with that disorder (537). On gross examination, SCT-NOS is usually firm, yellow to gray to white, well circumscribed, solid, and most commonly less than 4 cm in diameter, although occasionally quite large (Fig. 47.99). On microscopic examination, SCT-NOS typically forms solid or hollow tubules (Fig. 47.100) (536) and cords in a nested or diffuse arrangement with a variably prominent, sometimes hyalinized, intervening fibrous stroma (Fig. 47.101). Sometimes, the tubules have a retiform arrangement. A diffuse, solid pattern may also be seen. Cytoplasmic vacuolation may occur in some cases, causing a microcystic appearance (Fig. 47.101). The cytoplasm is often pale to clear, reflecting high lipid content (Fig. 47.102), and the nuclei are oval and may have a moderate-sized nucleolus, notches, or grooves (536). Mitotic activity and necrosis are generally inconspicuous but when present cause concern for malignant behavior. A lymphocytic infiltrate may be seen in some cases (Fig. 47.102), potentially causing confusion with seminoma when there is a diffuse arrangement and the cells are pale. Admixture of the Sertoli cell population with a significant component of granulosa cells or sex cord-stromal elements of indeterminate type places the neoplasm into a mixed sex cord-stromal category (417).

FIGURE 47.99 A Sertoli cell tumor is well circumscribed, solid, and gray white.

FIGURE 47.100 Solid and hollow tubules are formed by Sertoli cell tumor.

FIGURE 47.101 Prominently vacuolated cells cause a microcystic pattern in a Sertoli cell tumor with well-defined tubules.

FIGURE 47.102 Solid nests of pale cells and a lymphocytic infiltrate cause a Sertoli cell tumor to resemble seminoma.

The most frequently positive makers are SF-1 and (nuclear) β-catenin, which stain approximately 80% and 60% of SCT-NOS, respectively (513,538). FOXL2 and SOX9 are both reactive in approximately 50% of cases, but intensity tends to be weaker in SOX9. Inhibin-α is positive in 30% to 90% of cases (343,515,539), although our experience with its reactivity frequency is on the lower end and usually seen focally in those with prominent tubular differentiation. Calretinin is slightly more sensitive, but staining is often patchy. CK is positive in 60% to 80% (515,539,540); vimentin is positive in 90% to 100% (539,540); S100 is positive in 30% to 90% (515,516,539,541), and synaptophysin is positive in 45% (515). By ultrastructure, the cells are interconnected by desmosomes and have abundant smooth endoplasmic reticulum and lipid droplets (542). As in ovarian SCTs (543), it may be possible to demonstrate cytoplasmic Charcot-Böttcher filaments, which are considered pathognomonic of Sertoli cell differentiation. These structures, however, have more commonly been identified in LCCSCTs rather than in the “NOS” category (544,545). CTNNB1 gene mutations and nuclear localization of β-catenin via immunohistochemistry have been recently reported as characteristic molecular features in approximately 60% to 70% of SCTs-NOS (538,546). Occasional SCTs have a prominent, hyalinized stroma that represents the majority of the tumor (547550), termed sclerosing SCTs. Although classified separately previously, they are now considered a variant of SCT-NOS because they both harbor the same CTNNB1 gene mutations and show nuclear βcatenin expression in similar proportions (538,546,551). They occur over a wide age range (18-80 years), with a mean of 35 years. On gross examination, they are usually less than 2 cm (although some have been as large as 5 cm), well-circumscribed, firm, white to tan to yellow nodules. On microscopic examination, they are composed of tubules, both solid and hollow; nests; and cords of cells in a hypocellular, fibrous stroma (>50%) (Fig. 47.103). The cells have pale, eosinophilic cytoplasm, sometimes with lipid vacuoles. The nuclei are usually round to oval with finely granular chromatin and small nucleoli and show few mitotic figures. Of 31 with follow-up, only one exhibited malignant behavior (550), and this one had the unusual features of lymphovascular invasion and irregular, invasive growth in the testis. It appears, therefore, that the frequency of a malignant course in this variant may be lower than those without sclerosis, but similar criteria for such behavior likely apply. Considerations in the differential diagnosis include carcinoid tumor (which, however, usually has a mid-gut type of pattern rather than the cordlike pattern of sclerosing SCT), adenomatoid tumor (which is usually centered in paratesticular structures rather than the testis), and metastatic prostatic carcinoma (which does not form tubules and may be distinguished as well based on immunostains for NKX3.1 and PSA) (547). Inhibin and calretinin reactivity supports a diagnosis of SCT, but these stains have been negative in several cases.

FIGURE 47.103 A sclerosing variant of Sertoli cell tumor, NOS showing prominent cords of tumor cells in an abundant fibrous stroma.

Differential Diagnosis It is important to distinguish SCT-NOS from the rare seminomas with a tubular pattern (196) (Fig. 47.18). This distinction depends on the greater cytologic atypia in seminoma, the differences in the nature of the clear cytoplasm of the two tumors (being predominantly a result of glycogen in seminoma and lipid in SCT), and the association of seminoma with adjacent GCNIS, which is not seen in SCT. SF-1, Inhibin, PLAP, and OCT3/4 immunostains are helpful in problematic cases, as well as electron microscopy. In addition, some malignant SCTs have a solid nested to diffuse pattern of pale to clear cells and prominent inflammatory infiltrates that mimic the usual appearance of seminoma (552) (Fig. 47.102). The lower grade nature of the nuclei, absence of GCNIS, lower mitotic rates, and focal tubule formation in these cases are key in the recognition that they do not represent seminoma, with confirmation obtainable by positive immunostains for SF-1, inhibin-α, EMA, CKs (AE1/AE3), FOXL2, and calretinin and negativity for PLAP, OCT3/4, and SALL4. Hyperplastic Sertoli cell nodules occur commonly in cryptorchid testes and are not infrequent in non– cryptorchid testes (553,554). These are usually small, mostly microscopic lesions that consist of aggregates of variably sized tubules lined by immature Sertoli cells that occasionally have spermatogonia, unlike SCT. Central, hyalinized basement membrane material is commonly identified in the tubules (Fig. 47.104). Several cases of macroscopic Sertoli cell nodules have been reported with features identical to those described earlier, including admixed spermatogonia in the tubules, except for their greater size (Fig. 47.105) (555). The testes of patients with androgen-insensitivity syndrome may contain multiple hamartomatous nodules composed of small tubules lined by Sertoli cells with intervening Leydig cells (Fig. 47.106); in addition, Sertoli cell adenomas, consisting of a pure proliferation of Sertoli cells arranged in small tubules, may occur in approximately 25% of androgeninsensitivity syndrome cases (135). These are categorized separately from SCT-NOS because they have never metastasized.

FIGURE 47.104 A Sertoli cell nodule consists of an aggregate of tubules filled by small, fetal-type Sertoli cells and prominent basement membrane deposits.

FIGURE 47.105 A giant Sertoli cell nodule has a predominance of basement membrane deposits but also tubules that, on high power, showed fetal-type Sertoli cells and spermatogonia.

FIGURE 47.106 A hamartomatous nodule in a patient with androgen-insensitivity syndrome consists of Sertoli cell–lined tubules and intervening Leydig cells.

The adenomatoid tumor may resemble SCT, but there are almost always areas with the characteristic prominent vacuoles with flattening of the cells and the production of “thread-like bridging strands.” Rare adenomatoid tumors involve the testis to an appreciable degree, but they are primarily paratesticular, in contrast to SCT. Treatment and Prognosis It is reported that 10% of cases manifest malignant behavior (498,536,556,557), though this is probably an overestimate secondary to referral bias. These are more likely to be large tumors (tumor diameter >5 cm), have a mitotic rate of more than five mitotic figures per 10 high-power fields, show necrosis, have moderate-to-severe nuclear atypia, and/or demonstrate lymphatic/vascular invasion (536,557) (Fig. 47.107). A mainly diffuse pattern should also provoke concern (552). Only one of nine benign tumors with follow-up of 5 years or more had more than one of these features, whereas five of seven malignant cases had at least three features (536). In contrast to Leydig cell tumors, malignant behavior in children with SCTs has been reported (499,558).

FIGURE 47.107 A malignant Sertoli cell tumor showing significant atypia and focal necrosis (upper right).

Radical orchiectomy is the mainstay of treatment; radiation and chemotherapy have not proved effective. RPLND is indicated if the patient has apparent retroperitoneal involvement. For cases judged to be likely malignant based on pathologic criteria alone, in the absence of known metastatic disease, RPLND remains controversial. Large Cell Calcifying Sertoli Cell Tumor Approximately 30% of LCCSCTs occur in patients with the Carney complex (523,559-563). Presenting features may, therefore, include the stigmata of the syndrome, which, for the Carney complex, include myxomas of skin, soft tissue, and heart and myxoid lesions of the breast (564,565); lentigines of the face and lips; cutaneous blue nevi; Cushing syndrome secondary to pigmented adrenocortical nodular hyperplasia; pituitary somatotroph adenomas (564); and psammomatous melanotic schwannomas (566). LCCSCT usually occurs in young patients (525), with a mean age of 17 years for benign cases and a mean age of 39 years for the rare malignant tumors (523). Despite the associated syndromes, most patients present with a mass. The tumors are usually small, most commonly less than 2 cm, and sometimes bilateral (up to 25% (523,525) and multifocal (567). Bilateral and multifocal tumors are almost always syndrome associated, but solitary tumors may also be syndrome associated. They are typically yellow to tan with a gritty cut surface as a result of calcifications.

On microscopic examination, nodules composed of nests and cords of large, polygonal cells with abundant eosinophilic cytoplasm are identified in a myxoid to fibrous stroma, often with scattered neutrophils (Fig. 47.108). Intratubular tumor growth is common, but not as prominent a finding as in intratubular large cell hyalinizing Sertoli cell neoplasia (534,559,562,563). Calcifications, varying from small, psammomatous structures to larger, dystrophic-type calcifications or even ossification, occur within foci of intratubular and extratubular tumor. The tumor cells have round to oval nuclei and may occasionally have prominent nucleoli. Mitoses are rare, except in the uncommon malignant cases (523,525).

FIGURE 47.108 A large cell calcifying Sertoli cell tumor is composed of cords and nests of cells with abundant eosinophilic cytoplasm in a variably prominent stroma with focal calcification.

On immunohistochemical study, the tumors are often positive for inhibin, vimentin, S100 protein, EMA, desmin, and, focally, for CK (516,523,568-570). Several ultrastructural studies of these tumors support their Sertoli cell derivation (524,545,571), including the presence of Charcot-Böttcher filaments (544). Germline PRKAR1A gene mutations on chromosome 17q22-24 are seen in up to 70% of Carney complex–associated tumors but have also been reported in sporadic cases (572,573). FOXL2 is negative while SALL4 and SOX9 are frequently positive (513). Most cases are benign, but malignancy correlates with size greater than 4 cm, extratesticular growth, gross or microscopic necrosis, high-grade cytologic atypia, vascular space invasion, and a mitotic rate greater than three mitoses per 10 high-power fields. Malignant cases typically show at least two of these features, whereas the benign cases typically lack any of them (523). Malignant cases furthermore tend to occur in patients older than 25 years of age and are uncommonly syndrome associated (523). The problem of differentiating these tumors from Leydig cell tumors has been discussed in the section “Leydig Cell Tumor.” Intratubular Large Cell Hyalinizing Sertoli Cell Neoplasia Patients with Peutz-Jeghers syndrome may develop multifocal, intratubular, and, less commonly, invasive SCTs (534). The patients are usually young boys (mean age, 7 years) who present with gynecomastia and bilaterally firm testes that mostly lack a discrete mass (534). On microscopic examination, there are lobular clusters of enlarged seminiferous tubules that contain proliferations of large Sertoli cells and show thickened peritubular basement membrane that projects into the lumen as rounded deposits (Fig. 47.109), an appearance termed “intratubular large cell hyalinizing Sertoli cell neoplasia” (534). It has been recognized as a distinct entity associated with Peutz-Jeghers syndrome, with a characteristic STK11 gene mutation (534,574). An invasive component composed of cytologically

similar cells occurs in approximately one-quarter of the cases. In rare cases, the invasive tumor resembles the LCCSCT and was historically considered to be within the morphologic spectrum of LCCSCT. However, intratubular large cell Sertoli cell neoplasia differs from LCCSCT in several aspects: clinically features, almost exclusive intratubular growth, and different germline mutations. These tumors are multifocal, bilateral, and to date, there has been no evidence of malignant behavior (534).

FIGURE 47.109 There is a cluster of enlarged seminiferous tubules lined by large Sertoli cells and containing basement membrane deposits in a specimen from a Peutz-Jeghers patient.

GRANULOSA CELL TUMOR ADULT TYPE Granulosa cell tumors similar to the typical adult-type granulosa cell tumors of the ovary (see Chapter 55) are quite rare in the testis (575-580). They have occurred over an age range of 14 to 87 years (579,581) and have sometimes been associated with gynecomastia (417,582). They are typically circumscribed, yellow to gray, with a solid to partially cystic appearance. Microscopically, they have the typical patterns of ovarian granulosa cell tumors (Fig. 47.110) and the usual pale nuclei with occasional or frequent nuclear grooves. They are typically strongly reactive for inhibin and vimentin and may be reactive for CD99, CK8, and CK18 (576,578,580,583,584). Acquired FOXL2 mutations have been reported in a smaller percentage of these tumors than in their ovarian counterparts (585). Malignant behavior is rare (499,582) but should be suspected if the tumor is large (>4 cm), necrotic, or hemorrhagic, or shows vascular/lymphatic invasion or infiltrative borders (579,581).

FIGURE 47.110 An adult-type granulosa cell tumor showing islands with a peripheral palisade and prominent fibromatous stroma.

JUVENILE TYPE Juvenile granulosa cell tumors are similar in appearance to their ovarian counterpart and are more common than the adult form of testicular granulosa cell tumor. They, unlike ovarian cases, occur in infants less than 6 months of age in more than 90% of cases, some being congenital, with rare cases occurring in older children (442,444,586,587). Association with X/XY mosaicism has been described as well as occurrences in cryptorchid testes or dysgenetic gonads (442,444,540,588-590). Either a parent or physician in most cases finds a testicular mass. On gross examination, there is a variable admixture of gray to yellow solid areas and cystic foci filled with viscid fluid (Fig. 47.111). On microscopic examination, there is a typically lobular distribution of round, follicle-like structures filled with watery-appearing, often mucicarminophilic, fluid (442), with an intervening fibromuscular, sometimes hyalinized, stroma (Fig. 47.112). The follicles are lined by several layers of cells having an abundant amount of pale to eosinophilic cytoplasm and hyperchromatic, round nuclei with nucleoli. Mitotic figures and apoptotic cells may be numerous (Fig. 47.113). Immunohistochemical study shows positivity for inhibin, vimentin, CD99, and, occasionally, CK, smooth muscle actin, and desmin (343,540,587,591).

FIGURE 47.111 A gray-tan, solid, and cystic nodule is characteristic of juvenile granulosa cell tumor. Photograph courtesy of Dr. Carlos Galliani, Fort Worth, TX.

FIGURE 47.112 A juvenile granulosa cell tumor has follicle-like spaces filled with fluid and nests of cells with pale cytoplasm.

FIGURE 47.113 Juvenile granulosa cell tumor with mitotic figures and apoptotic bodies.

Despite worrisome histologic features, this tumor is benign (417,442,444). The major differential diagnosis is the distinction from yolk sac tumor, an issue that is addressed in the section “PrepubertalType Yolk Sac Tumor.”

TUMORS IN THE FIBROMA-THECOMA GROUP Intratesticular fibrothecomas of gonadal stromal origin have been reported but are rare (592). The tumors occur over a wide age range, from 16 to 69 years, and present as painless masses. On gross examination, they are circumscribed, yellow-white to white lesions that measure up to 7.6 cm in diameter. On histologic examination, they resemble ovarian fibromas, being composed of short, interlacing fascicles of spindle-shaped cells. Acellular plaques of hyalinized collagen are present in some cases. Immunohistochemically, the tumor cells are variable, with inhibin, calretinin, musclespecific actin, and melan-A being the most consistently positive markers. Although they may show atypical features such as minimal invasion into surrounding testis, high cellularity, and increased mitotic rate, no metastasis has been reported to date (592).

MYOID GONADAL STROMAL TUMOR Myoid gonadal stromal tumor is considered an emerging entity in the 2016 WHO classification and is composed of spindle cells with features of both smooth muscle and gonadal stroma that are thought to be derived from peritubular myoid cells (Fig. 47.114) (593-595). Less than 10 cases have been reported in the English literature, and these tumors occurred in mostly middle-aged men with an age range of 4 to 59 years (median 43 years) (595,596). The tumors are usually small (8 cm) and, after many years of neglect, may become locally destructive (giant or atypical condyloma) or may harbor foci of evolving carcinoma (11,71). It is important to keep in mind, however, that condylomata previously treated with podophyllin may display prominent degenerative changes such as vacuolization (pallor of the epithelium), nuclear enlargement, numerous necrotic keratinocytes in the lower half of the epidermis, and an increase in the number of mitotic figures (metaphase arrest) (72). Care should be taken not to confuse them with carcinoma. These degenerative changes tend to be focal, and atypical mitoses should not be seen. A high level of suspicion and clinical correlation is necessary to make the correct diagnosis. HERPES Herpes is a sexually transmitted disease caused by a DNA virus, the herpes simplex virus (HSV). Most genital infections are caused by HSV-2 (34). It clinically presents with multiple millimeter-sized vesicles that rupture and form painful ulcers. Atypical clinical presentations include fissures, furuncles, linear excoriations, and ulcerations (73). Histologically, there are usually intraepithelial vesicles containing prominent rounded acantholytic keratinocytes. These keratinocytes show the viral cytopathic changes that consist

of nuclear ground-glass appearance and molding (68). Multinucleated keratinocytes with the classic nuclear changes are common. Well-defined acidophilic inclusions can also be seen. The diagnosis can be made on Tzanck preparations. Immunohistochemical analysis to identify HSV-1, HSV-2, and herpes zoster viruses is now available. MOLLUSCUM CONTAGIOSUM This infection can be sexually transmitted and is caused by a DNA poxvirus. The lesion is a 3- to 6-mm, dome-shaped, pearly papule with central umbilication (Fig. 48.12) (34). Histologically, it shows lobular acanthosis of the epidermis, which causes an inverted pattern of epidermal hyperplasia. Intracytoplasmic eosinophilic inclusions called Henderson-Paterson bodies can be identified in the stratum spinosum and granulosum (68).

FIGURE 48.12 Clinical appearance of molluscum contagiosum characterized by pink pearly smooth and confluent papules on the foreskin cutaneous surface.

PENILE LESION IN AIDS

The retrovirus HIV-1 is the agent of AIDS in the United States and Europe. The selective replication of HIV within T-helper cells causes their depletion with consequent immunodeficiency. This severe defect in cell-mediated immunity makes the affected individual particularly susceptible to infections. AIDS patients are often concomitantly infected with several different types of microorganisms. Skin diseases (including genital lesions) are common manifestations of HIV infection. Such skin and genital lesions can be classified in noninfective dermatosis, infective disorders, and neoplasms (74-76). Patients with AIDS usually have atypical presentations and more severe dermatosis. Noninfective dermatoses include atopic dermatitis, seborrheic dermatitis–like eruption, psoriasis, genital (aphthous ulcers), and drug reactions. These conditions also tend to be more widespread and severe in patients with AIDS. Histologically, the findings are similar (although frequently more florid) to those seen in patients without AIDS. Plasma cells tend to be numerous in the cutaneous lesions of patients with AIDS. Infective dermatoses include bacterial, viral, and fungal disorders. HPV-related lesions such as verruca vulgaris and condyloma acuminata are common in such populations and may be widespread and often associated with preneoplastic and neoplastic HPV-related tumors. AIDS patients are also commonly affected by herpes viral infection (herpes simplex and herpes zoster), molluscum contagiosum, syphilis, Candida, and tinea cruris. Scabies is an infestations that can be seen and often presents as the Norwegian variant. Scraping of the lesions will show numerous mites. AIDS patients also have an increased incidence of neoplastic conditions. The most common tumors in these patients include Kaposi sarcoma and HPV-related squamous cell carcinomas (76,77). Precursor lesions of squamous cell carcinoma (such as bowenoid papulosis and penile intraepithelial neoplasia) are also more frequently found.

MISCELLANEOUS CONDITIONS PHIMOSIS This is a condition in which the prepuce cannot be retracted, usually as a consequence of nonspecific chronic bacterial infections, lichen sclerosus, or congenitally abnormally long foreskin (78). The accumulation of smegma induces a diffuse inflammation of all mucosal epithelial compartments of the glans and foreskin. The treatment of choice is surgical removal of the foreskin. Histologically, there is fibrosis of the lamina propria associated with a nonspecific lymphoid and plasmacytic infiltrate (79). It is important for the surgical pathologist to liberally sample phimotic foreskin specimens from adults to rule out dysplasia, carcinoma in situ, or occult early invasive carcinoma. Special attention to hyperkeratotic, thick, and slightly elevated or irregular foci is advised. Penile cancer has been reported to occur more frequently in patients with long phimotic foreskins (Fig. 48.13; see Fig. 48.28) (7). In paraphimosis, the prepuce cannot be advanced over the glans and becomes trapped in the space located between the coronal sulcus and the glans corona. Unusual cases of penile infarct secondary to arterial obstruction resulting from edema have been described.

FIGURE 48.13 Phimosis and cancer. A large destructive tumor of glans and foreskin is encased in a virtual sac produced by a phimotic foreskin. There is a narrow opening of the preputial orifice.

LICHEN SCLEROSUS (BALANITIS XEROTICA OBLITERANS) Lichen sclerosus (LS) is a chronic and atrophic mucocutaneous condition preferentially affecting anogenital areas of men and women. Extragenital location is less common. This condition was described in the penis as balanitis xerotica obliterans by Stühmer (80) in 1928; however, because some authors prefer to use the term balanitis xerotica obliterans for the end-stage condition and to unify gynecologic and urologic terminology, the use of LS is recommended (81). It may have an autoimmune etiology. LS is most commonly associated with non–HPV-related variants of penile squamous cell carcinoma, but they can also, rarely, be found in cases of HPVrelated PeIN or invasive cancers (81). Penile LS tends to affect middle-aged adults, but it can occur in boys (78). Grossly, the lesions appear as white-gray, irregular geographic and atrophic areas and most commonly compromise the inner aspect of the foreskin, glans, and perimeatal region. Erosion, ulceration, and elevated hyperkeratotic foci may also be seen. In advanced cases, the

preputial mucosa folds may disappear, resulting in acquired phimosis or paraphimosis (34). LS is a common cause of phimosis in countries with a high incidence of penile cancer (82), and it is the most common cause of phimosis in boys. Histological changes from the surface to deeper anatomic layers are as follows (83): 1. Squamous epithelium: normal, atrophic, or most commonly hyperplastic. The cornified layer usually shows hyperorthokeratosis. There is also vacuolar alteration of the basal layer (Fig. 48.14), which is the hallmark of all lichenoid/interface processes. This change progresses to tissue separation at the stromal-epithelial junction (clefts and bullae), which can result in erosion and ulceration (Fig. 48.15). 2. Lamina propria: thickened by edema hypervascularization and typically sclerosis/hyalinization (Figs. 48.14 and 48.15). Marked edema of the lamina propria may precede or coexist with the classic sclerotic changes. There are distinctive sclerotic patterns, and early changes include sclerosis at the surface (basement membrane), perivascular, globular, or linear. The presence of sclerotic globules is sufficient for a diagnosis of LS. Wellestablished lesions show the diffuse classic bandlike sclerosis/hyalinization in the lamina propria (Fig. 48.14). LS is a superficial mucosal disorder preferentially affecting the epithelium and lamina propria and typically sparing the preputial dartos and corpus spongiosum of the glans. The lesions, however, tend to be broad and multifocal, may affect more than one epithelial compartment, and may even extend to the epithelium and lamina propria of the distal urethra (81). 3. Lymphocytic infiltration: variable in degree and distribution. It can be superficial, at the interface of the epithelium and lamina propria (lymphocytes at the basal layer) or deeper in the lamina propria, underneath the well-established band of sclerosis (lymphocyte in deep lamina propria). In some lesions, the lymphocytes are very scarce (lymphocytic depletion). Depletion of lymphocytes is typical of LS associated with SCC.

FIGURE 48.14 Lichen sclerosus. Histologically, the lesion is characterized by an atrophic epithelium with vacuolar alteration of the basal layer and a thickened lamina propria with the classic hyalinization/sclerosis.

FIGURE 48.15 Bandlike sclerosis/hyalinization is the hallmark lesion in LS. It can be linear, perivascular, and globular (see arrow) at the dermo-epidermal interface.

LICHEN SCLEROSUS (LS) AND CARCINOMA Although extragenital LS appears to carry no risk for malignant transformation, the relationship of anogenital LS and squamous cell carcinoma (SCC) is well documented (82,84-89). In a prospective study, the incidence of SCC arising in the setting of long-standing LS of the penis was 9.3% (87,88). In a retrospective review of 200 penectomy specimens with penile invasive carcinoma, 33% of the cases were associated with LS (81), and this figure was much higher (69%) when considering carcinomas affecting exclusively the foreskin. When present adjacent to invasive carcinomas, LS is almost always associated with areas of epithelial hyperplasia and frequently shows squamous cell atypias (Fig. 48.16) (79). A significant association of LS with special (usually HPV-unrelated) variants of SCC such as usual, pseudohyperplastic, verrucous, and papillary SCCs has been demonstrated (Fig. 48.17). In one study, a distinct association of LS with differentiated (simplex) penile intraepithelial neoplasia was found (Fig. 48.16) (82). Atypical LS, with epithelial dysplastic changes (low grade or high grade), are currently classified as Differentiated PeIN (WHO 2016, AFIP 2020). The frequent coexistence of LS, squamous hyperplasia, differentiated PeIN, and low-grade SCC suggests a common nonHPV–related pathogenic pathway, especially for preputial lesions, and highlights the importance of circumcision in symptomatic patients for the prevention of penile cancer (82). An analogous lesion, lichen planus, has also been reported in association with SCC, but this relationship appears more controversial (90).

FIGURE 48.16 Lichen sclerosus with epithelial hyperplasia showing keratinocytic atypia. Such changes are frequently found adjacent to invasive carcinomas.

FIGURE 48.17 Lichen sclerosus (LS) adjacent to a verrucous carcinoma. There was a transition from classic atrophic areas of LS to hyperplastic and atypical foci.

There is a sequential spatial distribution of LS and precancerous and invasive carcinomas, suggesting LS is a precancerous condition (89). In a recent study (89), we found four spatial patterns of LS and precancerous or cancerous lesions: (a) LS next or contiguous to PeIN (33%), (b) LS next to and underneath PeIN (42%) (Fig. 48.16), (c) LS next to and underneath/admixed with PeIN/invasive penile carcinomas (32%), and (d) in a minority of cases (3%), LS was separate or discontinuous from PeIN and/or invasive carcinomas. The striking continuous spatial relationship among LS, PeIN, and invasive carcinoma, as shown in this study, may be an obligate (but not sufficient) condition for the hypothesis postulating LS as a penile precancerous lesion. BALANITIS CIRCUMSCRIPTA PLASMACELLULARIS (ZOON BALANITIS) This is an inflammatory condition of unknown etiology occurring preferentially in uncircumcised men (91,92). Grossly, they are solitary or multiple, well-defined, brown to reddish plaques. The clinical appearance can simulate PeIN. Histologically, there is edema and a dense plasma cell infiltrate associated with lymphocytes and usually siderophages. The epithelium may show mild reactive atypia, which needs to be distinguished from PeIN. The diagnosis of Zoon balanitis is largely one of exclusion. One must consider other specific entities such as lichen planus, syphilis, and pemphigoid before making this diagnosis, which is a diagnosis of exclusion. PEYRONIE DISEASE This unusual condition of unknown etiology is characterized by dense fibrosis with the formation of a plaquelike lesion affecting the tunica albuginea, penile fascia, and dermis. The fibrosis produces an abnormal curvature of the penis, which is painful during erection. In some cases, there are several firm nodules over the middorsal line.

Calcification and ossification can be present (93). The condition is more common than originally thought and may affect up to 9% of the population. Histologically, its appearance varies with the duration of the disease. Early lesions show a loose proliferation of plum fibroblasts/myofibroblasts admixed with some inflammatory cells (comparable to an early scar). In time, the lesions become more fibrotic and less cellular. Well-established lesions are characterized by hypocellular and hyalinized nodules. The changes in Peyronie disease recapitulate the sequence of events that characterize the development of tissue-scarring fibrosis. These are essentially an initial tissue insult (trauma, microtrauma, or local toxicity), followed by acute and then chronic inflammation that leads to deposition of excessive collagen and other extracellular matrix, fragmentation of elastin, and persistence of myofibroblasts (94,95). Recent experimental findings suggest that increased levels of the profibrotic factor transforming growth factor-β1 (TGF-b1) may play an important role in the pathogenesis of the disease. Injury to the erect penis is thought to trigger the condition by inducing extravasation of fibrin and subsequent synthesis of TGF-b1. Treatment is mainly surgical. Some cases show spontaneous regression. There are currently several promising molecular targets for antifibrotic treatments (94). TANCHO NODULES AND PARAFFINOMAS Tancho nodules refer to an unusual custom among some Asian populations to implant foreign material under the skin of the penis to improve sexual pleasure (96). Paraffinomas result from the injection of mineral oil in the penis, usually done for the purpose of penile enlargement. Histologically, these materials may cause foreign body reaction that may need surgical resection. Such foreign substances that are injected or inserted into the penis include paraffin, silicone, or wax. A characteristic foreign body reaction called paraffinoma is produced (97). This reaction may occur many years after the injection (98).

PAPILLOMATOSIS OF GLANS CORONA (HIRSUTOID PAPILLOMAS) This is an asymptomatic benign condition occurring in 20% to 30% of normal men characterized by multiple pearly gray-white fibroepithelial papules located in the dorsal aspect of the glans corona (99,100). These minute papillomas are characteristically arranged in two or three rows. The condition can be confused with condyloma acuminatum, although the diagnosis can be made easily because of the specific location of the lesions as well as the uniformity of the small papules. It was suggested that they may be related to excessive sexual activity. It has been noted that they are more frequently seen in male sexual partners of women carrying an HPV cervical lesion. It is likely that papillomas are not related to HPV infections, but, because more penises are scrutinized under peniscopy, more diagnoses are being made. PENILE MELANOSIS Also called melanotic macules or genital lentiginosis, this condition is clinically characterized by pigmented macules of variable size that may be multifocal and may show irregular borders. They may affect the glans and foreskin (34,101,102). Histologically, there is mild acanthosis with hyperpigmentation of the basal layer usually associated with mild lentiginous junctional melanocytic hyperplasia without atypia. Scattered melanophages are often present in the superficial dermis or lamina propria (101). It is considered a benign condition; however, the available information is insufficient to predict the natural history of genital lentiginosis or its relation to mucocutaneous melanoma (102). When associated with LS, there may be superficial fibrosis and pigment incontinence, mimicking regression, and these findings may be confused with melanoma in situ with regression (103). SCLEROSING LYMPHANGITIS OF THE PENIS

This unusual and usually self-limited condition, preferentially affecting young adults, is characterized by firm, subcutaneous, cordlike structure(s) located along the dorsal shaft of the penis or around the coronal sulcus (104,105). The etiopathogenesis is poorly understood, but it has been suggested that it may be associated with trauma and vigorous sexual activity. Concurrent infections, including herpes simplex infection, have been described, but, most likely, they are not the cause of the disease. Because of the difficulty in distinguishing between large lymphatic vessels and veins in this location, there is controversy about whether this condition preferentially affects large lymphatic vessels or veins, and perhaps either could be affected in different patients. This condition may show overlapping features with Mondor phlebitis. Histologically, one or more vessels of the superficial plexus show thickening of the wall, sometimes associated with thrombosis and various stages of recanalization. Inflammation is not prominent. OTHER RARE CONDITIONS There are other rare diseases that may present grossly as tumorlike conditions in the penis. Verruciform xanthoma shows a wartylike lesion characterized by acanthosis and hyperkeratosis and parakeratosis. A distinctive feature is the presence of foamy histiocytes between the elongated rete ridges (106). Rare cases of Wegener granulomatosis involving the penis have been reported (107,108). Other rare conditions affecting the penis are inflammatory pseudotumor secondary to chronic catheterization (109) and hyperplastic ectopic sebaceous glands in the foreskin mimicking molluscum contagiosum (110). Cutaneous horn is a clinical term that may be associated with different pathologic entities, including warts and SCC. Histologic examination is necessary to achieve a conclusive diagnosis (111,112).

DERMATOLOGIC CONDITIONS THAT INCIDENTALLY AFFECT THE PENIS

A variety of dermatoses can incidentally involve the penis (34). Usually, they are diagnosed and biopsied by dermatologists, not by urologists. Hence, the specimens are submitted to dermatopathologists for histologic evaluation and are rarely seen by the surgical pathologist. Some of these lesions include psoriasis vulgaris, lichen planus, lichen nitidus, fixed drug reactions, allergic contact dermatitis, atopic dermatitis, and lichen simplex chronicus. As part of the Reiter syndrome (urethritis, conjunctivitis, arthritis, and keratodermia blennorrhagica), a balanitis circinata can be noted. These are irregular plaques located in the corona of the penis. For a more detailed description of these entities, see Chapter 1.

NEOPLASMS AND PRECURSOR LESIONS SQUAMOUS HYPERPLASIA Squamous hyperplasia is characterized by acanthosis of the squamous epithelium without atypia (Fig. 48.18) (11),113,114). It may be seen as a reactive condition in the context of an inflammatory dermatitis or adjacent to SCCs, particularly usual (keratinizing), verrucous, and low-grade papillary variants. Squamous hyperplasia may involve the glans, sulcus, and foreskin. Grossly, the mucosa is flat, smooth, and pearly white with occasional slightly elevated to papillary configuration. The latter is preferentially seen in areas merging with the adjacent low-grade carcinoma. Microscopically, squamous hyperplasia usually has a flat surface; however, verrucous, papillary, and pseudoepitheliomatous lesions may also be observed. Squamous hyperplasia shows acanthosis, hyper-/orthokeratosis, and normal maturation of squamous cells. Parakeratosis, koilocytosis, deep keratin whorls, and cytologic atypia are not present. Some cases may be associated with LS. Pseudoepitheliomatous hyperplasia may be confused with SCC because the florid complex downward proliferation of squamous rete ridges may appear as detached from the epithelium in cut sections (Fig. 48.19). Important features that may be helpful to differentiate

pseudoepitheliomatous hyperplasia from carcinoma include the superficial nature of the lesion, absence of atypia, absence of deep keratin whorls (keratin pearls), and lack of stromal reaction or desmoplasia. Occasionally, hyperplasia of basal cells may be noted, especially in association with the basaloid variant of SCC. Another pattern is that of verrucous hyperplasia, which is typically found adjacent to verrucous carcinomas (Fig. 48.20). The frequent association of squamous hyperplasia with differentiated PeIN and invasive carcinomas, especially low-grade variants, and the usual continuity with the invasive tumor suggest that many lesions labeled as squamous hyperplasia are, in fact, hyperplasialike variants of differentiated PeIN (see Figs. 48.16 and 48.17).

FIGURE 48.18 Squamous hyperplasia, flat. On the right side of the picture, there is a classic example of squamous hyperplasia characterized by an acanthotic epithelium without atypia. There is hypergranulosis and compact orthokeratosis. On the left side of the picture, there is mild basal/parabasal atypia and parakeratosis suggestive of superimposed early differentiated penile intraepithelial neoplasia.

FIGURE 48.19 Squamous hyperplasia, pseudoepitheliomatous. Downward proliferation of rete pegs with few squamous nests detached from overlying epithelium.

FIGURE 48.20 Squamous hyperplasia, verrucous. (A) Diagrammatic representation of a verrucous carcinoma with an adjacent thickening of glans squamous mucosa (green), representing, respectively, verrucous (VH) and flat (SH) hyperplasia. (B) Histologic section of same specimen showing the area of verrucous hyperplasia. There is acanthosis with hyperorthokeratosis. The lesion has a spiky (slightly verrucous) surface. F, foreskin; U, urethra; VC, verrucous carcinoma.

PENILE INTRAEPITHELIAL NEOPLASIA

Penile carcinomas mainly show a bimodal pathway of carcinogenesis; therefore, precursor lesions can be broadly classified into two main groups: HPV related (HPV-associated) and HPV unrelated (HPV-independent). Taking into consideration the striking similarities in morphology and pathogenesis between vulvar and penile carcinomas (115-118) and in an attempt to have a simplified and more uniform terminology, we have recently proposed a slightly modified nomenclature for penile preinvasive lesions (118). The term penile intraepithelial neoplasia (PeIN) is preferred over old terms such as squamous intraepithelial lesion (SIL), erythroplasia of Queyrat, and Bowen disease (118-122). These latter two terms are synonymous with carcinoma in situ and have been used for lesions in the glans (erythroplasia of Queyrat) and skin of the shaft (Bowen disease). PeIN can be classified as HPV-unrelated/ HPVindependent (differentiated/simplex) and HPV-related/ HPVassociated (basaloid, warty and mixed variants). PeIN may be solitary or multifocal and tend to be associated with infiltrating SCC in approximately two-thirds of cases. In our experience, of these cases, approximately 65% are associated with differentiated PeIN and 35% with warty/basaloid PeIN (118). Differentiated PeIN tends to affect older patients, usually arises in the setting of a chronic scarring inflammatory dermatosis such as LS, and is more frequently located in the foreskin when compared with HPV-related variants. The latter tend to affect younger patients and are usually more centrally located in the glans and perimeatal region. The gross appearance of PeIN is heterogeneous and does not allow the distinction between the two main types. Lesions vary from flat to slightly elevated, pearly white or moist erythematous, dark brown or black macules, papules, or plaques. The contours may be sharp or ill-defined and irregular. Occasionally, a granular or low papillary appearance may be noted. Microscopically, differentiated (simplex) PeIN is characterized by the following: a thickened epithelium, usually associated with elongated and anastomosing rete ridges, subtle abnormal maturation (enlarged keratinocytes with abundant eosinophilic cytoplasm), whorling and keratin pearl formation (usually in deep rete ridges), prominent intercellular bridges (spongiosis and

sometimes acantholysis), and atypical basal cells with hyperchromatic nuclei (118). Parakeratosis is frequent (Fig. 48.21). At low power, the atypia seems to be present only in lower levels of the epidermis; however, at higher power, there is subtle but abnormal maturation in all levels of the epithelium. Despite the subtle changes, differentiated PeIN represents a squamous cell carcinoma in situ and may evolve to frank invasive carcinoma without showing more significant atypias than the already described (Fig. 48.22) (118,123). It is not surprising that the precursor lesions of welldifferentiated invasive tumors show such a high degree of differentiation. This lesion appears to be the most frequent precursor lesion of penile carcinomas, especially the keratinizing and welldifferentiated variants. There is a preferential association between LS and differentiated PeIN when compared with warty/basaloid variants (see Fig. 48.16). Therefore, it is important to keep a high level of suspicion when dealing with hyperkeratotic/hyperplastic lesions with subtle keratinocytic atypia arising in the setting of longstanding LS.

FIGURE 48.21 Differentiated penile intraepithelial neoplasia (PeIN). There is a thickened epithelium associated with elongated and anastomosing rete ridges, basilar atypia, enlarged keratinocytes with abundant eosinophilic cytoplasm, and keratin pearl formation. There is also spongiosis with acantholysis and parakeratosis.

FIGURE 48.22 Differentiated penile intraepithelial neoplasia (right) is seen adjacent to a well-differentiated invasive carcinoma (left).

The second major type of PeIN, the HPV-related type, shows more prominent morphologic changes. It can be subclassified as basaloid, warty, and mixed PeIN. In the basaloid variant, the epithelium is replaced by a monotonous population of small immature cells with a high nuclear-to-cytoplasmic ratio (Fig. 48.23) (113,114,118). Apoptosis and mitotic figures are numerous. Basaloid PeIN should be distinguished from urothelial carcinoma in situ of the urethra, which may secondarily involve the penile meatal region (124). In the warty pattern, the involved epithelium has an undulating/spiking surface with atypical parakeratosis. There is striking cellular pleomorphism and koilocytosis (multinucleation, nuclei with irregular contours, perinuclear halo, and dyskeratosis) (Fig. 48.24)

(113,114,118). Mitoses tend to be numerous. Frequently, lesions show overlapping features of both, namely warty/basaloid PeIN. These lesions tend to have a spiking surface with koilocytic changes, whereas the lower half of the epithelium is composed predominantly of small basaloid cells (Figs. 48.25 and 48.26). This is not a surprising finding considering that warty and basaloid carcinomas are both HPV-related tumors. Basaloid and warty PeIN can be divided into low-grade and high-grade lesions when the atypical cells occupy less than half or more than half of the epithelial thickness, respectively. Most of the warty and basaloid PeINs will fall within the high-grade category. Full-thickness atypia of the epithelium equals squamous cell carcinoma in situ. Low-grade lesions are exceptional and should be distinguished from benign condyloma, which is not considered a preneoplastic condition. Occasionally, separate differentiated and undifferentiated PeIN are found in the same specimen.

FIGURE 48.23 HPV-associated penile intraepithelial neoplasia, basaloid type. There is replacement of the epithelium by a monotonous proliferation of small cells with a high nuclear-to-cytoplasmic ratio. The diagnosis of carcinoma in situ is easy in such lesions.

FIGURE 48.24 HPV-associated penile intraepithelial neoplasia, warty type. The involved epithelium has an undulating/spiking surface with atypical parakeratosis. There is striking cellular pleomorphism and koilocytosis (multinucleation, nuclei with irregular contours, perinuclear halo, and dyskeratosis).

FIGURE 48.25 HPV-associated penile intraepithelial neoplasia (PeIN), mixed warty-basaloid type with flat surface. The lower part of the lesion shows classic features of basaloid PeIN. The top part shows atypical koilocytic changes characteristic of warty PeIN.

FIGURE 48.26 HPV-associated penile intraepithelial neoplasia, mixed wartybasaloid type with spiky surface. The lower half of the lesion is composed of atypical basaloid cells and the surface is composed of clear cells.

The distinction between differentiated and HPV-related PeIN is not as challenging as that between differentiated PeIN and other benign/reactive conditions. Because of the subtle histologic changes of differentiated PeIN, there is a considerable need for molecular and immunohistochemical markers to more easily identify this variant. The use of a triple p16/p53/Ki-67 immunohistochemical panel was found to be helpful in the classification of penile intraepithelial lesions. Squamous hyperplasia tends to be p16 and p53 negative, with variable Ki-67 positivity. Differentiated PeIN are p16 negative and Ki-67 positive, with variable p53 positivity. Basaloid and warty PeINs are p16 and Ki-67 positive (Fig. 48.27A,B), with variable p53

positivity (125-128). En bloc (diffuse) p16 expression is characteristic of high-risk HPV-related PeIN (basaloid/warty types).

FIGURE 48.27 (A) Basaloid penile intraepithelial neoplasia (left) and normal epithelium (right). (B) Note the overexpression of p16 by the basaloid carcinoma in situ.

With few exceptions, there is a good correlation between the microscopic appearance of the precursor (in situ) lesion and the associated invasive carcinoma, further supporting the concept of a bimodal pathway of penile tumorigenesis. BOWENOID PAPULOSIS Bowenoid papulosis represents a multifocal HPV-related clinicopathological condition affecting the anogenital region of young adults (129). Clinically, penile bowenoid papulosis is characterized by multiple soft papules or macules mostly affecting the skin of the shaft and, less frequently, the epithelium of the glans, sulcus, or foreskin. Despite the clinical benign-looking appearance of these papular lesions, histopathologic findings reveal features of a squamous cell carcinoma in situ. There is a proliferation of atypical cells with a high nuclear-to-cytoplasmic ratio that is indistinguishable from carcinoma in situ, especially undifferentiated (warty/basaloid) PeIN. The lesions often show hyperpigmentation of the basal layer. The atypical foci tend to be patchy and discontinuous, in contrast to carcinoma in situ, which tends to be more diffuse. However, a definitive distinction between carcinoma in situ and bowenoid papulosis is not possible on histology alone, and therefore clinical correlation is necessary to confirm this diagnosis. Lesions of bowenoid papulosis may regress spontaneously. In some cases (especially in immunocompromised patients), the lesions may be more widespread and may evolve to invasive carcinoma (130). Bowenoid papulosis is most frequently associated with high-risk HPV-16; however, other HPV types and mixed infections have also been reported (130,131). SQUAMOUS CELL CARCINOMA EPIDEMIOLOGY Penile cancer most frequently affects elderly patients, but age variations are seen with certain histologic subtypes. Basaloid and warty carcinomas appear in slightly younger patients than the other

variants. Although rare, penile carcinoma has been reported in young individuals (132-135). Familial cases have also been noted (136). There is no racial preference for penile carcinomas. They have been reported in whites from Europe and North America, Latin Americans, Asians, Africans, Jews, Arabs, native Indians of South America, and African Americans (137-140). Second primary tumors are common in black patients with penile cancer (17.7% of patients) (133). There is a wide geographic distribution, with low frequency in the United States and Europe to higher frequencies in Latin America, Africa, and Asia (except Japan), where it has been reported to comprise up to 20% of malignant tumors in males (140-145). In the United States, elevated rates among white males were seen in midwestern areas, whereas surprisingly low rates were noted in California and southern Florida, which are areas with large Hispanic populations. Although rates were higher among blacks than whites, the data for blacks are too limited for analysis. Reports from Asia and Central and South America are numerous (146-149). Regional differences in the prevalence of penile carcinoma are noted in northern and southern Brazil. According to the National Cancer Tumor Registry of Paraguay, penile cancer is the second most common urologic malignancy after prostate cancer. Clinicians in Sweden, Denmark, and Finland have performed extensive epidemiologic studies; no systematic geographic variation has been noted (150). Cancer of the penis is relatively common in Africa, being the most common male cancer in Uganda (140,151). There is wide variation in prevalence in India. The differences in this country do not appear to be related to circumcision status (Muslims practice circumcision, whereas Hindus do not) (152). RISK FACTORS Uncircumcised men develop penile carcinoma more frequently than those who have had early circumcision (153,154). The risk for penile cancer is 3.2 times greater among men who were never circumcised than in men circumcised at birth (153). Late circumcision does not seem to have the same preventive effect (154). Penile carcinoma

arising at the site of circumcision scars in cases of adult circumcision have been described (138). It has been shown that circumcision is associated with a reduced risk of penile HPV infection and, in the case of men with a history of multiple sexual partners, a reduced risk of cervical cancer in their current female partners (155). Uncircumcised male partners of women with HPV lesions have an increased incidence of HPV infections in the shaft-foreskin (156) and a higher incidence of PeIN compared with uncircumcised men (156). It is clear that when lacking hygienic habits, the foreskin provides a favorable microenvironment for infectious organisms and for the progression of HPV lesions. Penile cancers occur in circumcised males only rarely (129,138,157,158). A subset of cancer of the penis has been linked to HPV types, with a preponderance of HPV-16 (46-48,65-67,159-161). In a study of 194 penile lesions from the United States and Paraguay, HPV DNA was detected in 42% of penile carcinomas (67). HPV is more frequently associated with basaloid or warty carcinoma variants (85%). Most usual/keratinizing and verrucous carcinomas are HPV unrelated (66,162). There appears to be strong evidence that HPV16 is involved in penile carcinogenesis; the role of other HPV types remains controversial (46,47,159,160). Molecular studies have shown E6 transcriptional activity and a high viral load in HPV-16 DNA–positive SCCs. Additionally, HPV-16 molecular findings were strongly associated with HPV-16 L1-, E6-, and E7-antibody seropositivity (47). Numerous reports have linked LS to carcinoma of the penis (82,85-88,163-167). It is possible that LS represents a precancerous condition for a subset of SCCs, mainly the HPV-unrelated variants (71,86). For a more detailed discussion, please refer to the previous section on LS. The relation between lichen planus and penile cancer remains controversial (90,168). In a case control study, a significant association was found among smoking, chewing tobacco, and the use of snuff in patients with penile cancer, with a dose–response relationship for the first two. The use of more than one form of tobacco increases the risk (169). Daling et al. (170) found that the adjusted odds ratio for penile

cancer associated with current smoking was 2.8 times that of men who never smoked, and it was dose dependent. Secondary tumors after radiation have been described. They are usually more aggressive than the original tumor (171). Radiationinduced transformation of verrucous to anaplastic carcinoma has been reported (172). Psoralens and ultraviolet radiation (PUVA) were found to have a strong dose-dependent correlation with penile cancer. When followed for more than 12 years, 14 patients treated with PUVA developed 30 genital neoplasms. The incidence of cancer was 286 times higher than that in the general population and 16.3 times that in patients exposed to low levels. High-dose ultraviolet B radiation increased the risk of genital tumors by 4.6 times compared with controls (173). A syphilis history is not seen significantly more often in patients with penile cancer than in control patients. Syphilis is unrelated to penile carcinoma (174). Other anecdotal associations reported included Hailey-Hailey disease (175), burn scars (176), asbestos exposure (177), chronic sinus tract (178), hypospadias (179), mineral oil injection (180), and Zoon plasmacellular balanitis (18). PATHOLOGIC CLASSIFICATION Penile carcinomas can be classified according to patterns of growth and by histologic features. Growth patterns correlate with patients’ outcome. Whereas many penile SCCs are histologically similar to squamous cell neoplasms of other organs, only half are of the conventional type. The remainder are variants with distinctive clinical, morphologic, and behavioral features. The 2016 WHO classification of penile squamous cell carcinomas is based on the presence of HPV and defines histologic subtypes accordingly. See Table 48.1 (181,182). TABLE 48.1 Squamous Cell Carcinoma (SCC) Classification (Based on 2016 WHO Classification of Penile Malignant Epithelial Tumors)

NON–HPV-RELATED SCC SCC, usual type Pseudohyperplastic SCC Pseudoglandular SCC Verrucous carcinoma Carcinoma cuniculatum Papillary SCC, NOS Adenosquamous SCC Sarcomatoid SCC Mixed SCC HPV-RELATED SCC Basaloid SCC Papillary basaloid SCC Warty SCC Warty-basaloid SCC Clear cell SCC Lymphoepitheliomalike SCC Other rare carcinomas

PATTERNS OF GROWTH The classification of SCC of the penis according to growth patterns is justified because there is a correlation with prognosis (11,183-185). Approximately one-third of penile tumors show a mixed pattern of

growth, principally those of clinically advanced stages. There are four main growth patterns (Figs. 48.28 to 48.31).

FIGURE 48.28 Growth patterns of squamous cell carcinoma: superficial spreading. (A) Partial penectomy showing phimosis and a large neoplasm growing along the surfaces of penile anatomic compartments. (B) In the diagram, the tumor (ca) superficially involves the corpus spongiosum (cs) of the glans and extends to the coronal sulcus (cos) and foreskin. Skin (sk), urethra (u), albuginea (alb), and corpus cavernosum (cc) are spared.

FIGURE 48.29 Growth patterns of squamous cell carcinoma: vertical growth. (A) Cut surface with a solid tan neoplasm in glans ventral area. (B) A parallel section of the same specimen shows the relationship of the tumor (ca) to the urethra (u), albuginea (a), corpus spongiosum (cs), and corpus cavernosum (cc).

FIGURE 48.30 Growth patterns of squamous cell carcinoma: verruciform. (A) A white-tan papillomatous tumor replaces the distal glans and coronal sulcus. (B) Diagrammatic representation of the same tumor (ca) affecting the glans (gl) and coronal sulcus (cos). Other structures such as the foreskin (f) and corpora spongiosa and cavernosa (cs and cc) are not involved.

FIGURE 48.31 Growth patterns of squamous cell carcinoma: multicentric. (A,B) Diagram and cut section of a partial penectomy specimen showing multiple foci of tumor (MC) in the glans.

Superficial Spreading. The most common pattern of growth usually corresponds to neoplasms widely involving the superficial anatomic layers of glans, sulcus, or foreskin (Fig. 48.28) (183-185). There are two phases of growth: initially, horizontal (mainly involving the epithelium and lamina propria) and in more advanced stages vertical, with invasion of corpus spongiosum and cavernosum. More

than one anatomic compartment is usually affected (i.e., glans, foreskin, and coronal sulcus). Grossly, there is a slightly raised, white-gray, granular firm neoplasm involving epithelial surfaces that on cut surface is mostly limited to superficial layers (Fig. 48.28). Microscopically, the lesion is superficial with an extensive in situ component. Different presentations include (a) predominantly in situ carcinoma, with or without superficial infiltration into lamina propria; (b) predominantly in situ carcinoma, with a focal vertical pattern of growth (part of the tumor is nodular and deeply invasive); and (c) carcinoma that is horizontal and bandlike in growth and composed entirely of a mixture of in situ and superficially invasive carcinoma. The histologic variant usually associated with this pattern is SCC of usual type. Because of the centrifugal growth, tumors can extend to urethral or skin surgical margins of resection. Recurrent carcinoma in the glans is not uncommon years after an incomplete removal of a superficially spreading carcinoma of the foreskin. Vertical Growth. The classic presentation is that of a large, ulcerated, and fungating mass that on cut surface shows a solid, deeply invasive nodular appearance (see Figs. 48.29 and 48.36). Microscopically, subtypes associated with this pattern are the basaloid, sarcomatoid/anaplastic, and high-grade usual SCC. There is a high risk of regional metastasis and death (184). Verruciform. These are slowly growing, exophytic, welldifferentiated hyperkeratotic tumors with a papillary configuration (Fig. 48.30) (11,183-185). Approximately 25% of penile tumors are verruciform. They are superficial and rarely invade deep structures. This pattern of growth is preferentially associated with verrucous, condylomatous (warty), papillary, and cuniculatum carcinomas. Mixed forms are noted. Benign pseudotumors (verruciform xanthomas) and benign tumors (giant condylomas) may grossly simulate verruciform SCCs. Multicentric. These are defined as two or more independent foci of carcinoma separated by benign tissues (Fig. 48.31) (11,184,185).

They may be clinically evident or may be a microscopic finding, synchronous or metachronous, and appear in multiple compartments. Histologic subtypes may be similar or dissimilar. Patients are prone to recurrences unless all anatomic compartments are removed at surgery. Any tumor type may present in a multicentric fashion, but it is typical of pseudohyperplastic carcinomas. Mixed. It is not unusual to find a mixture of growths composed of superficially spreading, vertical, and verruciform patterns. Such lesions may also be multicentric. On histologic examination, a combination of low and high histologic grades may be found. A classic example is the hybrid verrucous/SCC of usual type. SUBTYPES OF INVASIVE SQUAMOUS CELL CARCINOMAS AND GIANT CONDYLOMAS Usual. These are malignant epithelial tumors composed of squamous cells with variable keratinization and differentiation. Lack of keratinization (pearls or cytoplasmic eosinophilia) serves as a sufficient criterion for exclusion. They harbor no special features. It is the most common type of penile cancer, accounting for 50% to 60% of all cases. The median age is approximately 58 years. Grossly, it appears as an irregular granular mass with a variably flat, exophytic, or even polypoid surface. Large lesions tend to be ulcerated. The cut surface shows a white to tan solid irregular tumor with either superficial or deep penetration into the various penile anatomic layers. Microscopically, there is an infiltrating keratinizing squamous carcinoma with three differentiation grades (186-188). Welldifferentiated (grade 1) tumors are characterized by squamous cells growing in sheets with almost normal to slightly enlarged nuclei and abundant eosinophilic cytoplasm. Intercellular bridges are easily identified, and keratinization is prominent. There is minimal pleomorphism, usually seen near the basal layer (Fig. 48.32). Moderately differentiated (grade 2) carcinomas show a more

disorganized growth compared with grade 1 lesions, higher nuclearto-cytoplasmic ratio, evident mitoses, and, although present, less prominent keratinization (Fig. 48.33). Poorly differentiated (grade 3) neoplasms show foci of solid sheets or irregular small aggregates, cords, or nests of cells with little or no keratinization, high nuclear-tocytoplasmic ratio, thick nuclear membranes, nuclear pleomorphism, clumped chromatin, prominent nucleoli, and numerous mitoses (Fig. 48.34). A tumor should be graded on the least differentiated element, even if this constitutes only a minor component of the neoplasm. Any proportion of grade 3 should be reported (188). When it is the predominant component, it may be difficult to establish the true nature of the neoplasm. In these cases, immunohistochemical studies may be necessary to differentiate these tumors from other less common malignancies such as melanoma and epithelioid angiosarcoma. Urothelial carcinoma of the urethra can also be confused with poorly differentiated solid variants of SCC. Urethral neoplasms usually affect the ventral portion of the penis and show no evidence of squamous intraepithelial atypias; the identification of urothelial carcinoma in situ in urethral epithelium or the history of a previous bladder cancer facilitates the diagnosis.

FIGURE 48.32 Squamous cell carcinoma, usual type, well differentiated.

FIGURE 48.33 Squamous cell carcinoma, usual type, moderately differentiated.

FIGURE 48.34 Squamous cell carcinoma, usual type, poorly differentiated.

Unusual patterns such as pseudohyperplastic, acantholytic, spindle cell, lymphoepitheliomalike, trabecular, giant cell/pleomorphic, small cell, rhabdoid, and clear cell may be focally seen (Fig. 48.35). The stroma shows a mild to severe inflammatory lymphoplasmacytic infiltrate. Eosinophils are occasionally prominent. Foreign body–type giant cell reaction to the keratin may be noted, especially in highly keratinized tumors. Desmoplasia is unusual. Associated PeIN is present in two-thirds of the cases. LS can be seen in the adjacent mucosa, especially associated with low-grade carcinomas of the foreskin (189).

FIGURE 48.35 Squamous cell carcinoma, poorly differentiated with rhabdoid features.

Basaloid. HPV-related, it is composed of small or intermediatesized basophilic cells with numerous mitoses (190). It accounts for 5% to 10% of all penile cancers; the median age at diagnosis is 52 years. More than half of patients show inguinal metastasis at the time of diagnosis. The glans is the main location, but primary preputial tumors also occur. Grossly, there is a rather flat, ulcerated irregular mass. The cut surface shows a solid, tan tissue usually replacing corpus spongiosum, with involvement of albuginea and corpora cavernosa. Necrotic foci within the tumor are noted using a magnifying lens (Fig. 48.36). Microscopically, characteristic solid nests composed of monotonous small tumor cells often with central comedonecrosis are noted (Figs. 48.37 to 48.39). Peripheral palisading may occur surrounded by peripheral clear spaces or clefts; the latter are caused by retraction artifact (similar to what is observed in cutaneous basal cell carcinomas). The cells are small, with a high nuclear-to-cytoplasmic ratio, similar to basal cells, and show inconspicuous nucleoli. Numerous mitoses are characteristic.

A starry sky appearance is due to apoptosis (Fig. 48.40). Occasionally, there are pleomorphic cells of larger size. Focal and abrupt keratinization in the central portion of the nest is characteristic. The adjacent epidermis usually shows basaloid PeIN and, less often, warty, warty-basaloid, or differentiated PeIN. The stroma may show chronic inflammatory cells or hyalinization. Perineural, lymphatic, and venous invasion tends to be prominent.

FIGURE 48.36 Basaloid carcinoma. (A) Partial penectomy with a solid beige-tan neoplasm replacing distal penis. (B) The tumor (ca), depicted in yellow, infiltrates the full thickness of the corpus spongiosum and partially extends to corpus cavernosum (cc) through the tunica albuginea (alb). Small red dots represent necrotic foci (n).

FIGURE 48.37 Basaloid carcinoma. Solid and confluent nests composed of small blue cells. The adjacent epithelium shows carcinoma in situ.

FIGURE 48.38 Basaloid carcinoma. Infiltrating nests composed of a monotonous population of small basaloid cells with central area of abrupt keratinization. Note the artifactual cleft between the tumor aggregate and the stroma.

FIGURE 48.39 Basaloid carcinoma. Higher power view illustrating the basaloid neoplastic cells and central comedolike necrosis. Mitotic figures are prominent.

FIGURE 48.40 Basaloid carcinoma. Solid nest composed of anaplastic small basaloid cells. There is a “starry sky” appearance as a result of apoptosis.

The differential diagnosis is with the usual SCC, distal urethral carcinomas, basal cell carcinomas of the skin, and small cell neuroendocrine carcinomas. Basaloid carcinoma differs from the usual carcinoma by having a distinctive nesting pattern with central comedonecrosis and smaller, more regular cells compared with the larger, more pleomorphic and keratinizing cells of the usual SCC. Keratinization in basaloid carcinomas is abrupt and focal within the center of the nest, whereas in the typical squamous carcinoma it matures gradually. Urothelial carcinomas of penile urethra may be composed of small cells and disclose a nesting pattern like basaloid carcinoma, but in the former, there is usually more pleomorphism than in the basaloid tumors. The association with a papillary or in situ urothelial neoplasm of the urothelial mucosa, or previous history of bladder cancer, may help. Most penile urethral carcinomas probably

represent intraluminal implants from a previous urothelial bladder or proximal urethral carcinomas. Immunohistochemical stains with urothelial cell markers (uroplakin II and thrombomodulin) may be used (191). As a consequence of the plastic potential of urothelium, basaloid SCCs may arise primarily in the urethra. They are identical to penile basaloid SCC, and distinction is not possible. The distinction of penile basaloid carcinomas from skin basal cell carcinomas may be problematic, but cells from basaloid carcinomas are more atypical in appearance than those of basal cell carcinomas, which characteristically occur in the skin of the shaft in contrast to the mucosal and usually glans location of basaloid carcinoma. An unusual basal cell carcinoma of foreskin inner mucosa has been reported (192). Basaloid carcinomas usually lack the peripheral palisading and myxoid stromal changes characteristic of basal cell carcinomas. Occasionally, in basaloid carcinomas, a small cell pattern with trabecular or solid growth in sheets resembling neuroendocrine or Merkel cell carcinoma predominates. The distinction may be difficult, and immunohistochemical stains for neuroendocrine differentiation may be necessary. Basaloid carcinomas are usually diffusely and strongly positive for p16INK4a, indicating their relation to HPV. CK 34βE12 (high– molecular weight cytokeratin) is positive in basaloid carcinoma, and it is useful for the differential diagnosis with other SCC variants (193). HPV-16 is detected in 71% to 81% of cases by whole tissue section PCR (66,67,194). Nodal metastases are frequent in patients with basaloid carcinomas. The mortality rate varied from 30% to 60% (190,195). Warty-Basaloid. They are heterogeneous penile neoplasms with features of basaloid and warty carcinomas in at least 20% of the tumor (Fig. 48.41). Evidence of HPV has been detected in most cases (194). Most tumors are located in the glans, but an extension to coronal sulcus or foreskin is present in about two-thirds of cases. Foreskin exclusive location may be noted. Tumors are large and destructive, measuring 2 to 12 cm (mean 5 cm). Grossly, they are exoendophytic. Microscopically, both basaloid and warty

components are present (Fig. 48.42A,B), but the basaloid predominates. Two main patterns are recognized: The most common, present in about two-thirds of cases, depicts warty features on the surface and basaloid carcinoma in the invasive portion of the tumor. Both components are separated, but mixtures may be present. The second pattern is represented by a nonpapillomatous infiltrative tumor composed of neoplastic nests showing basaloid features at the periphery and warty features at the center. Wartybasaloid carcinomas frequently invade deep erectile tissues. Tumors limited to lamina propria are rare. Vascular and perineural invasion are common (196).

FIGURE 48.41 Warty-basaloid carcinoma. Note the endophytic basaloid component underlying the exophytic component with warty pattern in the same tumor.

FIGURE 48.42 (A) High-power view of warty component showing a papilla with koilocytic changes. (B) Solid nests of basaloid neoplastic cells in the same tumor.

Warty-basaloid carcinomas should be distinguished from the pure forms of warty or basaloid carcinomas. Distinction is based on the relative proportions of the components. Basaloid carcinomas are usually nonpapillomatous and exhibit the typical nesting pattern of growth proper of this tumor. Warty features can be focally observed in basaloid carcinoma but should not represent more than 10% of the tumor mass (194). A papillary outgrowth is rarely noted on the surface of basaloid carcinomas. The papillae are condylomatous, that is, they have a central fibrovascular core, and lined exclusively by small basaloid cells. This unusual variant should not be confused with a warty-basaloid carcinoma because there is no warty component (196,197). As in the basaloid SCC, in pure warty carcinomas, a basaloid component can be focally present but should not account for more than 10% of the tumor mass. Most warty carcinomas exhibit an exophytic–endophytic pattern of growth, and morphologic features are similar in both components, a helpful feature.

Warty-basaloid carcinomas are more aggressive than pure warty carcinomas but less aggressive than typical basaloid carcinomas. Inguinal nodal metastases are found at presentation in about onehalf of all patients. The cancer-specific mortality rate in a study was 33%, and the estimated 5- and 10-year survival rates were about 65%. Clinical behavior is related to the basaloid component, with a high rate of regional metastasis (198). Papillary Basaloid Carcinoma. It is a distinctive HPV-related papillomatous tumor composed of basaloid cells, most likely a variant of basaloid carcinoma. Tumors may be completely papillary noninvasive or harbor an invasive component undistinguishable from classic basaloid carcinomas (199). Patients’ mean age is 66 years; patients with invasive tumors are older than those with pure in situ disease. Glans is the most common site, but the foreskin may also be exclusively affected. Grossly, tumors are exophytic and papillomatous. Microscopically, papillae are straight, foliaceous, or rounded, distinctively composed of a central fibrovascular core, resembling condylomatous tumors or high-grade papillary urothelial neoplasms (Fig. 48.43A,B). Although they are clear cells, koilocyticlike changes may be observed near the keratinized surface; unlike typical condylomata, these cells show malignant features. The cellular population lining the papillae is uniform, with scant pleomorphism throughout the epithelium, and tumor cells are similar to those observed in basaloid PeIN, high-grade papillary urothelial carcinoma, or invasive basaloid carcinoma. The interface between tumor and stroma is variable from well-defined noninvasive borders to in situ to deeply invasive. HPV is detected in most cases, and p16 is usually strongly positive.

FIGURE 48.43 Papillary basaloid carcinoma. (A) Papillomatous exophytic growth architecture with malignant features. (B) Basaloid neoplastic cells lining central fibrovascular core. These are indistinguishable from characteristic basaloid carcinoma cells.

Papillary basaloid carcinomas should be distinguished from other papillomatous tumors such as high-grade papillary urothelial carcinomas, warty carcinomas, and papillary NOS carcinomas. Highgrade papillary urothelial carcinomas usually show more nuclear pleomorphism, and cellular boundaries are sharper when compared with papillary basaloid carcinomas, but the distinction can be challenging on routine microscopy, especially in small biopsies. The presence of PeIN, usually of the warty/basaloid type; parakeratosis; and an underlying invasive basaloid carcinoma would suggest a papillary basaloid carcinoma. The identification of adjacent flat urothelial carcinoma in situ or high-grade invasive urothelial carcinoma would indicate a urothelial origin. A previous or concurrent history of urothelial neoplasms elsewhere in the urinary tract would also favor the latter. Because HPV infection does not seem to play a relevant role in urothelial oncogenesis (181), HPV presence can aid in the differential diagnosis. The differential diagnosis with urothelial lesions would be restricted to tumors involving the distal urethra and perimeatal regions of the penis because urothelial origin would be unlikely at other penile sites. Warty (200) and papillary carcinomas of the penis should also be excluded. Warty and papillary basaloid carcinomas are both exophytic tumors harboring a papilla centered by a fibrous and vascular core. Small basaloid cells may be observed in warty (or

warty-basaloid) carcinomas, but they are usually scant and restricted to the bottom layers of the papillae, whereas they occupy the full thickness of the epithelium in papillary basaloid carcinomas. Keratinized, eosinophilic cells are also a prominent feature in the former and are absent in the latter. Koilocytes may be present in both tumors, but they are scanty and restricted to the superficial parakeratotic layer in papillary basaloid carcinomas and conspicuous and scattered throughout the tumor in warty carcinomas. Papillary NOS carcinoma should also be ruled out. Because both lesions are quite different, caution should be employed given their similar denominations. Papillary carcinomas are of low grade and composed of keratinized cells with ample, eosinophilic cytoplasm, as opposed to the more immature basophilic appearance of tumor cells in papillary basaloid carcinomas. Warty (Condylomatous). Warty carcinoma is a verruciform, moderately differentiated tumor composed of condylomatous papillae with hyperparakeratosis, pleomorphic koilocytosis, and an irregular interface of tumor and underlying stroma. Despite its distinctive gross and microscopic features, the presence of HPV is variably and inconsistently reported (66,67,194). Patients’ age is about 10 years younger than usual SCC (200). Warty carcinoma is the most frequent variant of penile carcinoma seen in immunosuppressed patients. They are large tumors affecting glans, sulcus, or foreskin. Gross features are typical: a firm white-gray exophytic, cauliflowerlike tumor with a homogeneous, lobulated, cobblestone appearance measuring about 5 cm. The cut surface reveals a papillomatous growth, with frequent deep penetration into corpus spongiosum or cavernosum. The deep borders vary from irregular to broad and pushing (Figs. 48.44A,B and 48.45A,B). Microscopically, a papillary pattern is noted. Papillae are long, undulating, and rounded or spiky, with prominent fibrovascular cores. Cells are pleomorphic with prominent koilocytic changes (enlarged nuclei with irregular contours, binucleation, clear perinuclear halo, and dyskeratosis) (Figs. 48.46 to 48.48). Compared with benign condylomas, these HPV-related changes are not restricted to the

surface but are present throughout the tumor, including deep portions of it. The boundary between the neoplasm and stroma is infiltrative (jagged) in most cases; however, some cases may show a predominantly pushing border. Intraepithelial abscesses may be prominent, especially in the basal areas. In some cases, a deep endophytic growth pattern with well-circumscribed deep pseudocystic nodular aggregates can be seen (Fig. 48.49). We have seen cases of warty carcinoma with prominent clear cell features (Fig. 48.50).

FIGURE 48.44 (A,B) Warty (condylomatous) carcinoma. Gross and diagrammatic representation showing an exophytic tumor composed of confluent nodules with a cobblestone appearance affecting part of glans sulcus and foreskin.

FIGURE 48.45 Warty (condylomatous) carcinoma. (A) Cut surface showing a tan tumor involving all compartments. The pattern is flat with undulated surface in the foreskin and deeply invasive in the glans. (B) The tumor is shown in yellow. In the foreskin (F), the lesion is mostly in situ (warty squamous intraepithelial lesion

[WSIL] or warty penile intraepithelial neoplasia), whereas there are deeply invasive tumoral nests in the corpus spongiosum of the glans (G).

FIGURE 48.46 Warty carcinoma. Section corresponding to the preputial, noninvasive component of tumor shown in Figure 48.45. Note the arboriform and rounded condylomatous type of papillae with central fibrovascular cores and the broad base.

FIGURE 48.47 Warty carcinoma. Higher power view of a papilla with parakeratosis and clear cell koilocytotic changes. A central fibrovascular core is evident.

FIGURE 48.48 Warty carcinoma. There is nuclear pleomorphism and koilocytic changes throughout the neoplasm including the deeper invasive foci.

FIGURE 48.49 Warty carcinoma. Rounded nodules of carcinoma deep in the corpus spongiosum. Note the characteristic dark and clear appearance of this neoplasm. The clear areas correspond to koilocytic changes. Note the presence of vascular invasion.

FIGURE 48.50 Warty carcinoma is a human papillomavirus–related neoplasm; therefore, immunohistochemical stain with p16 usually shows diffuse expression, as illustrated in this figure.

Warty carcinomas should be distinguished from other verruciform neoplasms, especially giant condylomas, verrucous carcinomas, and complex tumors. Giant condylomas may be similar and quite atypical histologically, but the base of the tumor is broad and “pushing” rather than irregular as in warty carcinomas (200). Occasionally, warty carcinomas may present as a “noninvasive” tumor with pushing borders. The demonstration of the presence of high-risk HPVs, usually the genotype 16, in warty carcinoma and low-risk HPV, usually 6 or 11 in giant condyloma, may be necessary, using PCR or other methodologies. The distinction with verrucous carcinoma is usually straightforward because this tumor lacks condylomatous papillae and koilocytosis. Rarely, a tumor may harbor features of both warty and verrucous carcinomas and should be classified as such: mixed verrucous-warty carcinoma. Greatest diagnostic difficulties are in the differential diagnosis of low-grade warty and papillary carcinoma NOS. Both tumors may contain condylomatouslike papillae, but, typically, papillary carcinomas show no koilocytosis and are of lower grade and typically lack HPV (201).

Some usual SCC may show focal or extensive clear cell features strikingly similar to koilocytosis in deep invasive nests. It is not advisable to diagnose such lesions as warty carcinoma unless there is a condylomatous papillary configuration as the predominant feature. In our experience, these clear cell changes are not a reliable indication of viral presence (194,202). We have seen cases of warty carcinoma with prominent clear cell features, but usually such changes are more focal when compared with clear cell carcinoma. These clear cell SCCs should be differentiated from sweat gland carcinomas, clear cell carcinomas, and metastatic renal cell carcinomas to the penis (203). Warty carcinomas, similarly to identical tumors in other sites (204), are considered HPV-related neoplasms. HPV identification is usually performed using various modalities of the PCR technique. The most commonly found serotype in this tumor is HPV-16. However, there is some variability in the incidence of HPV from 22% to 100% (66,67,194,205). The causes of this variability may be related to differences in the sensitivity of the techniques utilized to detect the virus, to differences in the viral DNA degradation in old paraffin blocks or original inadequate fixation, or to different diagnostic pathologic criteria applied to select cases of warty carcinoma. It is also possible that some highly keratinized warty carcinomas are less likely to show positivity for HPV. For all verruciform tumors, p16INK4a was shown to be the best immunohistochemical tissue marker of warty carcinoma (Fig. 48.50) (206). Warty carcinomas may be associated with regional lymph node metastasis, and this appears to be more frequent in deeply invasive and high-grade tumors. Warty carcinomas have an intermediate biologic behavior between other types of low-grade verruciform tumors (verrucous, papillary) and the usual SCC of the penis. Clear Cell. Clear cell carcinoma is an extremely rare and aggressive HPV-related tumor that originates in the glans mucosa and affects elderly patients (207). Macroscopically, they are large white-yellowish irregular masses arising from the glans with extension to coronal sulcus and rarely to

foreskin. The cut surface is white with presence of yellow to gray foci corresponding to necrosis and invades deeply up to the corpora cavernosa. The main microscopic finding is the strikingly clear cell feature that dominates the tumor cytology. Architecturally, two patterns were observed: the nesting pattern with central comedolike necrosis resembling basaloid carcinomas but totally composed of clear cells (Fig. 48.51A) and the solid pattern composed of lobulated confluent sheets surrounded by delicate fibrous tissue. The cells contain ample clear cytoplasm with hyperchromatic, rounded to ovoid, or wrinkled nuclei resembling koilocytes (Fig. 48.51B). Binucleation, mitoses, and nuclear pleomorphism are seen. Focal areas of warty and basaloid invasive carcinomas comprising about 5% of the tumor have been described, and it is likely that the 3 tumors are closely related. HPV-related PeIN of warty/basaloid subtypes adjacent to the tumor supports the squamous epithelium origin of this neoplasm. GATA3 immunostaining is negative, whereas p16 immunostain and HPV16 in situ hybridization are positive. The differential diagnosis is with the most common warty (condylomatous) carcinoma with solid invasive pattern, skin adnexa sweat gland tumors, and metastatic renal cell carcinomas.

FIGURE 48.51 (A) Clear cell squamous cell carcinoma, low power. Note the neoplastic clear cells sheets with extensive geographic necrosis and the smaller nests with central abrupt keratinization and comedolike necrosis. (B) Clear cell squamous cell carcinoma, high-power. A neoplastic nest is observed with the characteristic central comedolike necrosis. The cells show ample clear cytoplasm with striking cellular borders. Most of the nuclei are hyperchromatic, round to ovoid, or wrinkled. Mitoses and binucleation can be seen. (C) Immunhistochemistry with p16 shows diffuse and strong expression supporting association with high-risk HPV.

Adverse factors characterize this tumor: deep invasion, necrosis, high histological grade, vascular and perineural invasion, and lymph node involvement at the diagnosis. In 2 of the 3 reported cases in a study, the patients were dead of disease by 9 and 12 months of follow-up. Verrucous. Verrucous carcinoma is an unusual, extremely differentiated, verruciform tumor with hyperkeratosis, papillomatosis, acanthosis, and broadly based interface between tumor and stroma. It can typically be mixed with the usual SCC, but basaloid and warty sarcomatoid carcinomas may also occur. They may affect any compartment, but they are most common in the glans. The median age at diagnosis is 57 years, and the average duration of the disease is 56 months, the longest among all penile malignant tumors

(185,186,208,209). Grossly, they are exophytic white-gray neoplasms measuring from 1 to 3 cm in diameter (Fig. 48.52); larger destructive lesions can be seen. Microscopically, the lesions are characterized by thick acanthotic papillae with thin and generally inconspicuous fibrovascular cores (Fig. 48.53). There is prominent piling up of keratin between the papillae, sometimes forming keratin craters (Figs. 48.17 and 48.53). The cross section of the tip of the papillae shows a central keratin plug, with peripherally located tumor cells. Orthokeratosis, with presence of keratohyaline granules, is more frequent than parakeratosis. Koilocytosis is not a feature of verrucous carcinoma; however, occasional vacuolated clear cells (likely not true koilocytes) may be seen at the surface. In any case, these changes are not prominent. The neoplastic cells are extremely well differentiated, with prominent intercellular bridges. There is minimal atypia at the base of the nests, as well as rare mitosis. The base of the tumor is broad and pushing and sometimes shows a characteristic club-shaped pattern. This feature defines the tumor as verrucous (Fig. 48.54). A dense inflammatory cell infiltrate may obscure the boundaries between tumor and stroma. Most verrucous carcinomas are limited to the lamina propria; deeper invasion is unusual. Irregular, non-broadly based microinvasion into lamina propria may occur, and we refer to these tumors as microinvasive verrucous carcinomas. Invasive nests should be in lamina propria (23 mm in depth). Most large and deep verrucous carcinomas undergo transformation into invasive carcinoma of the usual or sarcomatoid type (Fig. 48.55) (210,211). When more than 20% of a tumor is nonverrucous, we designate them as hybrid verrucous carcinomas. Verrucous carcinomas are usually HPV negative, with only a few exceptions that have been reported. The molecular profiling is generally lower expression levels of p16 and Ki-67, whereas p21, p53, and Rb expressions are no different compared with usual SCCs (212,213).

FIGURE 48.52 Verrucous carcinoma. (A) Two separate preputial verrucous carcinomas arising against a background of long-standing lichen sclerosus. (B) Diagram illustrating one of the verrucous tumors in blue.

FIGURE 48.53 Verrucous carcinoma: low-power view illustrating the verrucous surface and bulbous deep borders of this highly differentiated neoplasm. The papillae show thick acanthotic epithelium and thin fibrovascular cores.

FIGURE 48.54 Verrucous carcinoma. Higher power view illustrates the high degree of differentiation and bulbous, pushing deep borders.

FIGURE 48.55 Hybrid carcinoma, mixed verrucous and usual type. This tumor shows, in addition to areas of verrucous carcinoma, other less differentiated foci with infiltrative borders. These mixed tumors have a worse prognosis when compared with pure verrucous carcinoma.

Verrucous carcinomas should be distinguished from verrucous hyperplasia, other verruciform tumors, and from mixed verrucoususual SCC. There are no useful morphologic diagnostic clues to separate verrucous hyperplasia from a small verrucous carcinoma. One rather subjective criterion is the smaller size of the hyperplasias. Another may be the bulging of verrucous carcinoma front or base into the lamina propria and the flatness of hyperplasia, although there are unquestionable verrucous carcinomas with a flat base, the latter recognized by deeper penetration than the surrounding skin. It may be appropriate to designate small noninvasive lesions as hyperplasias with the knowledge that they may, in fact, represent an incipient form of a verrucous carcinoma. To distinguish giant condyloma from verrucous carcinoma, strict morphologic criteria

should be applied. Verrucous carcinomas lack koilocytosis, characteristically seen in giant condyloma and warty carcinoma. Also, the papillae are shorter and lack a central fibrovascular core, unlike giant condyloma. Papillary SCC, NOS exhibits more cytologic atypia than verrucous carcinoma and has an irregular invasive front but also lacks koilocytotic changes. In warty carcinoma, the interface of tumor and stroma is irregular, and the koilocytotic changes are identifiable in surface epithelium and in deep tumoral nests. Carcinoma cuniculatum, another verruciform tumor, is cytologically undistinguishable but grossly distinctive, with an endophytic burrowlike pattern of growth not seen in verrucous carcinomas. Verrucous carcinomas mixed with other SCC variants (hybrid) should be placed in the mixed category. The designation of verrucous carcinomas should be applied only to tumors that satisfy the strict morphologic criteria mentioned previously. Pure and microinvasive verrucous carcinomas have an excellent prognosis, whereas hybrid verrucous carcinomas are associated with regional metastasis in up to 25% of the cases (211). The presence of regional metastasis in verrucous carcinomas is related to higher grade areas and deeper infiltration. Verrucous carcinomas can be associated with local recurrence, and this may be related to insufficient surgery or a result of multicentricity. Pure verrucous carcinomas are not associated with nodal metastasis, and survival, when the tumor is correctly diagnosed, should be near 100%. Cuniculatum. This is a variant of verrucous carcinomas with deeply penetrating low-grade burrowing growth pattern. A macroand microscopic evaluation of tumor cut surface is required for the diagnosis: grossly, for the labyrinthine pattern identification; microscopically, for the verrucous appearance. Originally described by Ayrd et al. (214) in the plantar skin, carcinoma cuniculatum is characterized by a deep burrowing growth pattern mimicking rabbit burrows (cuniculum). Rare cases have been reported in the penis (215). The mean age of patients is 77 years. Grossly, the tumors are large, papillomatous lesions, usually affecting the glans and extending to the coronal sulcus and foreskin. Cut surface shows the

hallmark of the lesion represented by deep and complex tumor invaginations that connect to the surface through sinus tracts (Fig. 48.56). Microscopically, the bulk of the lesion has features of a verrucous carcinoma (extremely well differentiated with bulbous deep borders) usually associated with a minor component that is more infiltrative and less differentiated (Fig. 48.57).

FIGURE 48.56 Carcinoma cuniculatum. Longitudinal section of a partial penectomy specimen with a peculiar tumor showing a deep burrowing pattern.

FIGURE 48.57 Carcinoma cuniculatum. Microscopic appearance showing interanastomosing sinuslike structures. The tumor is well differentiated.

Carcinoma cuniculatum should be distinguished from classic verrucous carcinoma and mixed usual-verrucous (hybrid) carcinomas. Verrucous carcinoma is also an extremely welldifferentiated tumor with a broad base of invasion but rarely invades beyond the lamina propria. The identification of dedifferentiated areas of usual SCC, of identical or of higher grade, in an otherwise typical verrucous carcinoma, is characteristic of hybrid verrucous carcinoma (210,211). The burrowing pattern of invasion is typical of carcinoma cuniculatum and not of the aforementioned variants. The importance of the distinction of carcinoma cuniculatum from hybrid verrucous carcinomas is that the latter tumor has a potential for nodal metastasis and mortality (210,211). Higher grade and prominent koilocytic changes separate the endophytic variant of warty carcinomas (200). Carcinoma cuniculatum has a good prognosis. None of the reported cases metastasized (215).

Papillary, Not Otherwise Specified. It is a low-grade verruciform squamous invasive neoplasm with hyperkeratosis, papillomatosis, and an irregular interface between tumor and stroma; no koilocytosis is noted (11,184,185,201). The diagnosis is made by exclusion of the other more specific types of verruciform tumors. It is usually located in the glans, although other compartments also may be involved. Grossly, they are white-gray exophytic destructive lesions. The cut surface has a serrated papillomatous appearance and poor delineation of the limits between tumor and stroma (Fig. 48.58A,B). Microscopically, the appearance is that of a well-differentiated papillary squamous neoplasm. Hyperkeratosis and acanthosis are prominent. The papillae are variable, short or long, but usually broad and with a prominent fibrovascular core (Fig. 48.59). Keratin cysts or intraepithelial abscesses can be present. Koilocytoticlike changes are never prominent. The base of the lesion is irregular and infiltrative (Fig. 48.60). The differential diagnosis includes other verruciform tumors. The pleomorphic features and prominent koilocytosis characteristic of warty carcinoma are not seen in papillary carcinoma. Compared with verrucous carcinoma, papillary carcinoma is slightly less well differentiated and shows irregular infiltrative deep borders.

FIGURE 48.58 Papillary carcinoma. (A) Longitudinal section of a penectomy specimen with a tan-white irregular lesion in the ventral area. (B) Tumor (ca) shows invasion of lamina propria and corpus spongiosum (cs). Other anatomic structures (alb, albuginea; cc, corpus spongiosum; m, meatus) are uninvolved.

FIGURE 48.59 Papillary carcinoma. Complex papillae of a low-grade neoplasm. The papillae usually have a prominent fibrovascular core.

FIGURE 48.60 Papillary carcinoma. The interface between tumor and stroma is infiltrative. The tumor is less well differentiated than verrucous carcinomas.

The differential diagnosis is with other verruciform neoplasms (200). Papillae may be similar, but warty carcinomas have distinctive HPV-related pleomorphic koilocytosis in the majority of the cells. In cases of low-grade warty carcinomas, the distinction may be challenging, and HPV needs to be demonstrated to certify the case as warty carcinoma. The differential with verrucous carcinomas is possible after evaluating tumor fronts: Verrucous carcinomas show an acanthotic broad tumor base. This pattern contrasts with the jagged and irregular base of papillary carcinomas. Prognosis is excellent in patients with papillary carcinomas. Recurrent rate is about 12%, compared with 27% of usual SCCs. Inguinal lymph node metastasis is low, ranging from 0% to 12% (195,202). The mortality rate is also low, ranging from 0% to 6% (195,202). Sarcomatoid. These are large polypoid tumors composed of spindle cells and with at least focal or immunohistochemical evidence of squamous differentiation (216). At least 30% spindle cells are required for the diagnosis. Sarcomatoid carcinomas arise de novo, after recurrence of a usual SCC, or secondary to sarcomatoid transformation after radiation therapy of a primary penile carcinoma. Other synonyms are metaplastic carcinoma, carcinosarcoma, and spindle cell carcinoma. Sarcomatoid carcinomas are uncommon, representing approximately 4% of penile carcinomas (216-218). The mean patient age is 60 years. Grossly, most tumors are large, polypoid, and ulcerated masses frequently affecting the glans and deeply invading corpora spongiosa and cavernosa (Fig. 48.61) (216). Microscopically, the neoplasms are composed predominantly of atypical spindle cells disposed in interlacing fascicles, resembling fibrosarcoma or leiomyosarcoma (Fig. 48.62), sometimes admixed with pleomorphic giant cells mimicking malignant fibrous histiocytoma. Myxoid changes may be prominent. Lesions showing a prominent pseudovascular pattern

mimicking angiosarcoma have been described (Fig. 48.63) (216). Mitotic figures tend to be numerous and necrosis prominent. Foci of heterologous differentiation toward bone and cartilage (osteosarcomatous and chondrosarcomatous components) may be observed (Fig. 48.64). Associated foci of PeIN are frequent. Because keratinizing foci of invasive carcinoma are usually a minor component (Fig. 48.65), multiple sections and immunohistochemical studies may be necessary to make a correct diagnosis and rule out special types of sarcomas. The spindle cells are usually positive for vimentin, different cytokeratins, and p63. In our experience, cytokeratin 34βE12 and p63 appear to be the more sensitive markers to categorize these tumors as epithelial (Fig. 48.66A,B). AE1/AE3 and Cam 5.2 tend to be more variably and focally positive, sometimes highlighting only scattered single cells (216). Smooth muscle actin may be focally positive; however, desmin and S-100 are negative. Penile sarcomatoid carcinomas are aggressive tumors usually associated with lymph node metastasis and poor outcome (216,217).

FIGURE 48.61 Sarcomatoid carcinoma. Partial penectomy specimen showing a large, polypoid, and hemorrhagic tumor affecting the different anatomic compartments and deeply invading into corpus spongiosum. This tumor showed angiosarcomatoid features.

FIGURE 48.62 Sarcomatoid carcinoma. The neoplasm is predominantly composed of spindle cell neoplasm simulating a sarcoma. In other areas, foci of carcinoma were present.

FIGURE 48.63 Sarcomatoid carcinoma with angiosarcomatoid features. This is the microscopic appearance of the tumor depicted in Figure 48.61.

FIGURE 48.64 Sarcomatoid carcinoma with osteoid formation.

FIGURE 48.65 Sarcomatoid carcinoma. Spindle (sarcomatoid) areas are admixed with carcinomatous foci.

FIGURE 48.66 Sarcomatoid carcinoma, immunohistochemistry. Cytokeratins and p63 are useful markers to make this diagnosis. (A) This picture illustrates the expression of cytokeratin 34βE12 in the neoplastic spindle cells, supporting the

epithelial nature of the neoplasm. (B) Sarcomatoid carcinoma with angiosarcomatoid features. Immunohistochemical stain with p63 shows diffuse nuclear expression.

Pseudohyperplastic. These are low-grade SCCs preferentially affecting the foreskin of older patients (eighth decade) affected with LS. There is extreme differentiation, and in small biopsies, the tumors mimic pseudoepitheliomatous hyperplasia. They are often multicentric, and the second or third independent tumor may show some verrucous features. Grossly, they are flat or slightly elevated lesions measuring approximately 2 cm. Characteristic microscopic features are keratinizing nests of squamous cells with no or minimal atypia surrounded by a reactive stroma (Fig. 48.67A,B). This degree of differentiation is noted only in low-grade verruciform tumors such as verrucous or papillary cancers. The consistent association with LS suggests that this inflammatory condition may play a precancerous role.

FIGURE 48.67 (A,B) Pseudohyperplastic squamous cell carcinoma of the foreskin. The tumor is extremely well differentiated and should be distinguished from pseudoepitheliomatous hyperplasia.

Pseudohyperplastic carcinomas should be distinguished from usual SCC and pseudoepitheliomatous squamous hyperplasia. Extreme differentiation is rare in the usual SCCs, but these neoplasms are usually unicentric; affect glans, coronal sulcus, or foreskin of individuals in their 50s and early 60s; invade deeper; and do not resemble hyperplasias. Pseudoepitheliomatous hyperplasias may be difficult to distinguish from pseudohyperplastic carcinomas, especially in small biopsies. There are three distinguishing features of hyperplasia. The squamous nests are not really detached from the overlying squamous epithelium (multiple step sections will reveal their connection to the surface); they are superficial and do not affect tissues beyond lamina propria, and there is no stromal reaction around the pseudoinvasive nests. Pseudohyperplastic carcinomas are deeper, may affect dartos or corpus spongiosum, have reactive stroma, have at least minimal atypia, and in completely resected specimens there are usually at least focal areas of bona fide invasive SCC. The prognosis in pseudohyperplastic carcinomas is excellent. In a series of 10 cases, recurrence was noted in the glans of one patient who was circumcised for a multicentric carcinoma of the foreskin 2 years after diagnosis. No metastases were found in any of these cases (86,189). Acantholytic (Pseudoglandular Adenoid). This is an unusual variant of SCC characterized by prominent acantholysis and formation of pseudoglandular spaces (Fig. 48.68) (186,219). At least 30% of adenoid features are required for the diagnosis. Patient median age is 54 years. Tumors tend to be large, involve multiple penile anatomic compartments, and deeply invade into erectile corpora. The pseudoglandular spaces contain acantholytic neoplastic keratinocytes sometimes admixed with keratin material and necrotic debris. Carcinoembryonic antigen (CEA) and mucin stains are negative.

FIGURE 48.68 Squamous cell carcinoma with acantholytic (adenoid) features. Note the presence of acantholytic neoplastic cells within the pseudoglandular spaces.

The differential diagnosis includes true glandular penile tumors (surface adenosquamous (220,221), mucoepidermoid (222,223), and urethral adenocarcinomas) and the angiosarcomatoid variant of sarcomatoid carcinoma (224). Gland-forming tumors are more homogeneous and lack the variegated spectrum of morphologic changes typical of the pseudoglandular SCC. Sarcomatoid SCC can sometimes exhibit an alveolar pattern of growth around pseudovascular spaces, and this can simulate pseudoglands. A careful examination of these spaces and the identification of more typical areas of sarcomatoid features aid in the differential diagnosis.

Surface Adenosquamous. It is an exceedingly rare carcinoma that probably originates from misplaced glandular cells in the perimeatal region of the penis (220,221). Grossly, the tumors are usually large, firm, and granular. Deep invasion of the corpus spongiosum is usually present. Microscopically, a biphasic squamous cell and glandular pattern is noted (Figs. 48.69 and 48.70). The squamous component usually predominates. PeIN is frequently present in the glans mucosa. Immunostains for CEA are typically positive in the glandular component (Fig. 48.71); 34βE12 has also been reported positive in both components (221).

FIGURE 48.69 Adenosquamous carcinoma. Biphasic squamous and glandular infiltrating carcinoma. Note the presence of perineural invasion.

FIGURE 48.70 Adenosquamous carcinoma. Squamous carcinoma with central mucinous glandular features.

FIGURE 48.71 Adenosquamous carcinoma. Note the strong immunoreactivity for carcinoembryonic antigen in the glandular areas of the tumor.

Adenosquamous carcinomas of the penis should be distinguished from acantholytic SCC, adenosquamous (mucoepidermoid)

carcinomas of the urethra (11), adenocarcinoma arising in Littre glands, and metastatic adenocarcinomas. Urethral tumors are located ventrally in the penis, with the usual restriction to periurethral tissue and corpora cavernosa. Secondary invasion of Littre glands by SCC of the penis can simulate an adenosquamous carcinoma. In such cases, the glands appear benign. In adenocarcinoma metastatic to the penis, the corpora cavernosa is mainly affected or unexpected sites such as the penile fascia. Metastases show frequent tumor emboli and rarely involve the corpus spongiosum or lamina propria. The documentation of associated PeIN on surface epithelium argues against a metastasis. Medullary. Medullary carcinoma is an inflammatory cell-rich solid poorly differentiated (high-grade) HPV-related squamous cell carcinoma seen in older males (225,226). This tumor usually presents as a solid irregular mass of the distal penis comprising mainly glans, but some cases extended to coronal sulcus and foreskin. The cut surface is white-greyish and deeply invasive, comprising all penile compartments. Microscopically, tumor is characterized by large cohesive and confluent solid epithelial nests, sheets, or trabecula interspersed with dense inflammatory infiltrate within the tumor composed of a variable proportion of eosinophils and neutrophils (Fig. 48.72A,B). A less cohesive pattern could be noted in some peripheral isolated areas of large tumors. Necrosis of individual cells, foci or in geographic pattern is common. Peripherally, a less conspicuous lymphoplasmacytic infiltrate can be seen. The neoplastic cells are large, anaplastic with inconspicuous cellular borders and lacking maturation. Nuclei are polymorphic, usually large, vesicular with loose chromatin and prominent eosinophilic nucleoli. Adjacent HPV-related (warty/basaloid) PeIN and non-HPV-related (differentiated) PeIN as well as LS can be seen. Strong diffuse p16 immunostaining positivity is characteristic. HPV was found in all the reported tumors, HPV16 being the most frequent isolated genotype. The differential diagnosis includes HPVrelated tumors such as basaloid carcinoma and lymphoepitheliomalike carcinoma and non–HPV-related poorly

differentiated SCC. Basaloid carcinoma can show solid nests and sheets, but the cells are smaller with high nuclear cytoplasmic ration (blue cells) when compared with medullary carcinoma. Central comedolike necrosis is almost always at least focally present in basaloid carcinoma. Lymphoepitheliomalike carcinoma shares cytological features with medullary carcinoma, but architecturally it is not a solid tumor. Lymphoepitheliomalike carcinoma is composed of noncohesive tumoral cells obscured by a dense lymphoplasmacytic inflammatory infiltrate. Non–HPV-related, poorly differentiated SCC with solid pattern is the main differential because medullary carcinoma was initially classified in this category. Poorly differentiated SCC frequently show some foci with classic keratinizing/usual SCC features. Immunostain with p16 and HPV detection by PCR or in situ hybridization may be necessary for final diagnosis. There are no data regarding the outcome of these patients but considering the prognosis markers (deep invasion, highgrade, necrosis), it is expected to be somewhere among high-grade usual SCC, basaloid SCC, and sarcomatoid SCC.

FIGURE 48.72 Medullary carcinoma (A). Medullary carcinoma, low power. Solid growth pattern of a poorly differentiated carcinoma with abundant inflammatory peri and intratumoral infiltrate is shown. (B) Malignant epithelial cells nests

surrounded by inflammatory cells, composed predominantly of neutrophils and eosinophils. The tumor cell borders are inconspicuous, arranged in syncytium with minimal to no keratinization. Note the vesicular nuclei with prominent nucleoli.

Mixed. The denomination of mixed carcinoma is restricted to penile SCCs in which two or more clearly recognized histologic variants coexist. One of the subtypes should represent at least 20% of the tumor mass for the tumor to be placed in this category (227). Using this morphologic criterion, mixed SCCs comprise 28% to 37% of all penile carcinomas (195,227). The identification of mixed tumors is usually straightforward and related to the knowledge and experience of the pathologist to identify the special subtypes of SCC. Mixed tumors should be distinguished from the “pure” subtypes of penile SCC. SCCs of the usual type can be found in association with any other subtypes of penile cancer. The classic example is that of a verrucous carcinoma mixed with the usual SCC (Figs. 48.55 and 48.73) (210,211). The metastatic potential of these hybrid tumors is related to the grade and depth of invasion of the nonverrucous component (210,211). Another interesting tumor described earlier is the warty-basaloid, where an exophytic warty tumor is associated with a deeply invasive basaloid carcinoma. A rare tumor is the mixed squamous neuroendocrine carcinoma (Fig. 48.74) (11).

FIGURE 48.73 Mixed verrucous squamous cell carcinoma. (A,B) Gross view and corresponding diagram with tumor in pale color. The tumor is predominantly papillomatous verrucous (VEC), with solid areas corresponding to usual squamous cell carcinoma (SCC). U, urethra.

FIGURE 48.74 Mixed squamous–neuroendocrine carcinoma. A biphasic tumor with a moderately differentiated squamous cell carcinoma on the left and a small cell neuroendocrine carcinoma on the right.

Giant Condyloma. It is a benign, rare exophytic arborescent papillomatous tumor, sometimes referred to as Buschke-Löwenstein tumor (228). It occurs in patients slightly older than those with common condylomata and younger than those with condylomatous (warty) carcinoma. There is a long duration of the disease before pathologic diagnosis. Most commonly, giant condyloma affects the coronal sulcus and the foreskin, but the glans can also be involved. Grossly, they are large (5-10 cm), cauliflowerlike verruciform tumors, usually unicentric, with a surface showing a cobblestone or gyriform appearance (11). The cut surface shows a papillomatous growth, with a sharp separation of the lesion from the underlying stroma. Deep penetration of broad bands of tumor may be noted, with occasional skin fistulae. Microscopic features are identical to the common condyloma acuminatum, with arborescent papillae showing prominent fibrovascular cores, surface koilocytosis, and no significant atypia (Fig. 48.75A). However, there are some differences: Giant condylomata tend to show more exuberant growth and more atypia, and the base of the lesion shows a bulbous expansion into underlying tissues (Fig. 48.75B). The differential

diagnosis is with the condylomatous (warty) carcinoma, which is a clearly malignant lesion usually with infiltrative irregular borders. Occasionally, it is not possible to differentiate giant condylomas with atypia from noninvasive warty carcinomas. Negative p16 and demonstration of low-risk HPV would favor the diagnosis of giant condyloma. Although in the literature, giant condylomata and verrucous carcinomas have been considered the same tumor, it is now thought that they represent separate lesions (11). In verrucous carcinomas, the papillae tend to be acanthotic with thin fibrovascular cores; there is no evidence of koilocytosis. There are cases where distinguishing it from noninvasive warty or verrucous carcinoma is not possible, and HPV studies are necessary. HPV of low-risk genotypes are usually detected in giant condylomas. High-risk HPVs are present in warty carcinomas, and verrucous carcinomas are usually negative for HPV. We reserve the term giant condyloma for unusually large, long-standing variants of condyloma acuminata. In our experience, cell atypia from moderate to severe to foci of frank SCC, in situ or invasive, can be found in giant condylomata (Fig. 48.76A,B); therefore, extensive sampling and a high level of suspicion are advised when dealing with large and deep lesions. We avoid the term Buschke-Löwenstein tumor because it has been used for several different verruciform tumors, including benign and malignant.

FIGURE 48.75 Giant condyloma acuminatum. (A) The surface of the lesion shows large arborescent papillae with koilocytosis. (B) The base is broad and sharply delineated. The lesion shows similar features to classic condylomas but a deeper and more destructive base.

FIGURE 48.76 Condyloma coexisting with squamous cell carcinoma. (A,B) The foreskin (f) has been completely retracted to show the lesion. Condyloma (cdl) is in yellow and shows a cobblestone or cerebriform appearance. The invasive carcinoma (ca) is in purple. The glans (g) is uninvolved, and the sulcus (cos) is minimally affected.

HPV-POSITIVE AND HPV-NEGATIVE CARCINOMAS OF THE PENIS GENERAL FEATURES In a pioneering pathologic and virology study, Kurman et al. (229) established an association of HPV with special subtypes of vulvar cancer. Gregoire et al. (67) also found a relationship between morphology and HPV in penile SSCs. They demonstrated a preferential association of basaloid and mixtures of warty and basaloid carcinomas with HPV and a consistent negativity in verrucous and papillary NOS carcinomas. Despite the geographic coexistence of high incidence rates of cervical and penile cancer, most penile tumors are histologically similar to vulvar and not to cervical carcinomas. However, there is a subset of penile cancers, usually HPV positive, histologically identical to cervical cancer. Whereas almost 100% of cervical SCCs are HPV related (230), the virus is found in only about one-third to one-half of penile and vulvar cancers (204,231). For these reasons, a dual pathogenic and etiologic pathway has been postulated for these tumors (66,213,229). There are many reports emphasizing the association of special subtypes of penile cancer and HPV, but the prevalence figures are variable from 20% to 80%. A summary of these studies was recently reported by Miralles (232). The causes of the variations are not clear but may be related to (a) true geographical differences in the worldwide incidence of HPV-related tumors, along with other environmental factors related to habits, socioeconomic status, and/or cultural practices proper of the geographical region; (b) the existence of a false-negative rate attributable to technical failures in detecting HPV DNA or using inappropriate testing methods; (c) the existence of a false-negative rate attributable to poor DNA conservation in the

tissue samples. DNA detection rate in formalin-fixed, paraffinembedded tissues using PCR is highly sensitive to the fixation process. Fixation for more than 24 hours reduces the chances of successful high-quality DNA extraction; (d) the use of variable morphologic criteria for tumor subtyping resulting in moderate or low concordance rates when comparing different series of cases. DETECTION AND HUMAN PAPILLOMAVIRUS GENOTYPES HPV was detected in 64 of 202 tumors in a study using PCR (32%) (194). One genotype was identified in most cases (48 cases, 75%). As in another study (196), HPV-16 was the most commonly found genotype (72%) followed by HPV-6 (9%) and HPV-18 (6%), either in single or in multiple infections. No association of any of the genotypes with special subtypes of SCC was found, although in usual SCC, a greater variety of single genotypes (16, 6, 45, 11, and 18) was detected as compared with special variants of SCC. Lowrisk genotypes were found in four carcinomas: Two of them were usual SCCs (one with HPV-6 and the other with HPV-11) and the remainder two warty-basaloid carcinomas (both positive for HPV-6). Warty-basaloid carcinomas were more likely to be associated with multiple infections (33%) than warty (29%), basaloid (16%), or usual (11%) carcinomas. In 6 of 13 tumors with multiple viral infections (58%), there was a simultaneous presence of low- and high-risk HPVs (194). HUMAN PAPILLOMAVIRUS AND HISTOLOGIC SUBTYPES OF SQUAMOUS CELL CARCINOMA Higher positive rates were found in papillary basaloid (1 of 1), wartybasaloid (82%), basaloid (76%), and warty carcinomas (39%). In warty-basaloid carcinomas, most of the cells were small, uniform, and basophilic with scant nuclei; but they had a warty component with hyper- and parakeratosis, papillomatosis, and pleomorphic koilocytosis (196). In basaloid carcinomas, the hallmark was the presence of tumoral nests of small basophilic cells with central comedonecrosis (190). Among the HPV-positive basaloid

carcinomas, there was a papillary variant (papillary basaloid) resembling urothelial carcinoma (199). Despite the classic condylomatous pattern with abundant pleomorphic koilocytes (200), some warty carcinomas were negative for HPV. Less frequent presence of HPV was detected in usual (24%), sarcomatoid (17%), mixed (19%), and papillary NOS (15%) carcinomas. No HPV was found in verrucous, pseudohyperplastic, pseudoglandular, and cuniculatum carcinomas. Mixed carcinomas were separately analyzed: HPV positivity was associated with warty and/or basaloid (W/B) features; 6 of 15 mixed SCC with W/B features (40%) were HPV positive, whereas none of the 16 mixed tumors without W/B features showed evidence of HPV infection (p = 0.0068) (194). CELL TYPES AND HUMAN PAPILLOMAVIRUS In the same study, a strong association was found between the predominant cell type and the presence of HPV. HPV-positive tumors showed basaloid cells in 72% of the cases; clear koilocytic cells were found in 47% of HPV-positive tumors. Eosinophilic (pink) maturing keratinized cells were more common in HPV-negative tumors (81%). The association of HPV positivity and presence of basaloid cells was highly significant in univariate and multivariate analyses. On the other hand, the association of HPV and koilocytes, diagnostic tissue hallmark of warty carcinomas, was marginally significant on univariate and not significant on multivariate analyses. The increasing low frequency or negativity of HPV was also associated with an increase in the proportion of maturing, keratinizing, differentiated squamous cells. This cell pattern was associated with tumors consistently negative for HPV, such as verrucous, cuniculatum, and pseudohyperplastic carcinomas. Overall, there was an inverse relation of cell maturation and presence of HPV. No differences were found among tumors associated with different, single, or multiple genotypes (194). P16 IN SUBTYPES OF INVASIVE SQUAMOUS CELL CARCINOMAS OF PENIS

p16 immunohistochemical stain is commonly used as a surrogate for the presence of high-risk HPV in penile cancer and precancerous lesions. Similar to HPV, p16 expression is present in about a third of penile carcinomas. There is a strong association between coexpression of high-risk HPV and p16 with an overall concordance of 84%. The highest positivity is found in basaloid or mixed basaloid carcinomas. Intermediate rates are present in warty and usual carcinomas and negative rates in papillary NOS, sarcomatoid, verrucous, cuniculatum, and pseudohyperplastic carcinomas. Positive or negative concordant indexes are high for all subtypes (233).

PATHOLOGIC DIAGNOSTIC PROBLEMS GENERAL FEATURES Diagnosis of the usual variant of squamous cell carcinoma of the penis is usually not problematic. However, the heterogeneous pathologic presentation of penile cancer and its precursor lesions and mimics may pose diagnostic problems, particularly to pathologists in parts of the world where penile cancer is not common. In addition, there are areas of penile cancer pathology still not well studied. One of them corresponds to the fairly unclear diagnostic boundaries of squamous hyperplasias, flat condylomas, LS, and differentiated PeIN. Other somewhat problematic areas include the distinction between low-grade invasive neoplasms and pseudoepitheliomatous hyperplasia and the classification of verruciform tumors. At the other end of the spectrum, owing to their lack of differentiation, it is sometimes difficult to classify high-grade neoplasms. Penile tumors with glandular features should be distinguished from the pseudoglandular or adenoid variant of SCC, their most common mimic. The differential diagnosis of penile small cell carcinomas presents the same difficulties as in other sites. Penile carcinomas harboring more than one histologic grade (heterogeneous tumors) or more than one morphologic type (mixed

tumors) constitute a frequent finding, and the handling of these tumors may be difficult. The recognition of the different morphologic patterns, tumor type classification, and histologic grades is important because it has etiologic, prognostic, and therapeutic implications. SQUAMOUS HYPERPLASIAS, LICHEN SCLEROSUS, FLAT CONDYLOMAS, AND DIFFERENTIATED PENILE INTRAEPITHELIAL NEOPLASIA Squamous hyperplasias as a pathologic entity are overdiagnosed. In reality, most lesions under this designation may represent examples of differentiated PeIN in a background of LS or reactive thickening in various nonspecific inflammatory conditions. However, bona fide hyperplasias may be exuberant, simulating neoplastic processes, either clinically or microscopically. Hyperplasias are heterogeneous, and surgical pathologists should learn the spectrum of their morphologies. Microscopically, most squamous hyperplasias are flat, but verrucous, papillary, and pseudoepitheliomatous variants are also observed. SQUAMOUS HYPERPLASIA VERSUS FLAT CONDYLOMAS Penile condylomas often deviate from the classic description and present as flat acanthotic thickening of the squamous epithelium, with slight parakeratosis and no koilocytosis, especially in the skin of the shaft or the foreskin. That lesion is identical to squamous hyperplasia. There are two clues to diagnosis: If elsewhere in the foreskin or glans there are changes of typical condyloma acuminata, it helps to think about the flat lesion also being a condyloma, and, second, to detect low-risk HPV in these lesions using available molecular methods. Similar diagnostic problems are seen in the differential diagnosis of squamous papilloma–seborrheic keratosis (with no koilocytosis) versus typical condyloma. Detection of HPV is the only solution. It may be important in medicolegal cases. At the research level, PCR is the preferred technique, but we do not know which technique would be most appropriate for routine use.

SQUAMOUS HYPERPLASIAS VERSUS DIFFERENTIATED PENILE INTRAEPITHELIAL NEOPLASIA The main differential diagnosis of squamous hyperplasia is with differentiated PeIN. Hyperplasias show hyperkeratosis, hypergranulosis, occasional parakeratosis, orderly squamous maturation, and lack of atypias. In differentiated PeIN, there is surface parakeratosis, alteration of the cell maturation process, and frank atypia, more prominent or restricted to the basal or parabasal layers. There are three variants of differentiated PeIN; the classic, the pleomorphic, and the hyperplasialike, the latter with minimal atypias and most difficult to separate from hyperplasias. The differential diagnosis can be difficult, especially in small biopsies. In some cases, especially those associated with LS and/or prominent chronic inflammation, it may not be possible to distinguish squamous hyperplasias from differentiated PeIN. Negative p53 and Ki-67 by immunohistochemistry would favor a diagnosis of squamous hyperplasia (125). VERRUCOUS HYPERPLASIA VERSUS VERRUCOUS CARCINOMA Verrucous hyperplasia as an isolated lesion is not frequent. Most of them are found in tissues adjacent to verrucous carcinomas. The diagnosis is difficult because verrucous hyperplasia may represent an early stage of verrucous carcinomas. There is no single morphologic feature useful to separate these lesions. Hyperplasias are usually of small size, subclinical. When clinically evident, it would more likely represent a verrucous carcinoma. Morphologically, both lesions are identical. Pathologists are reluctant to diagnose small verrucous carcinomas, and this approach appears to be reasonable considering the lack of metastatic potential to these rare tumors. An extreme degree of differentiation characterizes both lesions, and atypias, if present in verrucous carcinomas, are subtle. If the microscopic evaluation of a biopsy is not sufficient for a definite diagnosis, a useful practice for the pathologist is to personally examine the patient. Often, a well-developed, clinically obvious, and

often large verruciform tumor can be macroscopically appreciated. In such a case, the advice to the urologist would be to resect the entire tumor with adequate surgical margins and to avoid another biopsy. PAPILLARY HYPERPLASIA VERSUS INVASIVE LOW-GRADE PAPILLARY CARCINOMAS The papillary variant of hyperplasias may simulate an invasive papillary carcinoma. Hyperplasias lack the complex architecture typical of papillary carcinomas. Atypias are absent in papillary hyperplasia, whereas in papillary carcinomas, neoplastic cells show mild-to-moderate atypias. Papillary hyperplasias are confined to lamina propria, whereas most papillary carcinomas invade penile erectile tissues or preputial dartos and are associated with a stromal reaction. PSEUDOEPITHELIOMATOUS HYPERPLASIA VERSUS PSEUDOHYPERPLASTIC INVASIVE SQUAMOUS CELL CARCINOMA Among the most challenging pathologic diagnostic problems in penile pathology is to distinguish pseudoepitheliomatous hyperplasia from pseudohyperplastic carcinomas. On limited biopsy materials, these lesions mimic each other, and, often, a resected specimen is necessary for pathologic diagnosis. Older age (70-90 years), presence of LS, multicentricity, and foreskin location would favor pseudohyperplastic carcinomas. The final diagnosis, however, should be based on pathologic findings. Deep invaginations of hyperplastic tongues or fingerlike prolongations of benign tissue can resemble invasive nests on tangential cuts, and the extreme differentiation of invasive pseudohyperplastic carcinomas may simulate nonneoplastic epithelial invaginations. The combination of small, verrucouslike lesions associated with typical SCCs in multicentric pseudohyperplastic carcinomas makes the distinction more difficult. Careful evaluation of the epithelial nests offers clues for the differential diagnosis. Regular, rounded epithelial nests, similar in shape and size, and recognizable peripheral palisading are

features in pseudoepitheliomatous hyperplasia, whereas in pseudohyperplastic carcinomas, the nests are irregular, with a variable architecture, and peripheral palisading is inconspicuous. Contrary to hyperplasias, tumor nests are frequently surrounded by reactive stroma. Intraepithelial squamous pearl formation is more typical of carcinoma and rarely found in hyperplasias. In the foreskin, hyperplasias are superficial and do not affect preputial smooth muscle dartos, which is typically invaded by pseudohyperplastic carcinomas. Perineural invasion does not occur in hyperplasia and is occasionally found, although rarely, in well-differentiated carcinomas. Several biopsies are sometimes required for a correct classification of invasive pseudohyperplastic carcinomas. Because of the rarity of these lesions, there is no sufficient published experience of the value of immunohistochemistry in the differential diagnosis. DIFFERENTIAL DIAGNOSIS OF VERRUCIFORM TUMORS Verruciform neoplasms of the penis are a group of exophytic, papillomatous, keratinizing, well-differentiated tumors. They share some features but special morphologic or viral characteristics separate them into special tumor entities: verrucous, warty (condylomatous), papillary, and giant condylomas (BuschkeLöwenstein tumor) (Fig. 48.77). Occasionally, there are complex tumors harboring more than one histologic feature. Tumors that are partially verruciform (cuniculatum, warty-basaloid, papillary basaloid carcinomas) are not included in this section. Differences among subtypes can be noted after evaluating the architecture of the papillae (condylomatous or noncondylomatous) and the interface between tumor and stroma (broadly based or irregular). Other secondary findings are the cell types, degree of differentiation, and presence of koilocytes. Additional immunohistochemical and molecular techniques may help in difficult cases. Warty carcinomas should be distinguished from papillary carcinomas. Papillae in warty carcinomas exhibit prominent fibrovascular cores, a spiky surface with prominent parakeratosis, and conspicuous and pleomorphic koilocytosis. Tumor base is jagged. Koilocytes are also found in

infiltrative tumor nests. They resemble typical condylomas, but cells are malignant. Papillary carcinomas may be similar to warty carcinomas, except that papillae are more complex and varied, with round, spiky, or blunt tips and absence of koilocytosis. Parakeratosis is not a typical feature. As in warty carcinomas, tumor base is jagged and irregular. Cells in papillary carcinomas are better differentiated. HPV is detected in 50% to 70% of warty carcinomas and usually absent in papillary carcinomas. p16 is usually positive in warty carcinomas and negative in papillary carcinomas. The main differential diagnosis of verrucous carcinoma is with giant condylomas. Both tumors are long standing, large, exophytic, papillomatous, low grade or benign, hyperkeratotic, and depict a broad base, separating it from the stroma. In verrucous carcinoma, papillae are acanthotic, whereas in giant condylomas, tumors are condylomatous. Fibrovascular cores are inconspicuous or absent in verrucous carcinomas, and intraepithelial keratin plugs are frequently found. Koilocytes, typical of giant condylomas, are absent in verrucous carcinomas. A feature of verrucous carcinoma, also present in giant condylomas, is its tumor base, which is broad and pushing, with a surrounding stromal reaction. Occasionally, fingerlike invaginations from the main mass as well as microinvasion can be observed in both tumors. Giant condyloma is characterized by papillomatosis, acanthosis, prominent fibrovascular cores, and koilocytosis. Nuclear atypia is not a usual feature, but there are cases with various degrees of atypias as well as those with in situ or invasive malignant transformation. Deep “noninvasive” tumor penetration can be observed, but morphology remains bland. Some unusual verrucous carcinomas may show focal or extensive condylomatous papillae, raising the possibility of a condyloma diagnosis. In these cases, detection of HPV may be necessary. HPV detection and p16 are usually negative or rare in verrucous carcinomas. Low-risk HPV is usually present in giant condylomas. p16 is usually negative in both verrucous carcinomas and giant condylomas.

FIGURE 48.77 Verruciform tumors: differential features. Epithelium is depicted in yellow, keratin in red, and stroma in blue, and koilocytic changes are shown as small white dots. (A) Verrucous carcinoma: hyperkeratotic and acanthotic papillae with characteristic piling up of keratin among them. The base is pushing. (B) Papillary carcinoma: Papillae are less regular and complex, and the interface between tumor and stroma is jagged. (C) Giant condyloma: arborescent and rounded papillae with surface koilocytosis and a broad base. (D) Warty carcinoma: Papillae tend to be long and undulated or rounded and condylomatous with prominent fibrovascular cores; there is koilocytosis throughout the tumor and usually jagged deep borders.

COMPLEX VERRUCIFORM TUMORS Some verruciform tumors are complex, showing more than one histologic pattern, and are difficult to classify. Instead of trying to classify into one type, it is advisable to describe the various tumor features emphasizing those pertinent to patients’ prognosis. In this context, identification and HPV genotyping can be helpful. DIFFERENTIAL DIAGNOSIS OF POORLY DIFFERENTIATED CARCINOMAS Basaloid carcinomas may be confused with high-grade usual SCCs. There is an underrecognition of the variegated microscopic spectrum of basaloid carcinomas and their potential to mix with other subtypes

of penile carcinomas. Classic features as described earlier predominate, but there are tumors deviating from the classicl description. Neoplastic cells can be larger, pleomorphic, or spindleshaped but retaining their basophilic cytoplasm. Pseudoglandular features may also be observed, and a papillary variant resembling urothelial carcinoma has been described. High-grade SCCs show gradual keratinization, whereas in basaloid carcinomas, keratinization, when present, is abrupt. Ample, eosinophilic cytoplasm with distinctive cellular borders and prominent intercellular bridges are more typical of high-grade usual SCC. There are unusual high-grade SCCs with trabecular, solid, or lymphoepitheliomalike features. Many of these tumors, like basaloid carcinomas, are HPV and p16 positive. These neoplasms do not resemble basaloid carcinomas and should not be classified as such. HETEROGENEOUS VERSUS MIXED CARCINOMAS Heterogeneous carcinomas are those usual or special subtypes harboring more than one histologic grade. They should not be confused with mixed tumors, where two or more histologic subtypes of various grades are present in the same specimen, intermixed, or in collision. About half penile carcinomas are heterogeneous, and a quarter show mixed histologic features. For the mixed tumors, instead of trying to classify them into one entity, it is advisable to designate them as mixed carcinomas, mentioning the morphologic components and their proportions. Warty-basaloid carcinomas is a frequent mixture, seen in about one-half of all mixed SCCs. Caution should be employed to avoid confusing warty-basaloid carcinomas with pure warty carcinomas because the metastatic rate of the former is higher than the latter. Considering the potential impact on prognosis, when greater than 10% of basaloid areas are observed in an otherwise classic warty carcinoma, the tumor should be classified as a warty-basaloid carcinoma. Hybrid verrucous carcinomas are a combination of typical verrucous and less differentiated SCC. The association of usual SCC with other subtypes is rare, although mixtures with papillary, warty, and basaloid carcinomas have been

reported. Sarcomatoid carcinomas also show a mixed morphology, with high-grade, spindle-shaped cells intermingled with other subtypes, usually as a minor component. These tumors should always be classified as sarcomatoid carcinomas, even if the other subtype represents more than 20% to 30% of tumor mass, as treatment and outcome will depend on the presence of sarcomatoid areas.

MOLECULAR BIOLOGY OF PENILE CARCINOMA Several molecular, immunohistochemical, and genetic studies support the notion that penile cancer occurs through mechanisms both dependent and independent of HPV (206,212,213,234-241). The overall prevalence of HPV DNA detection in penile cancers is approximately 40% to 45%, and this figure is very similar to that detected in vulvar carcinomas (66). No significant differences in HPV prevalence were seen when comparing cases from the United States and Paraguay (66). Although HPV reveals a remarkable array of different genotypes, only a limited number are associated with penile carcinomas. High-risk HPV-16 is by far the most frequent type associated with penile cancer, followed by HPV-18. Tumor types that are significantly related to HPV include the warty and basaloid variants. It is believed that HPV infection by itself is insufficient to induce malignant transformation, so that additional cellular changes are necessary in the tumorigenic process (47,234). Two viral oncoproteins, E6 and E7, appear crucial in the process of carcinogenesis. The HPV oncogenic product E6 interferes with the p53 pathway, causing the suppression of the p53 normal inhibitory function of the cell cycle (47). The retinoblastoma protein (pRB) is a target of the viral oncoprotein E7; thus, its inactivation appears to contribute to carcinogenic events, interfering with the p16INK4a/cyclin D/retinoblastoma pathway. Although E6 and E7 proteins may immortalize various types of human cells independently, their cooperative interaction leads to substantially enhanced immortalization efficiency. Several studies in cervix, vulvar,

and penile cancers support the hypothesis that p16INK4a is a specific marker for cells that express the viral E6 and E7 oncogenes (242,243). Because expression of p16INK4a underlies a negative feedback control through pRB, the enhanced expression of p16 is probably a result of reduced or lost pRB function. Binding of HPV-E7 oncoprotein to pRb causes degradation of pRb with consequent loss of Rb tumor suppressor function and p16 overexpression, which can be demonstrated immunohistochemically. We recently found a significant overexpression of p16 in warty and warty-basaloid tumors when compared with HPV-unrelated variants of verruciform lesions (Fig. 48.51) (206). Interestingly, the precursor lesions of HPV-related tumors (warty and warty-basaloid PeIN) were strongly positive for p16, whereas the precursor lesions of HPV-unrelated tumors (differentiated PeIN and LS) did not express p16, further supporting the concept of a bimodal pathway of tumor progression (Fig. 48.27) (206). It is important to recognize that most (~55% to 60%) penile cancers appear to be HPV unrelated. There are data indicating that both HPV-related and -unrelated modes of p16INK4a/cyclin D/Rb inactivation exist in penile carcinoma (235). The tumor suppressor gene Tp53 and its functional protein product p53 are believed to be involved in the HPV-unrelated pathway of carcinogenesis as well. Considering that mutant p53 accumulates in the cells, several studies have analyzed the expression of p53 in invasive penile carcinoma. The overall expression of the proteins varies between 40% and 89% (234,239). The evidence linking p53 expression and presence of HPV DNA in penile cancer is contradictory. Lam et al. (238) studied 42 penile carcinomas for HPV and p53 protein status and found that all HPV-positive cases showed p53 immunostaining. However, several other reports have shown an inverse or negative relation between these two factors. In a study on verruciform tumors, we found p53 expression to be independent from p16 expression (therefore independent from high-risk HPV infection), and its expression was preferentially seen in less differentiated areas (Fig. 48.78), supporting the concept that p53 expression is a marker of worse prognosis (206,244).

FIGURE 48.78 Poorly differentiated squamous cell carcinoma showing diffuse nuclear expression of p53.

Another study supporting the two carcinogenesis pathways with 148 penile SCCs examined for HPV by PCR and immunohistochemically for Rb, p16, p53, and p21 expression. Basaloid SCCs (10 of 13 positive for HPV) and verrucous carcinomas (3 of 13 positive for HPV) were in the extremes of each molecular profile. Rb protein correlated negatively, p16 and p21 correlated positively with HPV-positive tumors, whereas p53 did not correlate with HPV infection (213). Lopes et al. (244) showed that p53 expression was an independent predictor of lymph node metastasis on multivariate analysis. Patients with p53-negative tumors had a significantly better overall survival. Overall outcome was significantly worse if tumors were positive for p53 and HPV DNA. In this study, patients who were HPV positive but p53 negative had the best survival rates (244). The overexpression of p53 in intraepithelial neoplasia is unclear. The reports vary from almost no expression to 70% to 80% of expression (239,245). The concomitant presence of HPV and

immunohistochemical expression of p53 was associated with recurrences and progression in premalignant lesions in one study (245). It is important to keep in mind that overexpression of p53 does not necessarily indicate a p53 mutation. Castren et al. (236) found absence of p53 mutations in benign and premalignant lesions that overexpressed p53 by immunohistochemistry. These findings suggest that overexpression of p53 does not indicate a p53 mutation in these lesions and that p53 mutations are not important, or at least not early events, in male genital carcinogenesis (236). Many other molecular markers have been investigated. Considering that p21 and p53 interplay in the regulation of the cell cycle, Lam and Chan (240) analyzed the expression of both markers in penile carcinomas. p21 was found in 40% of invasive carcinomas, and its expression was also seen in the adjacent dysplastic epithelium. An inverse relationship between p21 and p53 was seen in 50% of the SCCs. The authors suggest that, in penile carcinoma, p21 expression is controlled by p53-dependent and p53-independent mechanisms. Such findings need confirmation by other studies. Ki67 was found preferentially expressed at the growing edge of verrucous carcinomas (246). Telomerase activity was found not only in 85% of invasive penile carcinomas but also in more than 80% of normal epithelium and corpus cavernosum (247). It has been found in a giant condyloma acuminatum as well (248). Cytogenetic changes have been described, but none are characteristic (249,250). Missense mutation in the c-RasHa codon 61 was found only in the second metastasis of a penile carcinoma with HPV-18. This suggests that changes in ras may be associated with late progression (251). PIK3CA gene mutations found in all grades and stages of SCCS and H-ras or K-ras gene mutations found in larger and more advanced tumors suggest these pathways are significant for development and progression of penile carcinoma (252). Upregulation of COX-2 and prostaglandin synthase 1 was shown in a small cohort of patients with penile cancer (253). ANXA1, an annexin superfamily group protein that inhibits COX-2 and cytosolic phospholipase A2, shows overexpression in all subtypes of penile SCCs, and this is the first report that describes association with high-

risk HPV infection (254). ABO blood group antigens are lost in most penile cancers (255). However, specific red cell adherence (SRCA) was found in 54%. Of those that metastasized, 91% were SCRA negative, suggesting an association with aggressive potential (256). Glucose transporter-1 is present in proliferative infiltrating areas, related to actively growing areas of the tumor (257). The use of DNA ploidy in penile cancer is controversial. Some studies found no prognostic significance (258), and others found that diploid cancers had a better prognosis than aneuploid tumors (249,259). Small nuclear size was also associated with poorer prognosis (259). The authors believe that this might result from the smaller nuclear size of basaloid carcinomas. With the aim of exploring the disease progression, Campos et al. (260) analyzed E-cadherin (involved in intercellular adhesion) and matrix metalloproteinases (MMP-2 and MMP-9; involved in the breakdown of extracellular matrix) in 125 patients with penile cancer. They demonstrated that low E-cadherin immunoreactivity is associated with greater risk of lymph node metastasis and that high MMP-9 expression appears to be an independent risk factor for disease recurrence (260). Next-generation sequencing techniques were applied in an effort to advance further in the comprehension of cancer-related genomics alteration. In one of such studies (261), 20 patients with advanced disease (stages III and IV) were examined for 3769 exons of 236 genes plus 47 introns from 19 genes frequently involved in cancer. Nineteen cases showed at least one genomic alteration (average: 5.45), and 40% of the gene alterations were linked to targeted therapies. TP53 mutation was the most common alteration. Among targetable alterations, CDKN2A (40%), NOTCH1 (25%), PIK3CA (25%), CCND1 (20%), EGFR amplification (20%), and BRCA2 insertions/deletions (10%) were the most frequently found in these patients. Regarding transcriptomics and epigenomics alterations, another integrative study (262) grouped 171 methylations hotspots and more than 4000 differentially expressed genes, reaching interesting conclusions about differential methylome and transcriptome patterns between HPV-negative and HPV-positive

carcinomas. Histological grade was also associated with distinctive methylation pattern. Some individual genes were targetable (PIK3CD, FGF1, IL1A, IL1B, and TNF), and MMP genes previously described were found differentially expressed. The study reached conclusions on altered pathways related to embryonic, cellular, and organism developmental functions for differential methylation and gene expression, as well as cell movement, migration, growth and proliferation, cell cycle, and angiogenesis only in gene expression analysis. A clearer picture is slowly emerging from these reports, with most cases of penile SCCs following a different and yet to be determined pathway involving p53, whereas HPV is involved in the tumorigenesis of a growing percentage of penile carcinomas, mainly basaloid and warty carcinomas. MOUSE MODEL IN PENILE CANCER The first mouse model for HPV-related penile cancer was recently reported (263). Ten-week-old mice expressing all the HPV16 early genes under control of the cytokeratin 14 gene promoter were exposed topically to dimethylbenz(o)anthracene (DMBA). HPV16transgenic mice developed intraepithelial lesions, including condylomas, PeIN, and invasive carcinomas. We found the latter to be heterogeneous, showing histological features closely resembling those of HPV-associated human penile cancers. These observations provide the first experimental evidence to support the etiological role of HPV16 in penile carcinogenesis. Importantly, this is the first mouse model to recapitulate key steps of HPV-related penile carcinogenesis and to reproduce morphological and molecular features of human penile cancer, providing a unique in vivo tool for studying its biology.

PROGNOSTIC FACTORS INFLUENCING REGIONAL NODAL METASTASIS AND OUTCOME

The most important prognostic factor related to outcome in penile carcinomas is nodal status (264-267). Patients with clinically nodenegative disease have cancer-specific survival probabilities between 75% and 93%, and those with pathologically proven negative nodes have 5-year cancer-specific survival probabilities ranging from 85% to 100%. Although patients with a single pathologically proven positive inguinal superficial lymph node have very good cancerrelated outcomes, patients with multiple involved lymph nodes have significantly less favorable outcomes (265). Pathologic prognostic factors influencing regional nodal metastasis and patients’ survival include sites and size of primary tumor, patterns of growth, histologic grade, depth of invasion or tumor thickness, anatomic level of invasion, perineural and vascular invasion, front of tumor invasion, histologic subtypes, presence of HPV and/or koilocytosis, status of resection margins, and presence of urethral invasion (268-270). SIZE The Union for International Cancer Control TNM Classification of Malignant Tumors staging system does not consider size of penile tumors as a criterion for classification. This is because metastasizing and nonmetastasizing tumors are of similar size (181,195-198,200206,208-224,227-260,264-271). The lack of correlation is attributable to the inclusion of verruciform and nonverruciform tumors as one group. But verruciform tumors are rarely associated with metastasis, irrespective of their size. In fact, they are usually significantly larger than other types except the sarcomatoid variant. Tumor size is a significant prognostic marker when verruciform neoplasms are excluded from the evaluation. ANATOMIC SITE Excluding large neoplasms where sites of involvement are not clear, most penile SCCs originate in the glans (80%), foreskin (15%), or coronal or balanopreputial sulcus (5%). Tumors arising in the skin of the shaft or outer surface of the foreskin are most unusual. To evaluate prognosis by anatomic site, only tumors compromising one

epithelial compartment should be compared. Carcinomas exclusively involving the foreskin are associated with better prognosis than those limited to the glans (82). Recurrences are less frequent in foreskin carcinomas than in those of glans (272). Tumors in the foreskin tend to be highly keratinized, associated with LS, and of low grade with superficial invasion, whereas those of the glans tend to be of higher grade, associated with HPV, and with infiltration of deeper anatomic layers. Carcinomas of the foreskin show a lower frequency of regional metastasis. PATTERNS OF GROWTH Verruciform tumors have the best prognosis followed by multicentric and superficially spreading penile carcinomas, whereas tumors with a vertical growth pattern carry the worst prognosis (184,273). HISTOLOGIC GRADE We recommend a three-tier grading system (187) with a clear definition at both ends of the spectrum: Grade 1: Tumors are composed of well-differentiated cells, almost indistinguishable from normal squamous cells except for the presence of minimal basal/parabasal cell atypias. Grade 2: This is an exclusion category for tumors not fitting into criteria described for grades 1 or 3. Cells have frequent keratinization, easily noted “keratinized” eosinophilic cytoplasm, minimal or moderate nuclear pleomorphism, evident or prominent nucleoli, and variable clumped chromatin; diffuse presence of keratin pearls is also noted. Grade 3: Tumors are composed predominantly of anaplastic cells with little or no keratinization; scanty or minimal amount of cytoplasm; and nuclear enlargement with thick nuclear membrane, nuclear pleomorphism, clumped chromatin, prominent nucleoli, and abundant mitotic figures. Any proportion of anaplastic cells should be sufficient to grade the carcinoma as high grade (274).

ANATOMIC LEVEL OF INVASION The levels in the glans are squamous epithelium, lamina propria, corpus spongiosum, and corpus cavernosum (8). The tunica albuginea divides corpus spongiosum from corpus cavernosum and is part of the corpus cavernosum. In the foreskin, the levels are squamous epithelium, lamina propria, dartos, and preputial skin (8). There is a significant correlation between tumor involvement of levels and incidence of inguinal metastasis. Superficial tumors (lamina propria and superficial corpus spongiosum up to 5 mm) are at low risk for metastasis. Deeply invasive tumors are at high risk. In a large clinicopathologic evaluation of 375 patients with penile cancer and long-term follow-up, the incidence of metastasis according to anatomic level revealed no metastasis for tumors invading up to lamina propria, 15% of metastasis for tumors invading corpus spongiosum or preputial dartos, and 34% of metastasis for tumors invading corpora cavernosa or preputial skin (p = 0.0001) (275). The corresponding cancer-specific mortality according to the level of invasion was 0% for tumors invading lamina propria, 11% for those invading corpus spongiosum/dartos, and 20% for tumors invading corpus cavernosum/preputial skin (p = 0.032) (195). DEPTH OF INVASION OR TUMOR THICKNESS To evaluate the maximum invasion depth in small penile carcinomas, perpendicular sectioning along the tumor’s largest diameter central axis is required. Measurement in these cases is recommended from the basal cell layer of adjacent normal squamous epithelium to the deepest point of tumor invasion. In large verruciform or other destructive bulky neoplasms, this method is not applicable, and thickness should be utilized. In our experience, depth of invasion and tumor thickness are of equivalent significance. We recommend measuring thickness from the granular cell layer of tumor (exclude keratin and parakeratin) to its deepest point of invasion. Many verruciform tumors have a broadly based tumor front, as seen in giant condylomas, verrucous and cuniculatum carcinomas, and rarely warty carcinomas. Thickness in these neoplasms should not

be compared with that of other subtypes of SCC. There is a correlation between depth of invasion and outcome. Minimal risk of metastasis has been reported for tumors invading 3.5, 4, and 5 mm (202,276). There is a high incidence of nodal metastasis (about 80%) in tumors invading more than 10 mm. The predictability of nodal metastasis in tumors invading from 5 to 10 mm has been problematic. In a study of 134 primary penile carcinomas 5 to 10 mm thick, perineural invasion and high histologic grade were more important than depth of invasion in predicting nodal metastasis (187). Thickness of tumors associated and not associated with groin metastasis were of 7.5 and 7.7 mm, respectively. PERINEURAL INVASION It is present in about one-third to one-half of patients with penile carcinomas (195). In a univariate and multivariate regression analysis of 375 uniformly treated patients in one institution, outcome was evaluated in relation to pathologic prognostic factors (age, site, size, grade, depth thickness, anatomic level of invasion, vascular invasion, and perineural invasion), and perineural invasion was the most significant independent mortality factor (275). Another study of 134 patients with penile cancers invading from 5 to 10 mm demonstrated perineural invasion and histologic grade as the most significant independent predictors of nodal metastasis (187). Riskgroup stratification systems that use perineural invasion as a component of the scoring methodology have also validated its usefulness as a prognostic factor (270). VASCULAR INVASION Vascular invasion, lymphatic or venous, adversely affects prognosis of penile cancer (268,269,277-280). In an outcome study of 375 patients, vascular (lymphatic) invasion was found to be an independent factor predictive of nodal metastasis, but perineural invasion was related to mortality (275). In a prognostic factor evaluation of 134 patients with penile carcinomas 5 to 10 mm thick, vascular (lymphatic) invasion was found in 39% and 61% of patients

with negative and positive nodes, respectively, but the difference was not statistically significant (187). Venous invasion, which is less frequent, indicates a more advanced stage of the disease and is related to the compromise of the specialized erectile venous structures of corpora spongiosa and cavernosa. FRONT OF INVASION It is the deepest part of an invasive carcinoma in relation to the underlying stroma. There are, according to Guimarães et al. (279), two patterns of tumor front of invasion designated as infiltrative and pushing patterns. Verrucous carcinomas are not to be included in this classification. The microscopic pattern of the front of tumor invasion has been found to be an independent risk factor for regional nodal metastasis of invasive penile SCC (279). HISTOLOGIC SUBTYPES There is a broad correlation between histologic subtypes of squamous carcinomas and rates of regional or systemic dissemination (281). Penile cancer subtypes can be classed in three prognostic groups: in the low risk are verrucous, papillary, warty (condylomatous), pseudohyperplastic, and cuniculatum carcinomas. The high-risk category is comprised of basaloid, sarcomatoid, adenosquamous, and solid poorly differentiated SCCs. Most of the neoplasms in the latter category present with high-grade histology, deep invasion of corpora cavernosa, and frequent nodal metastasis. There is a category of intermediate risk, comprising about one-half of penile carcinomas. To this group belong the usual SCCs, some mixed neoplasms, and pleomorphic condylomatous (warty) carcinomas. KOILOCYTOSIS It is considered a tissue marker for HPV-related lesions. There are conflicting studies on whether this finding is an independent prognostic factor (278,282). A caution in the diagnosis of koilocytosis

is related to the poor reproducibility of the lesion among pathologists when the evaluation depends solely on H&E histology, especially when the tissue changes are associated with subtypes of squamous carcinomas other than HPV-related tumors. A definite evaluation of HPV koilocytosis in penile SCC should be performed using immunohistochemical surrogates such as p16INK4a or more sophisticated techniques such as HPV ISH or PCR (126,206,283,284). POSITIVE MARGINS It adversely affects prognosis in patients with penile SCCs. It is probable that most local recurrences (intrapenile) are caused by either positive or insufficient resection margins. The prognosis of patients with recurrent penile carcinoma is dismal. This is most likely to occur in conservative penile-preserving procedures with less than adequate resections. Leijte et al. (285) reported a 28% recurrence rate in patients undergoing penile-preserving treatments compared with 5% after penile amputation. In another study of 25 patients with recurrent carcinomas, 6 had conservative local surgical resections of the tumor (272). In the same study, eight carcinomas presenting as primary foreskin tumors developed recurrences in the glans, most likely secondary to tumor involvement of surgical margins at the time of circumcision. URETHRAL INVASION Urethral involvement by penile cancer has been reported as an adverse prognostic factor, and it is mentioned with prostate invasion as a T3 in the TNM staging system. In our experience, histologic invasion of the distal urethra occurs in 25% of invasive SCCs (4) and is not necessarily associated with a poor outcome, as a T3 classification would suggest. Most studies indicating urethral involvement as an adverse prognostic factor lack a detailed description of the pattern (continuous or discontinuous with main tumor, in situ, or invasive) and urethral site of invasion (perimeatal, anterior at corpus spongiosum, anterior at penile shaft, or corpora

cavernosa involvement). Further work is needed on reevaluating the prognosis of urethral invasion. RISK FACTORS GROUPS The statistically more important pathologic factors related to regional metastases are histologic grade, depth of invasion, and perineural and vascular invasion. Because the combination of histologic grade, tumor thickness, and perineural invasion is thought to better predict metastasis and mortality (286,287), the use of a prognostic index has been proposed (270). The prognostic index is measured from 1 to 7, and it results from the addition of the numeric values given to the histologic grade, anatomic level of invasion, and perineural invasion. The numeric values that are given to histologic grade are 1, 2, and 3 (for well-, moderately, and poorly differentiated tumors); the numeric values given to anatomic level of invasion are also 1, 2, and 3 (corresponding to lamina propria, corpus spongiosum, and corpus cavernosum in the glans and lamina propria dartos and skin in the foreskin); and absence or presence of perineural invasion are 0 or 1, respectively (Fig. 48.79). When the sum of the values is less than 3 (low index), tumors are usually associated with no mortality. Metastasis and mortality rates are high in patients with indexes of 5 to 7 (270). The behavior of tumors with intermediate prognostic index is more difficult to predict.

FIGURE 48.79 Prognostic factors: perineural invasion (PNI). PNI has been shown to represent an independent predictor of regional metastasis.

An evaluation of clinical and pathologic variables using a nomogram was recently developed (288). The selected factors were clinical stage of lymph nodes, microscopic growth pattern, grade, vascular invasion, and invasion of corpora spongiosa and cavernosa and urethra. The probability of nodal metastasis as predicted by the nomogram was close to the real incidence of metastasis observed at follow-up. A second nomogram to estimate predictions of survival at 5 years using the same clinical and pathologic factors gave similar results (289). More recently, a nomogram using perineural invasion and histologic grade as predictors of mortality in penile tumors 5 to 10 mm thick was developed (287). TUMOR SPREAD LOCAL

Pathways of local tumor spread, shown in Figure 48.80, may be grossly or microscopically detected. Penile tumors may spread from one mucosal compartment to the other. Typically, foreskin carcinomas spread to the skin of the shaft, coronal sulcus, or glans; carcinomas originating in the glans may spread to the coronal sulcus and foreskin. Penile SCC may spread horizontally and externally to the skin of the shaft and internally to the proximal urethral margin of resection. This is the characteristic centrifugal spread of superficially spreading carcinomas. The vertical spread may sequentially involve surface to deep areas in (a) the glans, from lamina propria to corpus spongiosum, albuginea, and corpus cavernosum; (b) the foreskin, from lamina propria to dartos and outer skin; and (c) the sulcus, from lamina propria to fascia. This is the pattern of spread of vertical growth carcinoma. An important and underrecognized route of spread of penile carcinomas is the penile fascia, a common site of positive surgical margin of resection (5). The fascial involvement in glans tumors is usually through the coronal sulcus. Tumor compromising fascia may secondarily penetrate into corpora cavernosa via nutritional vessels and adipose tissue traversing the tunica albuginea (Fig. 48.80). It is not unusual to find rounded nodules of carcinoma separated from the main invasive tumor (“satellite” nodules) in penectomy or circumcision specimens of advanced stage (Fig. 48.81). These are strong indicators of regional metastasis and poor outcome. Macroscopic evaluation and inclusion of the entire urethral resection margin for microscopic evaluation are crucial. In a series of partial penectomies for penile cancer, urethral and periurethral tissues were found to be the second most frequent site of margin involvement (5). The tumor may compromise the urethral epithelium, lamina propria, periurethral corpus spongiosum, or fascia (Fig. 48.82A,B). Lymphatic or perineural invasion may also be seen at the margins. A pagetoid intraepithelial spread simulating Paget disease may be observed adjacent to the invasive tumors and sometimes extending to the squamous epithelium at the margins (Fig. 48.83). In clinically more advanced cases, penile carcinomas may spread directly to inguinal, pubic, or scrotal skin and soft tissues. These cases are usually associated with poor prognosis.

However, it is important to determine the tumor histologic type because, occasionally, giant condylomas and verrucous or low-grade warty carcinomas (which are less aggressive, albeit locally destructive lesions) may spread in this pattern and yet have a good outcome. Massive spread of long-standing carcinomas replacing the skin of ano-genito-scrotal-pubic areas is anecdotal.

FIGURE 48.80 Local spread of squamous cell carcinoma. Diagrammatic representation of possible routes of local spread from distal penis to shaft and urethral resection margins. The arrows indicate the different pathways. CA, carcinoma; CC, corpus cavernosum; COS, coronal sulcus; F, foreskin; PF, penile fascia; TA, tunica albuginea; U, urethra.

FIGURE 48.81 Local spread of squamous cell carcinoma. Large destructive distal recurrent penile carcinoma with central necrosis and a satellite nodule present in the corpus cavernosum. CA, carcinoma; CC, corpus cavernosum; N, necrosis; SAT, satellite lesion.

FIGURE 48.82 Local spread of squamous cell carcinoma. (A) Diagrammatic representation of the cut surface of positive urethral resection margin of a

penectomy specimen. Blue dots represent actual anatomic sites of involvement in 12 patients. Numbers 1 to 4 are usual sites of involvement by tumor at time of surgery. CS, corpus spongiosum; E, urethral epithelium; LP, lamina propria; PF, penile fascia. The fascia is the most common site. (B) High-grade intraepithelial neoplasia of the urethra, basaloid type at margin of resection. The patient had a basaloid carcinoma in the glans.

FIGURE 48.83 Local spread of squamous cell carcinoma. Intraepithelial pagetoid spread from an invasive basaloid carcinoma.

REGIONAL Regionally, penile carcinoma disseminates to inguinal lymph nodes bilaterally (Fig. 48.84). Rarely, skip metastases directly to deep inguinal or to pelvic nodes are found. The first site of metastasis is in the “sentinel” lymph node(s). Although originally thought to be in a constant anatomic location associated with the superficial epigastric vein in the superomedial quadrant of the inguinal field (13), subsequent studies found that anatomic variation exists in the position of the sentinel node(s) (290,291). Superficial inguinal lymph node dissection remains the most common practice among most urologists to ascertain inguinal node status for select patients at high risk (290). But tumor may directly involve deep inguinal nodes

sparing superficial ones. Results from preoperative lymphoscintigraphy and dynamic sentinel node biopsy appear promising (290-294). Some studies suggest that early detection of lymph node metastases by dynamic sentinel node biopsy and subsequent resection in clinically node-negative T2 or 3 penile carcinoma improves survival compared with a policy of surveillance (295). These techniques are successful in a few centers and are still in evolution and require further optimization for general application (290).

FIGURE 48.84 Regional spread of squamous cell carcinoma. Patient has a recurrent tumor and bilateral groin metastasis.

SYSTEMIC

Systemic dissemination of penile cancer may involve retroperitoneal nodes, heart, lungs, bone, and liver. Among histologic subtypes, sarcomatoid and basaloid carcinomas are prone to systemic spread.

OTHER RARE AND METASTATIC NEOPLASMS A series of five penile clear cell carcinomas with sweat gland differentiation that appear to be distinct from the clear cell squamous cell carcinoma described in the earlier section have been reported (203). The reported cases affected the inner side of the foreskin of middle-aged men. They were large, exophytic, partly ulcerated, and widely invasive tumors with sharp demarcation from the surrounding normal tissues. Histologically, they were composed of large clear cells with intracytoplasmic, periodic acid-Schiff with diastase-positive material and showed extensive lymphatic and blood vessel invasion (Fig. 48.85). The neoplastic cells were positive for cytokeratins, MUC1, epithelial membrane antigen (EMA), and CEA. All patients had extensive inguinal lymph node metastases. One of the patients died of metastatic disease. The histologic and immunohistochemical features and the occasional presence of an in situ component in the eccrine ducts support a sweat gland differentiation. Distinction of sweat gland clear cell carcinomas from clear cell SCC of the penis is important because of a potentially more aggressive course of sweat gland clear cell carcinomas (203).

FIGURE 48.85 Clear cell carcinoma of the penis. Solid tumor composed of clear cells arranged in large nests and trabecular structures. Courtesy of S. Regauer, Medical University Graz, Graz, Austria.

Extramammary Paget disease affecting the penis can be classified as primary or secondary. Primary Paget disease can be confined to the epidermis or may be associated with an underlying sweat gland adenocarcinoma (296,297). Dermal invasion from a predominantly epidermal lesion can also be seen. Secondary Paget disease usually represents an extension from a urethral or bladder carcinoma (124,298). Primary Paget disease may rarely show an exclusive penile location (297). Most frequently, however, it involves the skin of the shaft as part of a more extensive scrotal, inguinal, perineal, or perianal lesion. Secondary Paget disease tends to affect the glans, especially the perimeatal region (124,298). Typically, patients are in the sixth or seventh decade and present with thickened red-pale plaques with scaling or oozing. Microscopically, there is an intraepithelial proliferation of large atypical cells with abundant pale cytoplasm (Fig. 48.86). Nuclei are vesicular, and nucleoli are prominent. These cells may extend to the

epithelium of adnexal structures. Such in situ lesions have a favorable prognosis. The prognosis is much more serious in cases in which Paget cells invade the dermis from the epidermis or from an underlying sweat gland carcinoma (299). Primary Paget disease should be distinguished from SCC in situ with pagetoid pattern (Fig. 48.87), melanomas (Fig. 48.88), and pagetoid spread from an associated penile or urothelial carcinoma. Clear cell papulosis (300), pagetoid dyskeratosis (301), and mucinous metaplasia (302) should also be ruled out. Primary Paget disease is usually positive for mucins (303). Immunohistochemically, it frequently expresses CEA, low–molecular-weight cytokeratins, EMA, MUC1, and gross cystic disease fluid protein-15 (304). Low–molecular-weight cytokeratins, especially cytokeratin 7, are useful markers for Paget disease (304,305). Primary Paget disease is usually negative for cytokeratin 20. Secondary Paget disease associated with urothelial carcinoma will usually express cytokeratin 20 in addition to cytokeratin 7 and will usually be CEA negative. The lack of expression of melanocytic markers will allow the distinction of Paget disease from melanoma in situ.

FIGURE 48.86 Paget disease. Intraepithelial proliferation of large atypical (pagetoid) cells with pale cytoplasm. This lesion affected the scrotum and extended to the skin of the penile shaft.

FIGURE 48.87 Squamous cell carcinoma in situ (SCCIS) with pagetoid features. This lesion that affected the skin of the shaft shows atypical keratinocytes with pale cytoplasm reminiscent of Paget disease. The presence of intercellular bridges between the neoplastic cells in SCCIS and different immunohistochemical profile will help to make the distinction.

FIGURE 48.88 Malignant melanoma of the glans. This was an invasive lesion. The picture predominantly illustrates the in situ component of the tumor. There is

marked inflammatory response in the lamina propria.

Penile malignant melanoma is rare and mainly localized in the glans (Fig. 48.88) (306-308). Although classically associated with a poor prognosis, a recent study suggests that the prognosis of primary mucosal penile melanoma is not worse than that for cutaneous melanoma with comparable tumor thickness (308). Presence of ulceration, tumor depth of 3.5 mm or more, and tumor diameter greater than 15 mm have been shown to have a significantly adverse effect on prognosis (308). In two separate studies, all patients with nodal and/or distant metastases at presentation died of the disease (307,308). Melanoma in situ can mimic Paget disease, and the demonstration of immunohistochemical expression of melanocytic markers (i.e., S100, HMB-45, melan-A) may be necessary to make the correct diagnosis in difficult cases. Basal cell carcinomas arise on the skin of the shaft and, in general, show no metastatic potential (309,310). They are identical to basal cell carcinomas of the skin elsewhere (Fig. 48.89) and should be distinguished from basaloid carcinoma, a high-grade deeply invasive SCC arising from the mucosal surfaces. Sweat gland cysts such as hidrocystomas (Fig. 48.90) and other benign tumors (i.e., syringomas) (Fig. 48.91) have also been reported (311).

FIGURE 48.89 Basal cell carcinoma. The lesion is from the skin of the shaft.

FIGURE 48.90 Hidrocystoma of the foreskin. There is an ovoid regular mass in the foreskin.

FIGURE 48.91 Syringoma. There is a benign proliferation of double-layered eccrine ducts with characteristic “tadpole” appearance. The lesion was located in the preputial lamina propria.

Although rare, there are a great variety of benign and malignant penile soft tissue tumors (311-336). The most common benign soft tissue tumors that affect the penis are vascular neoplasms such as hemangiomas (314,315), angiokeratomas, and lymphangiomas (316), followed by tumors of neural (317-319), myoid, and fibrous origin (320,321). Granular cell tumors and glomus tumors have also been reported (322,323). An unusual benign vascular tumor designated as myointimoma has also been described (324). Among reported cases, the most frequent malignant penile soft tissue tumors are Kaposi sarcoma (328,330) and leiomyosarcoma (Figs. 48.92 and 48.93) (325-327). Other malignant soft tissue neoplasms reported are angiosarcoma, embryonal rhabdomyosarcoma (332), epithelioid sarcoma (333), clear cell sarcoma (334), dermatofibrosarcoma protuberans (335), and fibrosarcoma (336). Recently, a primary Ewing sarcoma/primitive neuroectodermical tumor (PNET) of the penis was reported (337). Correctly diagnosing penile soft tissue tumors is important because the biologic behavior and the clinical management of these

neoplasms vary considerably (313). Distinguishing sarcomas from sarcomatoid carcinoma (214) and melanoma is particularly important (313). Primary lymphomas of the penis (338-340) and histiocytosis (341) are extremely rare.

FIGURE 48.92 Kaposi sarcoma affecting the lamina propria and superficial corpus spongiosum of the glans.

FIGURE 48.93 Leiomyosarcoma. (A) Total penectomy specimen with a large and deeply located mass in the proximal portion of the penile shaft. The tumor replaces the corpora cavernosa. (B) Histologic features of this high-grade leiomyosarcoma.

Although the penis is a highly vascularized organ, metastasis to this organ is unusual (342). Metastatic tumors from the urogenital

area and neighboring organs are the most commonly reported (342). Urogenital, renal, urinary bladder, and prostatic carcinomas have been described (Figs. 48.94 and 48.95) (343,344). Metastatic tumors from distant organs are exceptional (345). A prominent clinical feature of metastatic carcinoma is the so-called malignant priapism, which is caused by massive replacement of corpora cavernosa by the neoplasm.

FIGURE 48.94 Metastatic bladder carcinoma. Transitional cell carcinoma infiltrating the corpus cavernosum.

FIGURE 48.95 Metastatic prostatic carcinoma to the penis. Gross view (A) and diagram (B) of a penectomy specimen from a patient who presented with priapism. The tumor (ca) is multinodular and preferentially affects the corpora cavernosa (cc). The glans corpus spongiosum (gcs) is free of tumor. alb, albuginea; f, foreskin.

HANDLING OF THE SPECIMEN

The methods described in this section were designed for SSCs but may be used for other tumor types as well. More details are in the recommendations on Reporting on Cancer Specimens, published by the College of American Pathologists (346). CIRCUMCISION Describe the specimen and take measurements. Identify and describe the tumor. Ink the mucosal and cutaneous margins with different colors. Most SCCs arise from the mucosal surface of the foreskin; therefore, the coronal sulcus (mucosal) margin is especially important (11). Lightly stretch and pin the specimen to a cardboard. Fix for several hours in formalin. Cut the whole specimen vertically, labeling from 1 to 12 clockwise. PENECTOMY—PARTIAL OR TOTAL Measure, describe, and identify the tumor. Most arise from the epithelium of the distal portion of the organ (glans, coronal sulcus, and mucosal surface of the prepuce; the tumor may involve one or more of these anatomic compartments) (11). If present, classify the foreskin as short, medium, long, and/or phimotic (7). Cut the proximal margin of resection en face, making sure to include the entire circumference of the urethra (Fig. 48.3). If the urethra has been retracted, it is important to identify its resection margin and submit it entirely. The resection margin can be divided into three important areas that need to be analyzed: the skin of the shaft with underlying dartos and penile fascia; corpora cavernosa with albuginea; and urethra with periurethral cylinder that includes lamina propria, corpus spongiosum, albuginea, and penile fascia (Figs. 48.3 and 48.7A,B). The urethra and periurethral cylinder can be placed in one cassette. The skin of the shaft with dartos and fascia can be included together with the corpora cavernosa. Because this is a large specimen, it may need to be included in several cassettes to include the entire resection margin. Fix the rest of the specimen overnight. Then, in the fixed state and if the tumor is large and involves most of the glans, cut longitudinally and centrally using the

meatus and the proximal urethra as reference points (Fig. 48.1). Do not probe the urethra. Separate the specimen in two halves, left and right (Figs. 48.1 and 48.2). Then cut two to six serial sections of each half. If the tumor is small and asymmetrically located in the dorsal or ventral area, the central portion of the tumor may be used as the axis of sectioning. If the tumor is large and involves multiple sites (glans, sulcus, and foreskin), it is important not to remove the foreskin, leaving the entire specimen intact for sectioning. In cases of small carcinomas exclusively located in the glans with no foreskin involvement, one may choose to remove the foreskin, leaving a 3-mm redundant edge around the sulcus. Proceed with cutting the foreskin as indicated for circumcision specimens. Even if the primary tumor is located in the glans, submit the foreskin serially and in orderly fashion labeled from 1 to 12 clockwise. Carcinomas may be multicentric and subclinical. The rest of the penectomy specimen should be handled as described earlier. PATHOLOGY REPORT A complete pathology report should include tumor anatomic site in the penis (glans, coronal sulcus, or foreskin), largest size (cm), histologic grade, histologic subtype of SCC, tumor depth or thickness (mm), anatomic level of invasion, tumor front, vascular and perineural invasion, and type of associated precancerous lesions or LS (346). Some pathologists or institutions are required to include the TNM classification in the pathology report (347). For an updated TNM classification and staging groups, see Tables 48.2 and 48.3. TABLE 48.2 Definition of Tumor-Node-Metastasis Primary Tumor (T) TX

Primary tumor cannot be assessed

T0

No evidence of primary tumor

Tis

Carcinoma in situ (Penile Intraepithelial Neoplasia [PeIN])

Ta

Noninvasive localized squamous cell carcinoma

T1

Glans: Tumor invades lamina propria Foreskin: Tumor invades dermis, lamina propria or dartos fascia Shaft: Tumor invades connective tissue between epidermis and corpora regardless of location All sites with or without lymphovascular invasion or perineural invasion and is or is not high grade

T1a

Tumor is without lymphovascular invasion or perineural invasion and is not high grade (i.e., grade 3 or sarcomatoid)

T1b

Tumor exhibits lymphovascular invasion and/or perineural invasion or is high grade (i.e., grade 3 or sarcomatoid)

T2

Tumor invades into corpus spongiosum (either glans or ventral shaft) with or without urethral invasion

T3

Tumor invades into corpus cavernosum (including tunica albuginea) with or without urethral invasion

T4

Tumor invades other adjacent structures (i.e., scrotum, prostate, pubic bone)

Regional Lymph Nodes (N) CLINICAL STAGE DEFINITION cNX

Regional lymph nodes cannot be assessed

cN0

No palpable or visibly enlarged inguinal lymph nodes

cN1

Palpable mobile unilateral inguinal lymph node

cN2

Palpable mobile ≥ 2 unilateral inguinal nodes or bilateral inguinal lymph nodes

cN3

Palpable fixed inguinal nodal mass or pelvic lymphadenopathy unilateral or bilateral

Pathologic Stage Definition pNX

Lymph node metastasis cannot be established

pN0

No lymph node metastasis

pN1

≤ Unilateral inguinal metastasis, no ENE

pN2

≥ 3 unilateral inguinal metastases or bilateral inguinal metastases

pN3

ENE of lymph node metastasis or pelvic lymph node metastases

Distant Metastasis (M) M0

No distant metastasis

M1

Distant metastasis present

ENE, extranodal extension. From AJCC Cancer Staging Manual. 8th ed. Springer; 2017:706-707; modified with permission of SNCSC.

TABLE 48.3 Anatomic Stage/Prognostic Groups for Penile Carcinoma Stage Group

T

N

M

0is

Tis

N0

M0

0a

Ta

N0

M0

I

T1a

N0

M0

IIA

T1b

N0

M0

IIA

T2

N0

M0

IIB

T3

N0

M0

IIIA

T1-T3

N1

M0

IIIB

T1-T3

N2

M0

IV

T4

Any N

M0

IV

Any T

N3

M0

IV

Any T

Any N

M1

From AJCC Cancer Staging Manual. 8th ed. Springer; 2017:707; modified with permission of SNCSC.

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X Female Reproductive System and Peritoneum SECTION

49

Gestational Trophoblastic Disease Pei Hui ■ Ie-Ming Shih

Gestational trophoblastic disease (GTD) constitutes a diverse group of lesions that include abnormally formed placentas (hydatidiform moles), benign tumor-like lesions, and trophoblastic neoplasms (Table 49.1). The diversity of lesions seen in GTD has been underscored by recent morphologic, epidemiologic, immunohistochemical, and cytogenetic studies. The various forms of GTD can be defined and related to discrete pathologic aberrations occurring at different trophoblastic subpopulations and stages of trophoblastic differentiation during placentation (1). The recognition and separation of the individual categories of GTD is important, as each disease entity has distinctive clinical manifestations and each requires different therapeutic approaches. Furthermore, these disorders mimic growth patterns encountered in early normal placental development, nonmolar abortions and a variety of nontrophoblastic lesions. Therefore, an appreciation of the morphologic manifestations of GTD is important to avoid confusing these trophoblastic lesions with their mimickers. TABLE 49.1 Classification of Gestational Trophoblastic Diseases Hydatidiform Mole (Abnormally Formed Placenta) Complete mole (CHM) Partial mole (PHM) Invasive and metastatic mole Abnormal (Nonmolar) Villous Lesion

Trophoblastic Tumor (Neoplastic Disease) Gestational choriocarcinoma (CC) Placental site trophoblastic tumor (PSTT) Epithelioid trophoblastic tumor (ETT) Mixed trophoblastic tumor Tumor-Like Condition (Benign Lesion) Exaggerated placental site reaction (EPS) Placental site nodule (PSN) Adapted from Kurman RJ, Carcangiu M-L, Herrington CS, et al., eds. WHO Classification of Tumors of Female Reproductive Organs. 4th ed. World Health Organization; 2014. With permission.

OVERVIEW OF TROPHOBLASTIC SUBPOPULATIONS AND PATHOGENESIS OF TROPHOBLASTIC NEOPLASIA Trophoblast plays a crucial role in implantation and placentation. It exhibits a number of unique properties given its wide range of metabolic, endocrine, and invasive functions. Prior to the development of villi, the primitive trophoblast is termed previllous trophoblast which may exhibit trophoblast stem cell features. Our knowledge of the previllous trophoblast is limited because few specimens are available for study. After formation of villi at approximately 2 weeks of gestational age, trophoblastic cells on chorionic villi are termed villous trophoblast, whereas trophoblastic cells in all other locations are designated extravillous trophoblast. The trophoblast in villous and extravillous locations can be broadly divided into three distinct populations based on morphologic, immunophenotypic, gene expression profiles and functional studies. The cytotrophoblast is a relatively small, mononucleate cell with a distinct cell border that contains a small amount of eosinophilic cytoplasm (Fig. 49.1). In early gestation, the cytotrophoblast on the villous surface differentiates along two main pathways, developing into either a villous or extravillous trophoblastic lineage (2). On the villous surface, cytotrophoblastic cells fuse to form

syncytiotrophoblastic cells that are terminally differentiated cells. This is made possible by the expression of syncytin-1 encoded by the ERVW-1 gene that is expressed on the surface of cytotrophoblast and syncytiotrophoblast where syntytin-1 mediates the fusion to expand the syncytiotrophoblast (3). This is accompanied by a drastic change in cell biology including tubulin detyrosination that promotes the syncytium formation (4). They synthesize and secrete a variety of pregnancy-associated hormones, including human chorionic gonadotropin (β-hCG) and human placental lactogen (hPL). The syncytiotrophoblast is multinucleated, and often the nuclei appear dark and pyknotic. There are numerous microvilli carpeting on the syncytiotrophoblastic cell surface to increase the surface area and to facilitate molecular exchange and transportation. The cytoplasm of syncytiotrophoblastic cells is dense and stains deeply eosinophilic to basophilic (Fig. 49.1). In contrast, cytotrophoblastic cells at the margin of what are destined to become anchoring villi display a morphologic spectrum of differentiation resulting in the development of intermediate trophoblastic cells within the trophoblastic columns (Fig. 49.1). This differentiation from cytotrophoblast to intermediate trophoblast is accompanied by gradual enlargement of the cells and their nuclei, accompanied by cytoplasmic clearing. From the trophoblastic columns, intermediate trophoblastic cells infiltrate the decidua and myometrium and invade and replace the spiral arteries of the basal plate to establish the maternal/fetal circulation. The differentiation of intermediate trophoblast toward the distal ends of the anchoring villi and in the endomyometrium is accompanied by a decrease in cellular proliferation (5). The term “intermediate trophoblast” is interchangeable with “extravillous trophoblast”; the former is used by most pathologists and the latter is only used by laboratory scientists and in basic research literatures.

FIGURE 49.1 Trophoblastic subpopulations according to different anatomic locations. Three distinct trophoblastic populations can be identified. The cytotrophoblast (CT) is a small mononucleate stem cell on the villous surface that fuses directly into the syncytiotrophoblast (ST). In contrast, cytotrophoblastic cells, at the margin of what is destined to become an anchoring villi, display a morphologic spectrum of differentiation merging imperceptibly into villous intermediate trophoblastic cells (IT) within a trophoblastic column. In contrast to CT, villous IT are larger and more polyhedral with abundant clear or amphophilic cytoplasm. From the trophoblastic columns, villous IT infiltrate the decidua and myometrium to become implantation site IT or presumably differentiate into chorionic-type IT, which are found in the chorion laeve (fetal membrane).

The morphologic features of intermediate trophoblastic cells are quite variable depending on their anatomic locations. In the trophoblastic columns, the intermediate trophoblastic cells are mononucleate, larger than cytotrophoblastic cells, and polygonal with clear cytoplasm. The intermediate trophoblastic cells that infiltrate the endomyometrium of the placental site are termed implantation site intermediate trophoblastic cells. In the endometrium, these cells are polygonal and contain abundant amphophilic cytoplasm closely resembling the decidual cells with which they are admixed, but their hyperchromatic nuclei with irregular outlines and frequent deep folds or clefts distinguish them from decidual cells. In the myometrium, implantation site intermediate trophoblastic cells are frequently

spindle-shaped and resemble the surrounding smooth muscle cells. Occasionally, mononucleate intermediate trophoblastic cells at the implantation site fuse to form multinucleated intermediate trophoblastic cells (6). The major function of implantation site intermediate trophoblastic cells is to attack spiral arteries and transform them into large-caliber and low-resistant vascular channels of which the maternal blood can be directed to placenta. The regulation of uterine spinal artery remodeling is believed to be critical in establishing the implantation site and for a successful pregnancy (6). The inadequacy of such process is associated with maternal hypertension. Last, in the chorion laeve there are two distinct populations of cells, one containing eosinophilic and the other containing clear cytoplasm. These trophoblastic cells likely represent a subpopulation of intermediate trophoblastic cells rather than a subtype of cytotrophoblastic cells and the designation chorionic-type intermediate trophoblastic cells is preferred for both cell types (1,2). The functional role of chorionic-type intermediate trophoblast has not been elucidated, but it may function as an immune barrier for maternal immune cells to access fetal tissues. Thus, at present the intermediate trophoblast are subdivided into three types based on its anatomical location: intermediate trophoblast in the trophoblastic column, implantation site intermediate trophoblast, and chorionictype intermediate trophoblast. The various trophoblastic subpopulations have different immunophenotypes (Table 49.2) (3,4). Identification and characterization of trophoblast-associated markers provide not only new insights into the physiology of different trophoblastic subpopulations but also immunohistochemical markers for the differential diagnosis in pathology practice. More recently, single-cell RNA sequencing to profile different types of cells in the placenta reveals unprecedented details in landscape on cell populations in the maternal-fetal interface, including different types of trophoblastic cells (5-7). For example, it has been shown that during extravillous trophoblastic differentiation, pathways involved in immunomodulation, cellular adhesion, and invasion are upregulated (5).

TABLE 49.2 Immunohistochemical Features of Trophoblastic Cells

Besides, the recognition of trophoblastic subpopulations and their unique gene expression profiles (Table 49.2) also help to understand the pathogenesis of various gestational trophoblastic lesions. Molecular analysis of gestational trophoblastic neoplasms is largely based on the characterization of gene expression profiles in various types of tumors and the reference of their unique gene expression patterns to different trophoblastic subpopulations in normal early placentas (2,3). It has been proposed that following neoplastic transformation of trophoblastic stem cells, presumably the cytotrophoblast, specific differentiation programs dictate the type of trophoblastic tumor to develop. These patterns of differentiation in gestational trophoblastic neoplasms recapitulate the stages of early placental development. Thus, choriocarcinoma is composed of variable amounts of neoplastic cytotrophoblast, syncytiotrophoblast, and intermediate trophoblast and resembles the previllous blastocyst that comprises a similar mixture of trophoblastic subpopulations. In contrast, the neoplastic cytotrophoblast in PSTT differentiates mainly into intermediate trophoblastic cells at an implantation site, whereas the neoplastic cytotrophoblast in epithelioid trophoblastic tumor (ETT) differentiates into chorionic-type intermediate trophoblastic

cells in chorion laeve. According to this model, choriocarcinoma is the most primitive trophoblastic tumor as compared to PSTT and ETT. This hypothesis explains the existence of gestational trophoblastic neoplasms with a mixed histological feature including choriocarcinoma and PSTT and/or ETT.

HYDATIDIFORM MOLES Hydatidiform moles are divided into sporadic and inherited forms. Sporadic hydatidiform moles are by far the most common and defined at the genetic level by the presence of excess paternal genomic complement along with variable trophoblastic proliferation and villous hydrops at histological level (8) and are subclassified into complete hydatidiform mole (CHM) and partial hydatidiform mole (PHM). Inherited hydatidiform moles are seen in a subset of familial biparental complete hydatidiform moles (FBCHMs) as a result of germ-line mutations involving maternal-effect genes including NRLP7 and KHDC3L. Patients with recurrent hydatidiform moles and no mutations in the known genes have a milder genetic susceptibility and/or a multifactorial etiology (9). Early clinical detection and intervention in recent decades have drastically changed the pathological practice in the diagnosis of hydatidiform moles. Molar pregnancies are now evacuated at much earlier stages of gestation with less well-developed lesions difficult to be detected and diagnosed accurately. Whereas an early-evacuated complete mole is frequently misdiagnosed as hydropic abortion clinically and pathologically, diagnosis of an early-evacuated partial mole is even more problematic. Over 50% of true partial moles cannot be accurately interpreted by routine histological evaluation (10) due to lack of specific histological features (11) with significant inter- and intra-observer variability even among expert gynecological pathologists (12). Nonetheless, the separation of hydatidiform moles from nonmolar gestations and precise subclassification of hydatidiform moles are critical for clinical patient management, as the risk of persistent trophoblastic disease or gestational

trophoblastic neoplasia (GTN) varies significantly among subtypes of hydatidiform moles and so does the clinical follow-up algorithm. In addition, without accurate classification of molar gestations, any epidemiological, clinical, and basic research will continue to suffer from a lack of high-quality study cohorts, ultimately leading to inappropriate conclusions. The discovery of p57 immunohistochemistry has significantly improved our diagnostic accuracy for complete moles. In the past decade, polymerase chain reaction (PCR)–based short tandem repeat (STR) DNA genotyping has emerged as the gold standard in the precise diagnosis of all types of sporadic hydatidiform moles. With increasing acquisition of molecular diagnostic capabilities at most medical centers, these new ancillary techniques have now been integrated into the routine diagnostic algorithm in the pathological work-up for hydatidiform moles (13). EPIDEMIOLOGY Although extensive data are available in the world literature, the epidemiology of hydatidiform moles is far from being perfect, primarily due to significant limitations in diagnostic accuracy, inclusion criteria in study cohorts, data collection methods, and data interpretation schemes. Hydatidiform mole is nonetheless the most prevalent GTD. Among plausible etiologies, maternal age, ethnicity, and genetic basis are the most convincing etiological factors of hydatidiform mole. The incidence of hydatidiform moles is generally reported in relation to the total number of pregnancies or deliveries in a study cohort, rather than the total population (14). It must be emphasized that epidemiological data curated from CHM are far more accurate than those including also PHM, as significant diagnostic problems exist in the routine evaluation of the latter (10,15), although many studies included CHM and PHM together in their study cohorts. A distinct world geographical distribution has long been observed in the prevalence of hydatidiform moles. The highest incidence is reported in southeastern Asia (3.8-13 cases/1000 pregnancies). The

incidence in the Middle East and South America ranges from 3.2 to 5.8 cases per 1000 pregnancies. Western Europe and North America have the lowest incidence of 0.8 to 1.5 cases per 1000 pregnancies (16-19). Although previous literatures showed predominance of CHM, a trend of increasing percentage of partial mole has been observed (16,20-22). Risk factors for the development of hydatidiform moles are essentially established by studies of complete mole, among which maternal age and history of molar pregnancy are the most relevant (17,20). Women over 35 years of age and teenagers have a significantly higher risk of molar gestation (23), with a 1.5- to 2-fold increase under the age of 20 (24), 2.5-fold increase in those over 35 years (25,26), and fivefold or more in those older than 40 years (2629). A previous CHM is associated with an increased risk of having another in 1% to 2% in Western counties and 2.5% to 9.4% in the Middle East (30-34). In women who already have had two molar pregnancies, the risk is increased to around 13% (27,34). The risk is diminished, however, if there are one or more normal pregnancies following a prior hydatidiform mole. Quality of dietary intake and socioeconomic status as risk factors for hydatidiform moles have been suggested by some studies (35), although controversies exist (29,36). Familial recurrent hydatidiform mole comprises 0.6% to 2.57% of all hydatidiform moles (37,38), among which FBCHM is inherited and characterized by multiple recurrent CHMs involving multiple family members, and mutations of NLRP7 or KHDC3L genes are the causal genetic etiology (39-42). GENETIC BASIS OF PATHOGENESIS Androgenetic Nature of Sporadic Hydatidiform Moles Fundamental discoveries in the 1970s solidified the genetic requirement for the development of hydatidiform moles (43,44). All cellular components derived from chorionic villi inherit an androgenic-only nuclear genome and a maternal-only mitochondrial DNA (45-47), with either 46, XX diploid karyotypes (homozygous,

80%-90%) or 46 XX or XY karyotypes (heterozygous, 10%-20%) (44,48-50). The prevailing hypothesis proposes that CHMs predominantly develop as a result of fertilization of an empty ovum (null genome) by a haploid sperm with duplication of the sperm DNA to reconstitute a diploid genome in the majority of the homozygous cases or by a diploid sperm resulting from failure of the second meiotic division (48). The heterozygous CHMs arise from fertilization of an empty ovum (null genome) by two independent haploid sperms (51). Diploidization of triploid ovum after abnormal fertilization is another proposed mechanism for the development of sporadic CHMs (52,53). In rare cases, androgenetic, tetraploid, and even triploid CHMs are possible as long as all the extra copies of the chromosomal sets are paternally derived without maternal genomic complement (54,55). Partial moles contain a triploid diandric-monogynic genome, arising from two sperms fertilizing an egg (heterozygous/dispermic) in over 95% of cases. The remaining less than 5% of cases arise from one haploid sperm fertilizing an egg followed by reduplication of the paternal chromosome set, or one sperm that is diploid due to failure of meiosis I or II (homozygous/monospermic) (56,57). As a result, approximately 70% of PHMs have a 69XXY karyotype, 27% are 69XXX, and 3% 69XYY (57,58). Tetraploid PHMs have been rarely reported, with three haploid paternal chromosome sets and a 92XXXX, 92XXYY, or 92XXXY karyotype (59-62), although some studies suggest nontriploid PHMs likely do not exist (50,63). In addition, the most recent investigations into large numbers of PHMs have observed that homozygous triploid PHMs are exceedingly rare (50) and that diandric triploids caused by postmeiotic errors perhaps do not occur (64). It should be noted that triploidy is one of the most common chromosomal abnormalities in human conceptions, occurring in up to 3% of all conceptuses (65,66) and representing up to 10% of all spontaneous abortions (67,68). As much as one-third of triploid abortuses are digynic nonmolar gestations, arising from meiotic nondisjunction of maternal chromosomes (10,57,65). Generally speaking, these digynic triploids do not have the biological, histological, or clinical profiles of diandric PHMs; however,

rare exceptions with overlapping histological features of PHM do occur and require careful histological and DNA genotyping analysis to confirm. Mutations of Maternal-Effect Genes in Familial Biparental Complete Hydatidiform Moles Familial recurrent hydatidiform moles represent 0.6% to 2.6% of all hydatidiform moles (38), among which Familial biparental complete mole (FBCHM) is a confirmed inheritable condition. More than 30 families of FBCHM have been reported (69-72). Candidate gene analysis led to the discovery of the NLRP7/NALP7 (Nucleotidebinding, Leucine-rich Repeat, Pyrin domains) gene on 19q13.4 in 2006 (39), of which about 50 homozygous or compound heterozygous mutations have been documented in FBCHM patients with various missense or truncating mutations involving all three functional domains of the protein (39,71-75). KHDC3L (KH domain containing 3-like, also known as C6orf221) is the second maternaleffect gene involved in FBCHM with its mutations responsible for 10% to 14% of cases (42,76). KHDC3L is mapped to chromosome 6q13 and is normally expressed at all oocyte stages and preimplantation embryos. To date, four homozygous or compound heterozygous mutations of KHDC3L have been identified in FBCHM patients (42). The expression of the NRLP7 gene is autosomalrecessive. As a maternal-effect gene, NLRP7 is expressed in the oocyte to support embryonic development until activation of the embryonic genome occurs (39). NLRP7 and KHDC3 appear to play an important role in controlling the timing of oocyte growth or in transducing signals for the initiation of imprint establishment (74) and work in a stage-dependent fashion during human preimplantation development. It has been argued that the global imprinting alteration at a pivotal time point in a preimplantation embryo is acquired by either the de novo absence of the maternal haploid genome in sporadic androgenetic CHMs or NLRP7 or KDC3L mutations shutting down the entire maternal imprinting gene expression in FBCHM. The net gain of paternal imprinting gene expression along with loss of counterpart maternal gene expression underscores the

pathogenesis of complete mole of both androgenetic and familial biparental nature (13). COMPLETE HYDATIDIFORM MOLE Historically, complete hydatidiform mole (CHM) is also known as “classic” hydatidiform mole. It is the prototype of molar gestation bearing the traditional description of “hydatid,” for example, grapelike structures at the time of evacuation, a term that was used throughout the previous century. Historical tales of “365 babies” of the Dutch’s Countess Margaret of Henneberg and “The Lump of Flesh in the King of Tars” are clearly documented cases of welldeveloped complete mole 700 years ago (77,78). Clinical Presentations (Table 19.3) Patients with well-developed CHM evacuated at late-first or earlysecond trimester present classic symptoms including vaginal bleeding, enlarged uterus, hyperemesis, hyperthyroidism, and preeclampsia as result of markedly elevated serum β-hCG (15,79). Vaginal bleeding is seen in more than 85% of the patients in the second trimester of pregnancy (80). Often vaginal bleeding is accompanied by passing tissues resembling grape-like structures, and 50% of the patients have an enlarged uterine size (81). Serum β-hCG was markedly elevated in over 50% of the patients and frequently reaching over 100,000 mIU per mL (82). Ultrasound shows no evidence of fetal development or fetal heartbeat; instead, the typical “snowstorm” or mixed echogenic appearance is seen due to well-developed molar vesicles admixed with blood (83). In clinical practice, the characteristic ultrasound findings combined with an elevated serum β-hCG inappropriate for the gestational age are highly suspicious for complete mole. Preeclampsia (pregnancyinduced hypertension, edema, and proteinuria) occurs in about 25% of the patients. Other symptoms including hyperemesis gravidarum, hyperthyroidism, and pulmonary embolization may also occur. Ovarian theca lutein cysts develop in a third of the cases leading to marked enlargement of the ovaries detectable by ultrasound (44,84).

Complete moles may rarely develop at ectopic gestational sites, including in the fallopian tube and ovary (85,86). In recent decades, the application of the highly sensitive serum βhCG detection and early ultrasound examination have drastically changed the clinical landscape of diagnosis and management of hydatidiform moles. An absence of fetal heart beat by ultrasound as early as 6 weeks of gestation can lead to therapeutic termination of pregnancy by dilatation and curettage (83,87). Consequently, the typical clinical presentation of CHM has become rare nowadays, and characteristic gross specimen is seldom encountered in a pathology lab (15,88-91). Most patients of early complete mole present with missed abortions, typically at a gestational age ranging from 6.5 to 12 weeks (87,90,92). Nonetheless, vaginal bleeding is still the most common presenting symptom seen in 46% of early complete moles, compared with 86% in the 1980s. (90). Macroscopic Pathology Gross appearance of a complete mole specimen depends on the gestational age at the time of evacuation. A well-developed complete mole evacuated at late-first trimester or during the second trimester usually consists of voluminous, bloody tissue aggregate with the hallmark of “hydatid” or grape-like appearance (Fig. 49.2). The hydropic changes are diffuse and uniformly transform chorionic villi into transparent vesicles of variable sizes ranging from a few millimeters to 3.0 cm with an average of 1.5 cm. There is no identifiable fetal tissue or normal placental structures except for the presence of coexisting normal twin gestation (93,94). The gross appearance of an early complete mole can be quite subtle, or there may be no gross evidence of abnormal edematous villi, indistinguishable from product of conception of a missed abortion. Not uncommonly, an early complete mole may be evacuated in an elective abortion procedure without any clinical suspicion for abnormal gestation.

FIGURE 49.2 Gross appearance of well-developed complete hydatidiform mole. Voluminous specimen with diffuse hydropic changes (hydropic vesicles) is characteristic. Courtesy of Dr. Peter E. Schwartz, Yale School of Medicine.

Microscopic Pathology Well-Developed Complete Hydatidiform Mole. Microscopically, diffuse villous hydrop and marked trophoblastic hyperplasia are hallmarks of a well-developed complete mole. The villous hydropic changes involve all chorionic villi with extensive edematous stroma and cistern formation. Cistern formation is defined as central cystic spaces that are completely devoid of cellular components occupying at least 50% of the villous stromal area (Fig. 49.3A). There is a significant increase in the villous size, and the shapes of the chorionic villi are round, oval, or irregular. Trophoblastic pseudoinclusions are generally present. Trophoblastic hyperplasia is characterized by irregular to diffuse trophoblastic proliferation involving a significant portion of chorionic villi. In contrast to a polarized distribution of trophoblasts in a nonmolar gestation, the trophoblastic hyperplasia in complete mole is nonpolar, multifocal, and frequently circumferential. Interconnections or bridges of

proliferating trophoblasts may be found between the villi (Fig. 49.3B). The proliferating trophoblasts form sheets or confluent aggregates of intermediate trophoblast admixed with cytotrophoblast and syncytiotrophoblast. Cytological atypia is almost always present in syncytiotrophoblast and intermediate trophoblast. Mitotic activity is frequent among cytotrophoblast and intermediate trophoblast. Although the villous stroma is generally hypocellular, cellular stroma can be found particularly in some smaller molar villi, where stellate spindle cells are embedded in a myxoid matrix along with numerous apoptotic bodies (karyorrhexis). Fetal parts or nonvillous placental structures, including amnion, yolk sac, and chorionic membrane, are essentially absent. Fetal nucleated red blood cells are not observed in a fully developed complete mole but can be rarely seen in very early complete mole (VECM; see below). Although well-formed villous vasculatures do not exist, immunohistochemistry (CD34 and QBEND10) may highlight the presence of distribution of endothelial cells in a linear fashion (95,96).

FIGURE 49.3 Histology of well-developed complete hydatidiform mole. Diffuse hydropic changes involve all chorionic villi with cistern formation (A). Abnormal circumferential trophoblastic hyperplasia consists of sheets or confluent aggregates of villous intermediate trophoblast admixed with cytotrophoblast and syncytiotrophoblast. Note the presence of trophoblastic bridging between villi (B).

Very Early Complete Hydatidiform Mole (VECM). Defined as a complete mole evacuated in less than 12 weeks of gestation, it was first documented by Keep who described 4 cases at 6.5 to 11 weeks

of gestation, and all were initially misdiagnosed as missed abortions based on clinical and ultrasound findings and only retrospectively confirmed as complete moles by DNA analysis (87). Such early complete moles may not be suspected by clinicians as well as pathologists due to their subtle clinical and pathological presentation. Although ultrasonography may detect an absence of fetal heartbeat, up to two-thirds of the cases demonstrate minimal abnormalities of molar gestation and are therefore unsuspected clinically, even with improved sonographic expertise (97,98). Likewise, in many cases, gross pathological findings distinguishing a VECM from a conventional missed abortion are not possible in a curettage specimen. Although the two diagnostic hallmarks of a well-developed complete mole—diffuse villous hydrop and exuberant abnormal trophoblastic proliferation—are generally absent, the histological changes of the villous stroma in VECMs are nonetheless highly characteristic (Fig. 49.4). Chorionic villi are small, relatively uniform, and display abnormal bulbous, polypoid to phyllodes-like configurations without significant stromal edema. The villous stroma frequently has a primitive appearance resembling mesenchymal villi during the early placental formation, including hypercellularity and the presence of stellate to plump fibroblasts embedded in a bluish myxoid matrix with prominent karyorrhexis or apoptotic bodies (Figs. 49.4C and 49.5A). Stromal hydropic changes may be present focally. Trophoblastic pseudoinclusions are uncommon. Abnormal surface trophoblastic proliferation in circumferential distribution is generally present to randomly involve some but not all villi (Figs. 49.4C,D). However, rare cases may show significant abnormal trophoblastic proliferation, even suggesting an early intramolar choriocarcinoma. Rudimentarily developed linear capillaries may be found, particularly by immunohistochemical staining of endothelial markers, and occasionally may contain identifiable fetal nucleated red blood cells (95,99,100), some of which may appear megaloblastic. So-called stunted embryos have been described in the literature as severely abnormal embryonic structures present in a VECM (101). However, a dead twin embryo with coexisting complete mole is possible (102).

FIGURE 49.4 Very early complete mole. Polypoid villous configurations are characteristically present in these four examples of very early complete moles (A, B, C, D). The villi are normal in size with primitive cellular myxoid stroma, stellate to plump stromal cells, and prominent karyorrhexis or apoptotic bodies (B, C). Abnormal trophoblastic hyperplasia is recognized by its random or circumstantial distribution (C, D).

Exaggerated implantation site reaction may be seen in an curettage specimen of complete mole, which is often more extensive than that associated with nonmolar gestations. The infiltrating intermediate trophoblast may show exuberant proliferation and remarkable cytological atypia, simulating PSTT. However, an absence of a mass lesion clinically or by imaging and the presence of concurrent molar gestation should easily confirm a nonneoplastic process. Ancillary Diagnostic Studies

P57 Immunohistochemistry. P57 encodes a cyclin-dependent kinase inhibitor protein, a paternally imprinted gene located on chromosome 11p15.5 (103). The gene is preferentially expressed from the maternal allele and is silent from the paternal allele. In both sporadic and familial biparental complete hydatidiform moles (FBCHMs), the cytotrophoblast and villous stromal cells loss the p57 nuclear staining that is diagnostically useful (Fig. 49.5) (104,105). All other gestations including partial moles, hydropic abortions, and trisomies contain maternal genomic complement and, therefore, have a normal p57 protein expression pattern: strong nuclear staining in cytotrophoblasts and villous stromal cells. It must be pointed out that in both complete and partial moles, intermediate trophoblasts, intervillous trophoblast islands, and maternal decidua normally express p57 nuclear staining, whereas syncytiotrophoblast are uniformly negative (105,106).

FIGURE 49.5 P57 immunostaining in complete mole. Nuclear staining of p57 is absent in cytotrophoblast and villous stromal cells in complete mole (A, B). Background maternal decidual cells demonstrate normal nuclear expression of p57 (B, lower right).

Although p57 immunostain is very helpful in confirming a diagnosis of CHM, several important pitfalls exist that can potentially interfere with the interpretation. Since p57 expression is retained in various cell types in CHM (i.e., intervillous intermediate trophoblasts, maternal decidua), care must be given to which cell types show positive staining. Cases of complete moles may show isolated

nuclear staining in less than 10% of the cytotrophoblast or stromal cells. The p57 staining pattern may be equivocal, with only 10% to 50% of target cells staining positive, which is insufficient to rule out a complete mole. Rare mosaic androgenetic/biparental mosaic/chimeric gestations may present discordant p57 immunostaining patterns (negative in cytotrophoblast but positive in villous stromal cells, and vice versa) (107). CHM arising from a twin gestation may show chorionic villi with absent p57 staining admixed with villi showing normal p57 pattern, that is, a divergent p57 expression pattern (108). Divergent p57 expression can also be observed, combined with p57 discordancy, in androgenetic/biparental mosaicism with a molar component. Additional pitfalls include rare cases of CHM with retention of maternal chromosome 11, resulting in normal p57 expression (109,110) and rare partial moles lacking p57 staining due to loss of maternal chromosome 11 (111). Loss of p57 expression in cytotrophoblast and villous stromal cells is rarely observed in gestations of paternal uniparental isodisomy of TH01 locus on chromosome 11 that may simulate complete mole histologically and clinically (112). Molecular Genotyping. STRs are repetitive DNA sequences of two to seven nucleotides that are highly prevalent and inheritably stable in the genome, mostly located in the noncoding regions of the genome. (113). The number of repeats at each STR locus may differ between individuals, and combined analysis at multiple specific STR loci offers a tremendous resolving power for human identity testing. In diagnostic pathology practice, STR genotyping is generally performed on DNA extracted from formalin-fixed paraffin-embedded tissue blocks. The assay is a single multiplex PCR amplification using one of the commercially STR genotyping kits. By comparing the allelic patterns of maternal and villous tissue at each STR locus, the presence and relative proportion (copy number) of maternal and paternal alleles in the villous tissue can be determined for precise diagnosis of hydatidiform moles (22,56,114-116). With increasing acquisition of molecular capability at many medical centers, STR

polymorphism analysis or STR genotyping is now considered the gold standard in the diagnosis and subclassification of hydatidiform moles (13). In contrast to a balanced biallelic profile of both maternal and paternal genetic contributions at all STR loci in nonmolar hydropic abortions, the diagnostic genotypic profile of a complete mole displays exclusively paternal alleles of either homozygous or heterozygous pattern consistently present at all STR loci (Fig. 49.6). Monospermic (homozygous) partial moles show one maternal allele and a duplicate quantity of one paternal allele consistently present at every STR locus, and two unique paternal alleles in addition to one maternal allele are diagnostic of dispermic (heterozygous) partial mole.

FIGURE 49.6 Short tandem repeat (STR) genotypes of complete mole. Homozygous complete mole (A) shows the presence of one paternal allele in chorionic villi at all STR loci (lower panel) compared with a biparental pattern in the

paired maternal endometrium (upper panel). Heterozygous complete mole (B) harbors two distinct paternal alleles at some STR loci, and other loci show duplicated quantity of one paternal allele (lower panel) compared with a biparental pattern in maternal endometrium (upper panel).

The major advantage of STR genotyping over other ancillary techniques is its ability to precisely classify molar gestations at the genetic level. Traditional karyotyping and ploidy analysis are unable to identify the exact parental genetic contribution in a triploid gestation and therefore cannot separate a true diandric-monogynic partial mole from a digynic-monoandric, nonmolar triploid gestation. Rare potential pitfalls of genotyping diagnosis of complete moles include the presence of rare FBCHM, in which a balanced paternal and maternal genetic profile is found, although histological features and p57 expression are indistinguishable from a sporadic diandric complete mole (69,70). Moreover, an egg donor gestation may post a significant diagnostic challenge for molecular pathologist when the fertility history is unknown and histological correlation is not readily available or considered. In this scenario, the villous tissue does not contain alleles from the recipient mother; hence, STR genotyping data simulate a heterozygous/dispermic complete mole (117). Rare cases of twin pregnancy with coexisting complete mole or gestations with mosaicism/chimerism can also pose significant challenge in the interpretation of genotyping data (102,118,119). Histological review in correlation with p57 immunohistochemistry and STR genotyping data is essential, and, if necessary, dissection of distinct villous populations for additional STR genotyping analysis (twin gestation with complete mole component) may resolve the diagnostic difficulty. Differential Diagnosis The differential diagnoses of CHMs include a variety of nonmolar gestations with overlapping morphologic features. Common differential diagnoses primarily occur in separation of VECM from its mimics. Partial Hydatidiform Mole. A partial hydatidiform mole (PHM) evacuated at an early stage of gestation may show stromal features

of VECM. The distinction between partial and very early CHMs may be facilitated by identification of fetal tissues, which are absent in complete moles. Fetal vessels and nucleated red blood cells, however, have been rarely described in very early CHM (51,100). A high index of suspicion at the time of morphologic evaluation is crucial to initiate p57 immunohistochemistry and/or STR genotyping for the correct diagnosis (Table 19.3). Nonmolar Hydropic/Spontaneous Abortions. Spontaneous nonmolar hydropic abortuses may show significant villous enlargement and edema, even with cistern formation, mimicking complete mole on the morphologic level. However, the villous contour is round or oval without invaginations, and trophoblastic hyperplasia is absent or mild and is polarized. In contrast, the villous configuration is polypoid or club-shaped and the trophoblastic proliferation when present is circumferential or random in VECM. The p57 expression pattern is normal, and genotyping shows a balanced biallelic profile in nonmolar gestation. Early Gestations. Often encountered in a curettage specimen, early placenta formation at the villous stage has primitive chorionic villi with cellular and myxoid villous stroma, simulating the stromal features of early complete mole. Ectopic pregnancy, particularly of tubal location, frequently terminates with gestational sac rupture and bleeding at an early stage, and the chorionic villi may show remarkable stromal hypercellularity and myxoid changes, greatly overlapping with VECM. However, the trophoblastic proliferation is focal and polarized at one end of the chorionic villi. Nevertheless, an early complete mole can still arise from an ectopic gestation. Immunohistochemistry of p57 or DNA genotyping may resolve the issue if in doubt. Intramolar Choriocarcinoma. Complete mole may present with areas of significant trophoblastic hyperplasia and marked cytological atypia in some cases, even in a VECM. In isolation, such exuberant trophoblastic proliferation may mimic choriocarcinoma histologically.

Although a full-blown choriocarcinoma requires the presence of tissue necrosis, destructive growth, and extensive hemorrhage, it has been increasingly recognized that such atypical trophoblastic proliferation may well represent an emerging or in situ choriocarcinoma arising from its concurrent complete mole. Similar to the well-recognized in situ or intraplacental choriocarcinoma arising in a normal placenta, endorsed by the 2020 WHO classification, the presence of sheets of highly atypical trophoblast with biphasic/triphasic arrangement embedded within a complete mole should prompt a consideration of in situ or intramolar choriocarcinoma. Prognosis and Treatment Complete moles have a 15% to 20% risk of developing postmolar GTD and a 2% to 3% risk of gestational choriocarcinoma, regardless of the gestational age at the initial evacuation (90,120,121). All patients with a diagnosis of complete mole will undergo the molar surveillance serum β-hCG monitoring program. Recent investigations have confirmed that heterozygous/dispermic complete moles are clinically more aggressive with a significantly higher risk for development of postmolar GTD than homozygous/monospermic complete moles (122-124). Genotyping classification of complete moles into precise subtypes based on their genetic zygosity is therefore important for risk assessment for postmolar GTD and subsequent patient management. PARTIAL HYDATIDIFORM MOLE Subclassification of hydatidiform moles into complete and partial moles did not occur until the late 1970s, and the initial logical basis for the division was the presence or absence of ascertainable embryonic or fetal tissue, hydropic changes affecting only some of the villi, focal moderate trophoblastic hyperplasia, villous “trophoblastic inclusions,” and maze-like central cisterns (84,125,126), although triploid genetic nature of partial mole had already been known in the 1960s (127).

Clinical Presentation (Table 19.3) Patients usually present in the late-first or early-second trimester with clinical impression of missed or incomplete abortion. Most cases are not suspected clinically for molar gestation prior to evacuation. The uterine size is small or appropriate for the gestational age. Vaginal bleeding is less common than with complete mole (91,128). Serum β-hCG is normal or only mildly elevated (15). Ultrasound may show focal placental cystic changes, and a fetus may be detectable (129,130). Partial mole may rarely develop from an ectopic gestation involving the fallopian tube (131). Macroscopic Pathology Generally, the volume of curettage specimens of partial mole is more copious than those of nonmolar missed abortions, and the villous tissue may show focal hydropic vesicles (Fig. 49.7). Gestational sac, fetal parts, or an intact fetus may be present and show intrauterine growth restriction (IUGR) and malformations (132,133). The fetus, if present, usually shows mild to moderate symmetrical IUGR and characteristic malformations including syndactyly involving fingers 34 and toes 2-3, spina bifida, cleft palate, cryptophthalmos, simian crease, and renal hypoplasia (10,84,132). It should be noted that partial moles evacuated earlier during the first trimester may show no grossly identifiable abnormalities. Although partial mole is generally incompatible with fetal survival, there have been rare cases of live-born triploid fetuses that died within a few hours of birth, some of which have been confirmed as partial moles (68,134).

FIGURE 49.7 Gross appearance of partial hydatidiform mole. A partial hydatidiform mole evacuated at 20 weeks of gestation displays semitransparent hydropic vesicles involving some but not all of the chorionic villi. Courtesy of Dr. Peter E. Schwartz, Yale School of Medicine.

Microscopic Pathology PHM typically demonstrates an admixture of two villous populations: a. small, fibrotic villi and enlarged, irregularly shaped villi with scalloped contours, a variable degree of hydrops, and mild to moderate circumferential trophoblastic proliferation (Fig. 49.8) (51,135,136). The size of the largest hydropic villi is greater than 0.5 mm (137). Cistern formation and trophoblastic pseudoinclusions are common (51). Cistern formation with a maze-like acellular pattern is usually seen in advanced cases. Round to oval trophoblastic pseudoinclusions due to invaginations of villous surface trophoblast are frequent findings (Fig. 49.8B). Single-cell trophoblastic inclusions (“wandering trophoblast”) are also commonly seen in the villous stroma (137,138). Mild to moderate circumferential—nonpolar— trophoblastic hyperplasia is usually present, generally without significant cytological atypia (84,125,138). Focally prominent syncytiotrophoblast “knuckles” (or “sprouts”) with intracytoplasmic

lacunae are also a characteristic finding in partial mole. Fetal blood vessels and nucleated red blood cells are often present.

FIGURE 49.8 Histological characteristics of partial hydatidiform mole. There are two populations of chorionic villi: the enlarged hydropic villi with cistern formation and irregular contours (surface scalloping), and the smaller villi with fibrotic stroma (A). Round to oval trophoblastic pseudoinclusions are frequently seen (B).

However, the above histological findings of PHM are not diagnostically specific and significantly overlap with those seen in CHM, hydropic abortion, trisomy syndromes and other chromosomal abnormalities, placental mesenchymal dysplasia, and CHM arising in twin gestation (11,139). The following morphologic parameters are important for the initial screening for PHM: villi size enlargement, presence of two villous populations, round or oval trophoblastic pseudoinclusions, at least moderate villous hydrops, cistern formation, and abnormal trophoblastic hyperplasia. The most sensitive morphologic features included villous hydrops (86% sensitivity) or the presence of at least one of the following three parameters: two villous populations, round or oval pseudoinclusions, and cisterns (84% sensitivity). The presence of cisterns and villous size greater than or equal to 2.5 mm has the highest positive predictive value (90%) for PHM, but the sensitivity is only 30%. The presence of any one of the following histologic findings should prompt DNA genotyping study to rule out PHM: round or oval pseudoinclusions, cistern formation, two populations of villi, and a villous size of greater than or equal to 2.5 mm (11).

Ancillary Diagnostic Studies With the presence of maternal genome in partial moles, immunohistochemistry for p57 shows normal nuclear staining in cytotrophoblast and villous stromal cells (Fig. 49.9) (104,140). Although a retained nuclear p57 expression is useful to rule out a complete mole, it neither confirms partial mole nor separates partial mole from many of its histological mimics.

FIGURE 49.9 P57 immunohistochemistry of partial mole. Partial mole demonstrates normal nuclear expression of p57 in cytotrophoblasts, intermediate trophoblasts, villous stromal cells, and maternal decidual cells.

The gold standard in the diagnosis of partial mole is DNA genotyping (13). STR DNA genotyping provides precise diagnosis by identifying diandric triploidy (Fig. 49.10) (11,139,141). It is necessary to point out that digynic nonmolar triploid gestation may also mimic PHM microscopically in rare cases and DNA ploidy analysis or karyotyping cannot be used to separate it from a true diandric partial mole.

FIGURE 49.10 Short tandem repeat (STR) genotypes of partial mole. (A) Heterozygous partial mole demonstrates the presence of two distinct paternal alleles in addition to one maternal allele at several microsatellite loci (lower panel), compared to a balanced biparental allelic pattern of the corresponding gestational endometrium (upper panel). (B) Homozygous partial mole demonstrates the presence of duplicated quantity of one paternal allele in addition to one maternal allele at all STR loci (lower panel).

Differential Diagnosis The differential diagnoses of PHMs include a variety of nonmolar gestations with overlapping morphologic features. Nonmolar Hydropic Abortions. Hydropic abortion may show significant villous hydrop including cistern formation. However, the

villi have a smooth contour without abnormal trophoblastic hyperplasia, trophoblastic atypia, or pseudoinclusions. Genotyping shows a balanced biallelic genetic profile in contrast to the diandricmonogynic triploid genome in partial mole. Digynic Nonmolar Triploid Gestations. Representing one-third of triploid missed abortions, triploid digynic-monoandric gestations may mimic partial moles at both the morphologic level and DNA ploidy level. Although most cases are without abnormal villous morphology, in rare cases, digynic triploidy may simulate a PHM (Fig. 49.11), mainly due to the presence of hydropic villi and, less commonly, cistern formation and irregular villous shape with trophoblastic pseudoinclusions and syncytiotrophoblastic knuckles. One common scenario requires attention of a pathologist to rule out partial mole is the karyotyping findings of triploidy after a nonmolar diagnosis has been reported by pathologists. It is important to separate PHM from a nonmolar digynic triploidy, as the latter is not associated with increased risk of persistent GTD or GTN. STR genotyping is perhaps the only ancillary study to separate the two entities (Fig. 49.12).

FIGURE 49.11 Digynic nonmolar triploidy. This digynic triploid gestation demonstrates villous hydrop and abnormal villous shapes (A) and the presence of round to oval trophoblastic pseudoinclusions, remarkably simulating a true partial mole (B).

FIGURE 49.12 Short tandem repeat (STR) genotyping profile of digynic nonmolar triploidy. The nonmolar triploid nature is confirmed by the presence of two matching maternal alleles (lower panel) to the two alleles in the corresponding gestational endometrium (upper panel).

CHM Arising in Twin Gestation or Mosaic Gestation. In twin gestation with complete mole arising from one of the twin placentas, distinct complete molar villi are admixed with normal nonmolar villi in a curettage specimen (10,100). Mosaic conceptions with a molar component (CHM) may result in the presence of two morphologically distinct villous populations (nonmolar villi without trophoblastic hyperplasia and molar villi with trophoblastic hyperplasia and hydropic changes), therefore simulating PHM (107). Careful morphological assessment is crucial to ascertain the true molar hydropic villi, which may be confirmed by abnormal p57 immunohistochemistry or comparative DNA genotyping of the two villous populations. Trisomy Gestations. Chromosomal trisomies are common findings among missed abortions, particularly trisomies 16 and 21.

Trisomy gestions can be microscopically indistinguishable from PHM, as they often show abnormal villous configurations with trophoblastic pseudoinclusions and villous hydrops (Fig. 49.13A) (11,22,142-144). Trophoblastic hyperplasia, although mild, may also be found, more commonly seen in trisomy involving chromosomes 7, 15, 21, and 22. P57 immunostaining offers no value in this setting, as the staining pattern is similar to partial mole. STR genotyping offers a definitive separation of partial mole from chromosome trisomies. Chromosome trisomy demonstrates an allelic gain (three allelic copies) only at the involved chromosomal locus (Fig. 49.13B) in contrast to three allelic copies present at all STR loci in partial mole.

FIGURE 49.13 Trisomy 21 syndrome. (A) Irregular villous shapes, hydropic changes, and trophoblastic pseudoinclusions are present in this product of conception of trisomy 21. (B) Three allelic copies at D21S11 locus (chromosome

21) are present as the only abnormal STR locus (the second locus at the lower panel), and a balanced biparental allelic pattern is seen at all other STR loci.

Placental Mesenchymal Dysplasia. Also known as “pseudopartial mole with angiomatous proliferation,” the lesion is usually evacuated during the second or third trimester. Villous hydrops primarily involve the stem villi with cyst formation, size enlargement, and vascular aneurysmal changes with thrombosis (10). Terminal villi are usually normal but may show villous immaturity, including villous spindle and stellate mesenchymal cells in a myxoid background. Angiomatous change and chorangiomas are common. In contrast to partial mole, there is usually no significant trophoblastic hyperplasia and trophoblastic pseudoinclusions. The fetus may show IUGR or signs of Beckwith-Wiedemann syndrome (i.e., macrosomia, visceromegaly, hemihypertrophy, macroglossia, omphalocele, and adrenal cytomegaly) (10). Molecular genotyping definitively separates placental mesenchymal dysplasia from partial mole. A few case reports of placental mesenchymal dysplasia found an abnormal/equivocal p57 staining pattern due to androgenetic/biparental mosaicism (145,146). Diagnostic Algorithms of Hydatidiform Moles Although the recent introduction of p57 immunohistochemistry and STR genotyping has drastically changed the diagnostic accuracy with significant improvement of intra- and inter-observer variabilities, careful microscopic evaluation of morphologic features on hematoxylin and eosin (HE)–stained slides remains the foundation in daily diagnostic practice. Microscopic evaluation of HE slides serves the initial screening tool in identifying and triaging abnormal cases to various ancillary studies. It is also crucial for correlative interpretation of p57 and STR genotyping data for final diagnosis. Currently, any case that shows microscopic features suspicious for hydatidiform moles should be further evaluated by ancillary techniques. In consideration of availability, cost efficiency, turnaround time, and prognostic implications of ancillary studies, algorithmic approaches have been proposed in recent years to combine histology, p57

immunohistochemistry, and DNA genotyping in the various workflows (50,108,147). One algorithmic approach advocates for genotyping on all morphologically suspicious cases and use of p57 immunostain only when there is a discrepancy between the morphology and the genotyping result, for example, rare cases of biparental CHM, mosaicism/chimerism, or CHM arising from a twin gestation (147). The second approach will subject the specimens with morphologic suspicion for CHM for p57 immunostain, and cases with histologic suspicion for PHM for DNA genotyping evaluation. The third algorithm subjects all suspicious cases for p57 immunohistochemistry to identify complete moles and triage the remaining cases for STR genotyping (50,108). It is important to note that ploidy analysis is no longer recommended and it plays no role in modern diagnostic algorithms for hydatidiform moles. The second and third algorithms may provide a cost-effective approach in the diagnosis of complete mole and are more acceptable to laboratories with limited accessibility to molecular genotyping techniques. Although an accurate diagnosis of complete mole can be achieved by histological and p57 immunostaining, the first one-step genotyping approach provides an overall efficient way to precisely diagnose various molar gestations, provided that an access to molecular genotyping is feasible (22). Moreover, recent investigations have confirmed that heterozygous/dispermic complete moles are clinically more aggressive with a significantly higher risk for the development of postmolar GTD than homozygous/monospermic complete moles (122-124). Therefore, genotyping classification of complete moles into precise subtypes based on their genetic zygosity is important for risk assessment for postmolar GTD. Prognosis and Treatment Approximately 0% to 5% of PHMs are followed by persistent GTD, mostly invasive hydatidiform moles (91,120,148,149). The risk of choriocarcinoma after PHM is 0.15% according to a recent study of genotypically confirmed partial moles (150). All patients with a

diagnosis of partial mole should undergo the molar surveillance serum β-hCG monitoring program, though with a shorter duration compared to those with complete mole. INVASIVE AND METASTATIC HYDATIDIFORM MOLES Invasive hydatidiform mole is defined as either a complete mole or much less often a partial mole that demonstrates invasion into the uterine myometrium and/or vasculatures (Fig. 49.14) (151). Invasive hydropic villi may be visible grossly, and uterine perforation may occur. Most invasive hydatidiform moles are complete moles and retain the villous histological characteristics. Invasive partial moles also occur (152,153). Metastatic mole is diagnosed when the molar chorionic villi is identified at extrauterine sites, mostly commonly involving the vaginal wall or pelvis. Vaginal bleeding may occur with persistent elevation of serum β-hCG after the initial evacuation of the primary hydatidiform mole (152,154,155). Both invasive and metastatic hydatidiform moles are lesions included in the category of postmolar or persistent GTD or GTN. In the absence of immediate complications (e.g., severe hemorrhage), chemotherapy is highly effective, with a cure rate in over 80% of the cases, depending on the extent of the disease (15,151,155).

FIGURE 49.14 Invasive complete mole. Hysterectomy specimen shows invading molar villi in direct contact with uterine myometrium without intervening maternal decidual (A) and the presence of molar villi plugging the myometrial vascular space (B).

ABNORMAL (NONMOLAR) VILLOUS LESIONS Abnormal (nonmolar) villous lesions are various nonmolar lesions with histological features overlapping with the PHM (10). They have diverse origins related to the specific diagnosis causing the abnormal villous morphology. Although various chromosomal or genetic alterations may be found (11,112), in many cases, the etiology is unknown. Hydropic (nonmolar) abortions and chromosomal trisomy syndromes are often responsible for atypical/dysmorphic villous morphology. Less common causes include digynic triploid conception and placental mesenchymal dysplasia. They may present normal gross findings or discernible vesicle formation. Microscopically, chorionic villi often display some degree of irregularity in size and shape, with enlargement and focal mild trophoblastic proliferation (sometimes manifesting as syncytiotrophoblastic snouts) and occasional trophoblastic pseudoinclusions. Such atypical/dysmorphic villous morphology shares features within the spectrum of histological alterations of PHM. Placental mesenchymal dysplasia, which is characterized by cystic dilatation of the stem villi, is usually seen later in gestation and is associated with IUGR, nonmolar mosaic conceptions, and Beckwith-Wiedemann syndrome (156). P57 immunohistochemistry is generally not helpful. DNA genotyping analysis effectively distinguishes these lesions from partial mole and may detect aneuploid conceptuses (22,56,114). These are nonmolar entities and therefore have the same low risk of persistent GTD as any other morphologically unremarkable nonmolar conceptions. When definitive genotyping studies are not available, women in whom PHM is suspected because of the presence of atypical/dysmorphic villous morphology may be monitored by β-hCG surveillance (141).

EXAGGERATED PLACENTAL SITE

Exaggerated placental site (EPS) is a benign, nonneoplastic lesion characterized by an increased number of implantation site intermediate trophoblastic cells that extensively infiltrate the endometrium and underlying myometrium. An EPS may occur in association with normal pregnancy, an abortion, or a hydatidiform mole. An EPS is composed of implantation site intermediate trophoblastic cells, which display an identical immunophenotypic profile to the intermediate trophoblastic cells found in the normal implantation site. It has been thought that this lesion represents an exaggeration of a normal physiologic process. MORPHOLOGIC FEATURES The EPS is defined as an increased number of implantation site intermediate trophoblastic cells exceeding those normally present in the implantation site from an early pregnancy. The lesion is composed predominantly of mononucleate implantation site intermediate trophoblastic cells and variable numbers of multinucleated intermediate trophoblastic cells that extensively infiltrate the endomyometrium (Fig. 49.15). Despite the extensive infiltration by intermediate trophoblastic cells, the overall architecture of the implantation site is not disturbed. Endometrial glands may be surrounded by trophoblastic cells but are not destroyed, and similarly the smooth muscle cells of the myometrium are separated by cords, nests, and individual implantation site intermediate trophoblastic cells that diffusely infiltrate the myometrium without producing necrosis (157). The surrounding decidua, however, may show degeneration and necrosis typical of a spontaneous abortion. Other features associated with gestation are usually present, including hyalinized spiral arteries, hypersecretory glands, and chorionic villi. EPS is associated with hydatidiform moles, especially complete moles. Despite the profuse infiltration of trophoblastic cells, the Ki-67 index of these cells is near zero (except those associated with CHMs), suggesting that the increased number of trophoblastic cells is not the result of de novo proliferation at the implantation site (157,158).

FIGURE 49.15 An exaggerated placental site showing nests of implantation site intermediate trophoblastic cells infiltrating the superficial myometrium. The pattern of invasion closely simulates placental site trophoblastic tumors but is focal and not confluent.

DIFFERENTIAL DIAGNOSIS At times, EPS may be difficult to distinguish from PSTT, particularly in a curetting, as the diagnostic criteria for an EPS are subjective (159). There are no reliable data quantifying the amount and extent of infiltration of implantation site intermediate trophoblasts at different

stages of normal gestation. Thus, to report EPS is somewhat subjective, but the importance to have this diagnosis is to rule out PSTT, the most important differential diagnosis of PSTT. The features that support the diagnosis of EPS rather than PSTT include the following: (1) presence of chorionic villi or fetal parts, (2) the lack of confluent/sheet-like growth, (3) the absence of a macroscopic lesion, (4) preserved endomyometrial architecture, (5) lack of mitoses and extremely low Ki-67 proliferation index (near zero), and (6) more multinucleated trophoblastic cells (157). It should be emphasized that the “degenerated” appearance of nuclei in intermediate trophoblastic cells makes it difficult to identify an unequivocal mitotic figure on HE sections, and a high morphological stringency should be applied to call mitotic figures. Ki-67 nuclear labeling index using a Ki-67–specific (MIB-1) antibody is superior to the mitotic index as a diagnostic aid in the differential diagnosis of EPS versus PSTT (158). Specifically, the Ki-67 index (mean ± standard deviation) of the trophoblastic cells in an EPS is near zero in contrast to 14% ± 6.9% in a PSTT. There are two cautionary points that should be highlighted in evaluating the Ki-67 index in the differential diagnosis. First, only the implantation site intermediate trophoblastic cells should be counted because the fragments of villous trophoblastic cells and the junctional areas where the anchoring villi attach to the endometrium may contain highly proliferative trophoblastic cells. Second, because Ki-67 labeling may occur in the lymphoid cells normally present at the placental site, it is important to be certain that Ki-67 labeling is assessed only in intermediate trophoblastic cells using strict cytologic criteria. In difficult cases, double immunostaining utilizing an antibody against Ki-67 and intermediate trophoblastic markers including HSD3B1, HLA-G, and CD146 can assist in this distinction (4,160-163). The trophoblastic cells in an EPS demonstrate an identical expression profile of trophoblast-associated markers to the intermediate trophoblastic cells in an early implantation site (Table 49.2). The application of immunohistochemistry in assisting the diagnosis of EPS is described in the section of “Immunohistochemistry Approach for Differential Diagnosis.”

BEHAVIOR AND TREATMENT EPS is a benign trophoblastic lesion that involutes following curettage. EPS is not associated with increased risk of persistent GTD. No specific treatment or follow-up is necessary. When an EPS cannot be confidently distinguished from PSTT by morphology and immunohistochemistry, close follow-up with serial β-hCG titers is advisable.

PLACENTAL SITE NODULE A placental site nodule is a well-circumscribed hyalinized lesion composed of chorionic-type intermediate trophoblastic cells that are normally located in chorion laeve (fetal membrane) (Fig. 49.1). Typically, it is incidentally found in an endometrial biopsy, endocervical curetting, or cervical biopsy from a woman in the reproductive age group. In one study, 40% of placental site nodules were present in the endocervix, 56% in the endometrium, and 4% in the fallopian tube (1). In this latter scenario, one must postulate that the tubes were incompletely transected or that they recanalized after surgery. It is generally thought that placental site nodule represents a retained noninvoluted placental site. However, morphologic and immunohistochemical studies have shown that placental site nodules are more closely related to intermediate trophoblastic cells from the chorion laeve (fetal membrane) than from the placental site (1). PATHOLOGIC FEATURES The placental site nodule is a small, often microscopic finding (Fig. 49.16). When grossly visible, it presents as a yellow, tan, or hemorrhagic nodule measuring up to 1 cm and located in the endometrium or superficial myometrium. Rarely, extrauterine placental site nodules can be found in the fallopian tube, peritoneal wall, vagina and, postcesarean section scar (164,165). Microscopically, placental site nodule has a discrete, wellcircumscribed, lobulated border sometimes showing small irregular

nests of cells projecting into the surrounding tissue. A thin layer of chronic inflammatory cells and decidual cells often encompasses the lesion. The chorionic-type intermediate trophoblastic cells within the placental site nodule are arranged singly, in nests and cords (157). They are embedded in abundant eosinophilic fibrillar extracellular matrix protein. The latter finding is a prominent feature of placental site nodules. The cells vary in size; many have relatively small uniform nuclei but others are large with mononucleate, irregular, and hyperchromatic nuclei (Fig. 49.17). Occasionally, scattered multinucleated cells are present. The cytoplasm of chorionic-type intermediate trophoblastic cells is abundant and eosinophilic to amphophilic, sometimes with conspicuous vacuolation. Morphologically and immunohistochemically these cells resemble those in the chorion leave; therefore, they have been designated as chorionic-type intermediate trophoblastic cells. Gene expression of intermediate trophoblastic cells in the placental site nodules and in chorion laeve (fetal membrane) is the same. For example, they are only focally positive for hPL and CD146 (Mel-CAM), and are negative for mucin-4, in contrast to the intermediate trophoblastic cells at the implantation site, where these antigens are highly and diffusely expressed (Table 49.2) (1,166-168). Immunoreactivity for βhCG is usually absent. In contrast to implantation site intermediate trophoblastic cells in the first trimester, the chorionic-type intermediate trophoblastic cells in placental site nodules are positive for p63 (Table 49.2) (168,169). Similar to the chorionic-type intermediate trophoblastic cells found in the fetal membranes of normal pregnancy, there is a low level of proliferation in the cells of placental site nodules as indicated by a few scattered Ki-67-labeled nuclei (1). In contrast, in the normal implantation site and even in EPSs, the Ki-67 index of intermediate trophoblastic cells is zero (158).

FIGURE 49.16 A placental site nodule in endometrial curettage samples showing the typical nodular and hyalinized appearance of the lesion at low magnification.

FIGURE 49.17 A placental site nodule with a cluster of hyperchromatic and vacuolated chorionic-type intermediate trophoblastic cells in a hyaline matrix. They are not as crowded as in ETTs.

DIFFERENTIAL DIAGNOSIS Placental site nodules may be microscopically confused with PSTTs, ETTs, and certain nontrophoblastic lesions, notably invasive keratinizing squamous carcinoma of the cervix (157,170). The small size, circumscription, extensive eosinophilic extracellular matrix, and paucity of mitotic figures distinguish this lesion from PSTT, ETT, and

cervical squamous carcinoma. The distinction of a placental site nodule from an ETT is usually not difficult as placental site nodules are microscopic lesions with sharply circumscribed borders (1). ETTs are larger and display substantial necrosis and focal calcification. Moreover, the placental site nodule is much less cellular than ETT. In addition, the trophoblastic cells in a placental site nodule in contrast to an ETT are bland and mitotic activity is low or absent. The Ki-67 index in ETTs is significantly higher (>12%) than in placental site nodules (5 mm depth of invasion; limited to cervix with size measured by maximum tumor diameter

T1b1

IB1

Invasive carcinoma >5 mm depth of invasion and ≤2 cm greatest diameter

T1b2

IB2

Invasive carcinoma >2 cm and ≤4 cm in greatest dimension

T1b3

IB3

Clinically visible lesion >4.0 cm in greatest dimension

T2

II

Cervical carcinoma invades beyond uterus, but not to pelvic wall or lower third of vagina

T2a

IIA

Tumor without parametrial invasion

T2a1

IIA1

Clinically visible lesion ≤4.0 cm in greatest dimension

T2a2

IIA2

Clinically visible lesion >4.0 cm in greatest dimension

T2b

IIB

Tumor with parametrial invasion

T3

III

Tumor extends to pelvic wall and/or involves lower third of vagina, and/or causes hydronephrosis or nonfunctioning kidney and/or involves pelvic and/or paraaortic lymph nodes

T3a

IIIA

Tumor involves lower third of vagina, no extension to pelvic wall

T3b

IIIB

Tumor extends to pelvic wall and/or causes hydronephrosis or nonfunctioning kidney

IIIC

Involvement of pelvic and/or paraaortic lymph nodes (with r and p notations)c

IIIC1

Pelvic lymph node metastasis only

-

IIIC2

Paraaortic lymph node metastasis

T4

IV

Tumor invades mucosa of bladder or rectum and/or extends beyond true pelvis (bullous edema is not sufficient to classify a tumor as T4)

T4

IVA

Involves adjacent organs

IVB

Spread to distant organs

NX

Regional lymph nodes cannot be assessed

N0

No regional lymph node metastasis

N0(i+)

Regional lymph nodes with isolated tumor cells 4.0 cm) that clinically mimic malignancy have also been reported but are rare (122). Endocervical polyps occur in the transformation zone and consist of an elongated or rounded mass connected to the endocervix by a narrow stalk. The most common morphologic appearance is adenomatous with mucinous endocervical-type glands admixed with variable amounts of fibrovascular stroma (Fig. 52.45). Mucus retention within the endocervical glands can lead to a cystic appearance, whereas a predominance of fibrous stroma will lead to a fibrotic appearance. The surface may also be notable for areas of squamous metaplasia, tuboendometrial metaplasia, or microglandular hyperplasia. Inflammation is variable, and in cases of traumatized polyps, surface erosion, ulceration, or infarction may be seen. The surface epithelium may exhibit reactive changes, such as nuclear enlargement, prominent nucleoli, and multinucleation. Although these changes may be marked, noting the presence of low N:C ratios is helpful in supporting a benign process. Endocervical polyps occurring in the setting of pregnancy can also exhibit features of Arias-Stella reaction or stromal decidualization. Carcinomas arising in an endocervical polyp are rare (0.3%) (121).

FIGURE 52.45 Endocervical polyp. Elongated mass with mucinous endocervical-type glands and central fibrovascular stroma.

Papillary Endocervicitis Inflammation of the cervix commonly causes the mucosal surface to assume a papillary appearance that is referred to as papillary endocervicitis (Fig. 52.46). In most cases, the architectural changes are mild and easily recognized as reactive. However, florid cases may raise concern for the possibility of a villoglandular or well-differentiated endocervical adenocarcinoma. The presence of minimal nuclear atypia and lack of mitotic activity help point to a benign process.

FIGURE 52.46 Papillary endocervicitis. Papillary projections of endocervical mucosa with a chronic inflammatory infiltrate.

Arias-Stella Reaction

Arias-Stella reaction typically occurs in the endometrium in the setting of pregnancy or hormonal therapy but can less commonly involve endocervical glands (123). When it occurs in sites other than the endometrium, the changes can be mistaken for a malignant process, such as clear cell carcinoma. Regardless of site, the morphologic changes are the same: vacuolated clear or oxyphilic cytoplasm, intraglandular tufting, hobnail cells, and intranuclear inclusions (123) (Fig. 52.47). Delicate papillary structures or cribriform architecture may also be seen. The nuclei are variably pleomorphic and have a characteristic smudged, dense appearance. Mitotic figures and apoptotic bodies are rare. The changes are typically a focal finding and not associated with a mass lesion. A history of pregnancy or identifying a stromal decidual reaction is helpful in supporting a diagnosis of Arias-Stella reaction.

FIGURE 52.47 Arias-Stella reaction. Intraglandular tufts of cells with clear to oxyphilic cytoplasm and smudged nuclei.

Radiation Change Radiation change can lead to significant nuclear atypia that persists for more than a decade after treatment. Although knowledge of prior radiation therapy is an essential piece of information, it is often missing, especially for patients with a remote history of radiation therapy. Consequently, it is important to recognize morphologic features that are characteristic of radiation-induced atypia. Radiation therapy can lead to marked nuclear enlargement with multinucleated or bizarre forms, hyperchromasia, and prominent eosinophilic nucleoli (124) (Fig. 52.48). However, the cells lack nuclear crowding and have low N:C ratios with abundant eosinophilic cytoplasm that may be bubbly or vacuolated. The nuclei often appear smudged or degenerated, and mitotic activity is uncommon. The finding is usually focal with atypical cells intermixed with unremarkable endocervical cells and maintenance of normal glandular architecture. Stromal cells may also exhibit radiation atypia with similar nuclear changes, as well as fibrosis, hyalinization, edema, chronic inflammation, and calcifications. Vessels may appear ectatic with intimal thickening. If p16 is used to aid in distinguishing radiation atypia from an HPV-associated

endocervical adenocarcinoma, it is important to recognize that, although uncommon, diffuse strong nuclear and cytoplasmic staining of benign endocervical glands has been reported in postradiation cervical biopsies (125).

FIGURE 52.48 Radiation change. (A) Endocervical gland with enlarged, irregular nuclei and abundant eosinophilic cytoplasm; apoptosis is present, but no nuclear crowding or mitotic activity. (B) p16 stain is extensive but discontinuous, which should be interpreted as negative.

Viral Infection Although uncommon, cytomegalovirus (CMV) may affect the cervix in nonimmunocompromised women where it is detected as an incidental finding. Characteristic eosinophilic nuclear and cytoplasmic inclusions occur predominantly within endocervical cells but can also be seen in endothelial and stromal cells (126). Mild nuclear atypia, fibrin thrombi, and associated inflammation, including lymphoid follicles, may also be present.

MALIGNANT GLANDULAR LESIONS The incidence of endocervical adenocarcinoma and adenosquamous carcinoma has increased considerably, whereas the overall age-adjusted incidence of cervical squamous cell carcinoma has declined in countries such as the United States, Canada, and most European and some Asian countries that have implemented organized cervical cytology screening programs (127,128). Worldwide, 82% of endocervical adenocarcinomas are associated with HPV types 16 and 18; the next most common HPV type is 45 (129). A higher percentage of adenocarcinoma harbor HPV-18 as compared with HSIL where HPV-16 predominates (130). Detection of HPV-18 as a result of Pap cotesting is associated with precancerous lesions that are missed by cytology and that often represent glandular abnormalities (131). However, less common morphologic subtypes of cervical adenocarcinoma, such as gastric, clear cell, and mesonephric, are unrelated to HPV (132-134). The 2020 WHO Classification of Female Genital Tumours (fifth edition) divides cervical adenocarcinomas and AIS into those that are HPV-associated vs. HPV-independent (54). It is based on the International Endocervical Adenocarcinoma Criteria and Classification (IECC) system that resulted from an international, interinstitutional study of invasive

endocervical adenocarcinomas (135). Glandular dysplasia, previously defined by the WHO as glandular lesions with significant nuclear abnormalities that are more striking than benign glandular atypia but fall short of the criteria for AIS, is no longer recognized as an entity. It is a poorly reproducible diagnosis where the morphologic criteria and clinical implications of glandular dysplasia are not well defined (136). In the United Kingdom, the term cervical glandular intraepithelial neoplasia (CGIN) is in widespread use with low-grade CGIN corresponding to glandular dysplasia and high-grade CGIN to AIS (137). ENDOCERVICAL ADENOCARCINOMA IN SITU AIS of the cervix was first described in 1953 as a precursor lesion for endocervical adenocarcinomas (138). Although it occurs most commonly during the reproductive years (mean age, 37 years), it can also be seen in older or younger women, and as would be expected for a precursor lesion, it occurs in women aged 10 to 15 years younger than those with invasive endocervical adenocarcinomas (54). AIS is typically asymptomatic and detected during evaluation for an abnormal Pap test or as an incidental finding in a biopsy or excision of a squamous lesion. AIS often occurs high in the endocervical canal where it cannot be visualized by colposcopy. The use of new collection devices such as the cytobrush has increased the yield of endocervical cells for evaluation in cervical cytology specimens. However, despite the effectiveness of cervical cytology as a screening tool, the false-negative rate is 52% on average, with reported rates ranging from 0% to 94% (139). In the Kaiser Permanente Northern California population-based study, 17 of 27 endocervical adenocarcinomas were detected in women who were HPV positive but had negative Pap test results (140). Although HPV testing is more sensitive than cervical cytology (18), it will not detect glandular abnormalities due to endometrial carcinomas or HPV-independent endocervical carcinomas. However, HPV testing may be helpful in stratifying risk of endometrial cancer vs. cervical cancer, especially in women aged 50 years or older with AGCs detected by cervical cytology (141). Histology It is best to initially evaluate cervical biopsies on low scanning power (×4 or ×10) and to make a conscious effort to look at the endocervical glands, as well as the squamous mucosa. Frequently, so much focus is placed on evaluating the squamous mucosa that areas of AIS are missed. AIS usually occurs at or close to the transformation zone and often coexists with SIL. On scanning power, areas of AIS will be apparent as darker, more basophilic glands that abruptly transition from normal endocervical mucinous epithelium (Fig. 52.49). The abnormal glands are confined to the preexisting “normal” endocervical glandular architecture that, by itself, may be quite complicated with accentuation of the lobular architecture. Usually, both the surface and crypt epithelium are involved, but surface involvement may be absent. It was previously considered that both skip lesions and extension high up the endocervical canal were common in AIS, but although these sometimes occur, both are relatively uncommon (142).

FIGURE 52.49 Endocervical adenocarcinoma in situ (AIS). (A) Abrupt transition of normal endocervical mucosa (left) to AIS (right) with crowded, stratified nuclei and mucin depletion. (B) Positive p16 stain with continuous nuclear (required) and cytoplasmic staining of virtually every cell in the focus of AIS.

On higher power, the glands exhibit floating (or luminal) mitotic figures and apoptotic bodies, which are usually nonluminal (Fig. 52.50). The nuclei are enlarged and elongate with crowding and stratification, as well as hyperchromasia and fine to coarsely clumped chromatin. The cytoplasm is typically eosinophilic but may be pale and abundant. Focal intraglandular papillae and cribriform architecture may be seen with AIS, but if these changes are prominent and widespread, consideration should be given to the possibility of invasive adenocarcinoma (Fig. 52.51). AIS should exhibit smooth glandular contours and lack a desmoplastic reaction, although surrounding stromal inflammation may be present.

FIGURE 52.50 Endocervical adenocarcinoma in situ (AIS). (A) Enlarged and elongated nuclei with hyperchromasia, crowding, and stratification; floating mitotic figures are luminal, whereas apoptotic bodies are often nonluminal. (B) Ki-67 shows extensive nuclear staining in the areas of AIS but is essentially negative in the benign endocervical mucosa.

FIGURE 52.51 Endocervical adenocarcinoma in situ (AIS). (A) Focal intraglandular papillary or (B) cribriform architecture may be seen in AIS.

HPV-Associated. The majority of AIS diagnosed by cytology or cervical biopsy is HPVassociated. However, this may simply reflect lack of recognition of the more recently described HPV-independent AIS, as well as the fact that they can appear morphologically similar. S E . The term superficial or early AIS has been used for lesions confined to the surface mucosa and crypt openings (143). It is thought to represent an early form of AIS that occurs in a younger age group (mean, 26 years) and exhibits less pronounced nuclear atypia with rare to absent apoptotic bodies (Fig. 52.52). Given its superficial nature and less marked cytologic abnormalities, the lesion is easily missed if one does not specifically look for this entity. Immunohistochemical stain for p16 can be helpful in confirming the diagnosis (see section “Molecular Markers”).

FIGURE 52.52 Superficial (early) adenocarcinoma in situ (AIS). (A) The surface mucosa and crypt openings are involved by a single layer of abnormal glandular epithelium with crowded, stratified nuclei and mucin depletion; (B) p16 shows continuous, strong nuclear (required) and cytoplasmic staining that highlights the focus of AIS.

U T . The majority of AIS resembles endocervical-type mucinous adenocarcinoma, which is the most common variant referred to as usual type. However, AIS can be mucin depleted and resemble endometrioid adenocarcinoma but retain an

endocervical phenotype (see section “Molecular Markers”) (Fig. 52.53). When HPVassociated, these mucin-depleted AIS are classified as usual type.

FIGURE 52.53 Mucin-depleted adenocarcinoma in situ resembles endometrial adenocarcinoma due to marked depletion of intracytoplasmic mucin.

I

T . Intestinal differentiation in AIS is not rare and is often found adjacent to areas of usual-type AIS. It is characterized by prominent goblet cells and, less commonly, Paneth cells or neuroendocrine cells (144). Cellular stratification may be minimal, and the nuclear features of malignancy can be subtle because of compression by intracytoplasmic mucin globules (Fig. 52.54). Although the majority of intestinal AIS are associated with HPV, a subset occurring in older women has been shown to be unrelated to HPV and is postulated to occur through an alternate pathway (133,145).

FIGURE 52.54 Intestinal-type adenocarcinoma in situ (AIS). AIS with prominent goblet cells.

T C T . Tubal- or ciliated-type AIS is rare and difficult to diagnose because of admixed ciliated cells that are usually considered the hallmark of a benign process. Diagnosis relies on identifying unequivocal cytologic features of AIS, that is, enlarged, stratified nuclei with coarse chromatin and loss of polarity, mitotic figures, and apoptosis. Immunohistochemical stain for p16 may be helpful with diffuse nuclear positive p16, supporting a diagnosis of AIS. However, given the rarity of tubal-type AIS, it should be noted that its immunophenotype has not been studied. S M -P I L . Stratified mucin-producing intraepithelial lesion resembles HSIL but is distinguished by the presence of cells with cytoplasmic mucin in the form of discrete vacuoles or cytoplasmic clearing dispersed throughout the lower to middle epithelial layers (Fig. 52.55) (28,29). Mucicarmine stain can be helpful in highlighting the distribution of mucin-producing cells. Stratified mucinproducing intraepithelial lesion is thought to likely arise from reserve cells, but because it exhibits more glandular than squamous features by immunohistochemical and ultrastructural studies, it is classified as a variant of endocervical AIS. Areas of conventional HSIL and/or AIS are often present.

FIGURE 52.55 Stratified mucin-producing intraepithelial lesion. Stratified epithelium with peripheral cuff of basaloid cells and scattered cells with mucinous cytoplasm; classified as adenocarcinoma in situ.

HPV-Independent. With the recognition of HPV-independent adenocarcinomas as a distinct entity, more attention has been focused on identifying HPV-independent precursor lesions. HPV-independent AIS with gastric and, at times, intestinal (goblet cell) differentiation is termed gastric-type AIS (gAIS) (103,146). Normal endocervical glands are replaced by cells with distinct cell borders and abundant eosinophilic to pale pink cytoplasm or, less commonly, foamy or clear cytoplasm (Fig. 52.56). The cells resemble gastric foveolar epithelium. Goblet cells may also be present but typically involve less than half the cells. Nuclear atypia ranges from minimal to severe. Mitotic activity and apoptosis can be inconspicuous and require close examination to identify. In contrast to

lobular endocervical glandular hyperplasia, gAIS retains normal endocervical gland architecture.

FIGURE 52.56 Gastric-type adenocarcinoma in situ (gAIS). Human papillomavirus–independent gAIS with abundant pale pink cytoplasm and distinct cell borders.

Scoring System In 2003, Ioffe et al (136) proposed a scoring system for classifying noninvasive endocervical glandular lesions, which can be useful as an objective tool, especially when evaluating challenging cases (Table 52.6). Three components—nuclear stratification, nuclear atypia, and mitotic/apoptotic index—are graded and summed to yield the final score: score 0 to 3 = benign, 4 or 5 = endocervical glandular dysplasia, and 6 to 9 = AIS. Using this system, the authors agreed on the diagnosis of AIS in 95% of problematic cases (κ = 0.8), which was an improvement from 62% agreement (κ = 0.7) in the pretest phase. There was lower reproducibility for the diagnosis of glandular dysplasia (43%, κ = 0.6), but when these equivocal lesions were collapsed into the benign/reactive category, overall diagnostic concordance in distinguishing AIS (scores 6-9) from less than AIS (scores 0-5) was high (94%). The use of biomarkers such as p16 can be helpful in evaluating equivocal lesions and providing additional information that supports definitive classification of the lesion as benign or AIS (see section “Molecular Markers”). TABLE 52.6 Scoring System for Evaluating Noninvasive Endocervical Glandular Lesions Points

Nuclear Stratification (Scores 0-3)

Nuclear Atypia (Scores 0-3)

Enlargement

Hyperchromasia

Mitoses and Apoptosis (Scores 03) Pleomorphism

Nucleoli

Average per

Glanda 0

None

(−)

(−)

(−)

(−)

0

1

Mild

Slight

+

+

(−)

×3 normal

+++

+++

Prominent

>3.0

Total score: 0-3, benign; 4-5, equivocal; 6-9, endocervical adenocarcinoma in situ. aCount mitoses and apoptotic bodies in two most active glands; determine average per gland. +, minimal; ++, moderate; +++, marked. Adapted with permission from Ioffe OB, Sagae S, Moritani S, et al. Proposal of a new scoring scheme for the diagnosis of noninvasive endocervical glandular lesions. Am J Surg Pathol. 2003;27(4):452-460.

Differential Diagnosis Many of the entities discussed in the section “Benign Endocervical Glandular Lesions” can mimic AIS. The most likely to cause a diagnostic dilemma are tuboendometrioid metaplasia and superficial endometriosis. Other reactive changes and entities that can mimic AIS are discussed in the following sections. Postbiopsy Changes and Cautery Artifact. Following an endometrial biopsy or endocervical curettage, the endocervical glands may exhibit nuclear stratification with micropapillary architecture, squamoid change, hobnail cells, and/or mild cytologic atypia (147). Benign glands can become entrapped within submucosal fibrosis, and a granulation tissue response as well as surface erosion and fibrin deposition can also be seen. Thermal damage is often seen in LEEP specimens where the electrocautery causes distortion of the endocervical glands. The nuclei are elongated and stream, lending a crushed appearance. The cervical stroma appears homogenized and dense, and signetring alteration of the stroma may also be seen (148). In cases where cytologic detail is difficult to evaluate owing to extensive cautery artifact and there is concern for the possibility of a malignancy such as endocervical AIS, immunohistochemical stain for p16 can be useful because the distorted glands will maintain their characteristic staining patterns (Fig. 52.57).

FIGURE 52.57 Cautery artifact. (A) Distorted endocervical gland with elongated and streaming nuclei due to cautery. (B) Negative p16 stain with focal, discontinuous nuclear reactivity confirms the benign nature of the process.

Reactive Endocervical Cell Atypia. Reactive endocervical cells are notable for enlarged nuclei with prominent nucleoli and occasional multinucleation but low N:C ratios (Fig. 52.58). Inflammation may be sparse or absent. Mitotic figures can be seen, and Ki67 proliferation rate may be high, but the nuclear chromatin should remain fine and evenly dispersed. The lack of diffuse p16 immunohistochemical reactivity would also support a benign, reactive process.

FIGURE 52.58 Reactive endocervical cells. Enlarged nuclei with prominent nucleoli, low nuclear-tocytoplasmic ratios, and occasional multinucleation.

Mitotically Active Endocervical Cells. It is unusual to see mitotic figures in benign endocervical glands. However, increased mitotic activity can be found in some cases, raising concern for the possibility of very well-differentiated gastric-type adenocarcinoma. The benign nature of the glands can be discerned by normal gland contours, lack of

nuclear atypia, and lack of nuclear stratification. The presence of abnormal mitotic figures or nuclear atypia should raise concern for a well-differentiated adenocarcinoma. Molecular Markers Immunohistochemical stains may be useful in distinguishing benign endocervical glandular lesions from AIS. However, the stains have to be interpreted carefully and in the context of the morphologic findings because the results can overlap. p16 Immunohistochemistry. The most useful immunohistochemical stain is p16, which is a surrogate marker of high-risk HPV. Because the majority of AIS and endocervical adenocarcinomas are HPV-associated, p16 will be overexpressed as exhibited by continuous, strong nuclear reactivity involving virtually all the cells; cytoplasmic staining may or may not be present (Fig. 52.49). In contrast, benign lesions are typically negative or exhibit patchy (noncontinuous) reactivity that can be quite extensive in entities, such as tuboendometrioid metaplasia or endometriosis (Fig. 52.34). HPV-independent gAIS is also expected to be p16 negative with absent or patchy reactivity (146). Human Papillomavirus In Situ Hybridization. ISH for HPV RNA can be helpful when there is a discordance between the pattern of p16 reactivity and morphology, for example, extensive p16 reactivity in tuboendometrioid metaplasia or patchy p16 reactivity in a lesion that meets morphologic criteria for endocervical AIS. Positive signal is characterized by punctate dark brown signal in the cytoplasm and, sometimes, nucleus (42). Because positive signal can be focal, it is important to evaluate the slide at high power before concluding it is negative. Comparison with negative control tissue can be helpful when there is a question of background reactivity characterized by pale brown globules. Ki-67 Immunohistochemistry. Ki-67 (MIB-1) typically highlights a low proliferation index of less than 10% in tuboendometrioid metaplasia, whereas AIS has a much higher proliferation index, in excess of 30% (97,149). However, some AIS cases can have a low proliferation index, and endometriosis or tuboendometrioid metaplasia with prominent endometrioid differentiation can exhibit a high proliferation index. Consequently, Ki-67 (MIB-1) should not be used as a single marker but may be helpful as an adjunct to p16 in challenging cases. p53 Immunohistochemistry. p53 expression is aberrant in a subset of HPVindependent gAIS, exhibiting no staining (null phenotype; requires positive internal control) or diffusely positive nuclear reactivity (103,146). In contrast, HPV-associated usual-type AIS typically shows normal (wild-type) p53 expression with patchy reactivity. Other Markers. Other markers such as BCL2, PAX2, ProEx C, CEA, vimentin, and ER have been reported in literature as having some utility in distinguishing AIS from benign mimics (81,97,150-152). However, each stain has been shown to have overlap in the staining patterns of benign and malignant endocervical glandular lesions, limiting their reliability in an individual case.

Management AIS can exhibit minimal colposcopic changes, hampering the ability of the colposcopist to determine the extent of disease. After a biopsy diagnosis of AIS, a diagnostic excisional procedure (i.e., cold knife cone biopsy or large loop excision) is recommended to exclude the possibility of invasive cancer. For women who wish to maintain fertility, observation after conservative local excision with negative margins is an option. However, for women who have completed childbearing, total hysterectomy is recommended as the treatment of choice for AIS (23). INVASIVE ENDOCERVICAL ADENOCARCINOMA The majority (98%) of endocervical adenocarcinomas can be subclassified as HPVassociated and HPV-independent (Table 52.7) (54). HPV-associated adenocarcinomas encompass usual type, accounting for approximately 75% of cervical adenocarcinomas, and mucinous type, which is less common (~10%). HPV-independent adenocarcinomas include gastric type (10%-15%), clear cell (3%-4%), and mesonephric (2.0 mm, with or without positive pelvic nodes

DISTANT METASTASES M0

No distant metastases

M1

IVB

Distant metastases (including metastases to inguinal lymph nodes, intraperitoneal disease, or lung, liver, or bone metastases; excludes metastases to paraaortic lymph nodes, vagina, pelvic serosa, or adnexa)

International Federation of Gynecology and Obstetrics (FIGO) (2015), and from AJCC Cancer Staging Manual. 8th ed. Springer; 2017:666; reproduced with permission of SNCSC.

The most recent iterations of both staging systems have several important improvements from prior versions. First, there is no longer a recognized “in situ” (stage Tis) version of endometrial carcinoma: nonmyoinvasive cancers and those demonstrating less than 50% myometrial invasion are now collapsed into the IA/T1a stage. Endocervical mucosal involvement is also no longer considered significant for staging nor is peritoneal washing status. Finally, lymph node assessment has now been expanded to include isolated tumor cells (2.0 mm). Although these updates address some controversies in endometrial carcinoma staging, others persist. For instance, it is unclear whether lymphovascular involvement in ovarian vessels or cervical stroma merits upstaging. Existing guidelines suggest that this is not the case, but one can easily make a biologically sound argument that the contrary should be true. Furthermore, cervical stromal involvement remains poorly reproducible, and the staging of tumors confined to polyps presents challenges. Finally, attempts to subdivide stage III/T3 tumors into prognostically relevant subdivisions have been fraught, as variables such as tumor grade, histologic subtype, and the adequacy of nodal staging complicate the predictive power of these stage groupings (267). Myometrial Invasion Myometrial invasion is measured from the normal endometrial-myometrial junction to the point of deepest tumor infiltration and is compared to the overall uterine wall thickness to determine the percentage of myometrial involvement. Its assessment, therefore, requires a full-thickness section of the endometrium, myometrium, and uterine serosa. If the uterine wall thickness precludes submission in a single cassette, the section should be bisected and submitted in two cassettes. Myometrial invasion is a critical staging criterion as tumors with less than 50% invasion (including noninvasive tumors) are classified as IA/T1a, whereas those that infiltrate the outer half of the myometrium are staged as IB/T1b. Neither the most recent AJCC nor the current FIGO staging system recognizes an “in situ” or Tis stage for endometrial carcinoma. Notably, EIC should be considered T1a, even though true invasion is not appreciated; this is because these lesions can show malignant behavior with extrauterine spread even in the absence of overt infiltration (161,169,265,266,268). Although simple in concept, measuring myometrial invasion can be challenging in practice. These difficulties are reflected in the high degree of interobserver variability with afflicts pathologists’ assessment of myometrial invasion, with interpretative disagreements in approximately 30% of cases (269-271). These disagreements can usually be attributed to one of three causes: difficulty localizing the endometrial-myometrial junction, adenomyosis, and problematic patterns of invasion. Localization of the endometrial-myometrial junction is necessary for measuring myometrial invasion. The normal junction is somewhat irregular on close assessment but should be relatively even and smooth from low power. A normal junction also demonstrates intact stroma between its deepest glands and the underlying myometrium. Identifying the endometrial-myometrial junction becomes challenging, however, when normal architecture is disrupted or effaced by tumor. We often encounter bulky, exophytic tumors without adjacent normal areas to aid in orientation. In these cases, it is critical that tumor thickness not be conflated with depth of invasion, as most of the tumor growth may be within the endometrial cavity rather than into the myometrial wall. Even

if an exact measurement cannot be provided owing to distortion or loss of normal anatomy, we should endeavor to assess whether the invasion extends into the inner vs. the outer half for staging purposes. Importantly, the presence of tumor immediately adjacent to thick-walled blood vessels raises concern for outer half involvement. Myometrial invasion measurements may also be confounded by adenomyosis. Adenomyosis is defined as benign endometrium embedded within the myometrium. However, adenomyosis may be populated by carcinoma, and the cancerous glands should not be considered invasive in this setting (Figs. 53.83 and 53.84). Although this process is readily recognizable when the constituent glands are surrounded by obvious endometrial stroma, it may mimic invasive carcinoma when the stromal component becomes scant or indistinct. “Astromal adenomyosis” refers to cases where the glands have no apparent associated stroma. Hormonal influences may exacerbate the interpretative problems surrounding adenomyosis by rendering stroma eosinophilic and spindled and, therefore, hard to differentiate from the background myometrium. Adenomyosis can be particularly problematic during intraoperative assessments as frozensection artifacts can render both benign endometrial stroma and desmoplastic reactions supportive of invasion difficult to visualize on a gland-by-gland basis. Appreciating the background is critical in such cases: if adenomyosis is prominent throughout the myometrium or near a gland of interest, we should be cautious before classifying that gland as invasive. Conversely, we should worry about invasion if we see scattered glands involving the myometrium without evidence of associated adenomyosis or if adenomyosis is present but in a dissimilar geographic distribution. Importantly, when evaluating adenomyosis, benign cytology can help exclude invasion, but malignant cytology does not confirm invasion, as carcinoma can grow along preexisting adenomyosis without changing the tumor stage. In some cases, true invasion will emerge from an involved adenomyosis gland, and in such instances, the depth of invasion should be measured from the originating adenomyosis rather than the endometrial-myometrial junction (269,270). Unfortunately, immunohistochemical stains for stromal markers such as CD10 are limited value in this assessment as the myometrium may also be positive (272). Immunostaining for interferon-induced transmembrane protein 1 (IFITM1), however, has been shown to be a fairly sensitive and very specific marker for endometrial stroma in this setting and may be of value in challenging cases (273).

FIGURE 53.82 Mesonephric-like carcinoma. Eosinophilic intraluminal secretions are common in mesonephric-like carcinoma.

FIGURE 53.83 Adenomyosis involved by grade 1 endometrioid carcinoma. The adenomyosis in this case is populated and expanded by carcinoma and atypical hyperplasia. However, true myoinvasion can be excluded based on the presence of associated stroma.

FIGURE 53.84 Adenomyosis involved by an atypical endometrioid proliferation. This focus of adenomyosis shows scant residual normal glands and a florid proliferation of atypical glands. The atypical glands show focal cribriforming concerning for International Federation of Gynecologic and Obstetrics grade 1 endometrioid carcinoma but are bordered by a rind of endometrial stroma, supporting classification as involvement of adenomyosis rather than true invasion.

The pattern of invasion can also complicate staging assessments. Infiltrative tumors flaunting irregular glands and nests that elicit a desmoplastic response are easiest to evaluate. Broad, pushing tumors can be more problematic because they often eliminate the normal endometrial-

myometrial junction, as described earlier. In addition to these fairly common patterns, endometrioid carcinomas may show two other types of invasion that can complicate assessment for endometrioid tumors in particular: the diffusely infiltrative pattern and the MELF pattern. The diffusely infiltrative or adenoma malignum–like pattern (also called the “melter” or “melting” pattern) is difficult because its cytologically banal glands percolate throughout the stroma without provoking stromal changes, making differentiation from stroma-poor adenomyosis complicated (134). In such tumors, the extensive nature of the proliferation represents an important clue to its malignant nature. The MELF pattern displays small, jagged glands with attenuated lining and intraluminal sloughed tumor cells, which often mimic the appearance of lymphovascular invasion. The presence of a surrounding myxoinflammatory stromal reaction helps confirm that these are indeed glands infiltrating the myometrium rather than involved vessels (137,274-277). These patterns are discussed in further detail in the section “Endometrioid Carcinoma Patterns of Invasion.” Intraoperative Evaluation Intraoperative assessments of endometrial carcinomas are done primarily to evaluate whether or not staging should be performed and include a frozen section evaluation of the tumor histology and depth of invasion. Staging via lymphadenectomy is indicated for all tumors with high-risk histologic features, including serous carcinomas, clear cell carcinomas, FIGO grade 3 endometrioid carcinomas, undifferentiated/dedifferentiated carcinomas, and carcinosarcomas. For FIGO grade 1 to 2 endometrioid tumors, forgoing lymph node dissection can be considered in some patients, provided the tumor is restricted to the inner half of the myometrium. Outer half involvement should trigger strong consideration for staging even for low- to intermediate-grade tumors because this suggests that these malignancies have a higher risk of extrauterine spread. Frozen section results provide accurate data and appropriately guide surgical therapy in the majority of cases; however, approximately 5% of the time the final interpretation will differ significantly from the diagnosis rendered on frozen (278,279). Disagreements most often hinge on the depth of myometrial invasion owing to the problems that can attend localization of the endometrial-myometrial junction, differentiating adenomyosis from invasion, and recognizing problematic patterns of invasion. These issues, which are detailed earlier, can all be exacerbated by frozen-section artifact. Furthermore, nuclear features can be difficult to appreciate through frozen artifact, and a tumor that appeared to be endometrioid at the time of surgery may occasionally prove serous on permanent sections. Finally, frozen section evaluation is inherently limited by sampling, and review of the permanent material may demonstrate areas of increased solid growth or regions of deeper invasion, which were not appreciated on initial sections. Thus, although intraoperative evaluation by frozen section is critical for managing patients with endometrial carcinoma, it should be approached with caution and an awareness of common pitfalls. If FIGO 1 and FIGO 2 prove close to the 50% invasion threshold on initial sections, one could consider submitting additional areas of potential invasion for intraoperative review. When in doubt, however, a conversation with the surgeon discussing areas of uncertainty is always prudent, as their appreciation for the patient’s preferences and complete clinical picture may inform your actions. For instance, if you are concerned that a low-grade endometrioid tumor approaches the outer half of the endometrium but the surgeon does not plan to perform a lymphadenectomy for borderline cases because of the patient’s comorbidities, then you can save yourself sweat and spare the patient additional time under anesthesia. Cervical Invasion Cervical invasion is defined as infiltration of the cervical stroma and upstages the tumor to stage T2/II. Importantly, under current staging systems, involvement of the endocervical mucosa does not qualify as cervical invasion (265,280). It can be challenging to differentiate colonization of

endocervical glands from true stromal involvement. If the tumor appears in a similar distribution to the background endocervical glands and does not invoke a desmoplastic response, it likely represents intraglandular extension rather than invasion. Parametrial Invasion Parametrial invasion occurs when tumor infiltrates the fibrofatty connective tissue that surrounds the uterus. The parametria are typically only collected in radical hysterectomies and are associated with a true surgical margin. Although radical hysterectomies are most often performed for cervical carcinoma, they may occasionally also be done for endometrial cancer, particularly when it involves the endocervix. Parametrial involvement is a negative prognostic risk factor for endometrial carcinoma (281-283). Lymphovascular Invasion Lymphovascular invasion is characterized by pathologic tumor deposits within lymphatics or blood vessels. Lymphovascular invasion occurs in around one-quarter of endometrial cancers and is more common in high-grade tumors (284-286). It serves as an independent risk factor for low-grade cancers and may inform clinical therapy for these cases (284,286). Although lymphovascular invasion often appears as cohesive clusters of tumor cells within vessels, malignant cells may also invade singly and appear admixed with native hematopoietic elements (the “histiocytoid” pattern of lymphovascular invasion). Assessment for nuclear features and cytologic atypia is critical to avoid missing such cases, and immunostaining to confirm the epithelial nature of the cells of interest may be warranted. It may sometimes be challenging to distinguish true lymphovascular invasion from technical artifacts (pseudoinvasion). Tumor may be artificially implanted in vessels during laparoscopic hysterectomy as well as at the grossing bench (287,288). The presence of detached tumor fragments in large vessels, particularly those near a cut edge, is highly suggestive of gross knife artifact. Adherence of the tumor deposit to the vessel wall or conformation of the deposit to the shape of the vascular space suggests true invasion over artifactual implantation. Histologic mimics of lymphovascular invasion can also present challenges: for instance, tumoradjacent stroma often retracts, creating a cleft around the tumor that falsely suggests a vascular lumen. Retraction artifact can be excluded by the identification of hematopoietic elements and the identification of an endothelial lining, which can be confirmed by vascular or lymphovascular immunohistochemical markers such as CD31, D2-40, or ERG. The identification of adjacent blood vessels also favors lymphatic involvement over retraction, as lymphatics travel in parallel with veins. The MELF pattern of invasion also frequently masquerades as lymphovascular invasion due to the attenuation of the malignant lining cells and sloughing of tumor cells into the gland lumen. In these cases, the characteristic surrounding fibromyxoid stroma helps classify the lesion as MELF invasion, rather than lymphovascular involvement. Lymph Node Involvement Lymph node involvement is a critical component of endometrial cancer staging (265,280) (Table 53.4). The most recent iteration of the AJCC staging system subdivides nodal involvement on the basis of size, with metastases greater than 0.2 mm but less than 2 mm qualifying as micrometastases and warranting designation as either N1mi (for pelvic nodes) and N2mic (for paraaortic nodes). Metastatic deposits of or exceeding 2 mm are considered macrometastatic and do not receive any special designation. Involvement of less than 0.2 mm is designated as isolated tumor cells and should be designated “pN0i+.” Importantly, isolated tumor cells do not impact stage groupings.

Peritoneal Washings Peritoneal washing status does not impact stage under the current AJCC and FIGO staging systems (265,280). This stems from the fact that positive washings have not been shown to independently impact prognosis for endometrial cancers overall, although there is an association between positive washings and poor outcome in high-grade, nonendometrioid tumor subtypes (289-293). For low- and intermediate-grade endometrioid tumors, tumor cells identified in fluid do not seem to bear the biologic potential for implantation and, in many cases, are postulated to be present as a result of uterine manipulation (294). Margin Status In total hysterectomy specimens, the paracervical soft-tissue margin represents the only true surgical margin. In radical hysterectomies, the vaginal and parametrial margins are true margins. ENDOMETRIAL CARCINOMA ASSOCIATED WITH OVARIAN CARCINOMA It is not uncommon for endometrial and ovarian cancers to occur simultaneously, provoking questions about whether they represent synchronous primaries vs. metastases. Differentiating between the two can be challenging when the tumors have similar histology and the diagnostic approach is informed by whether the histology is endometrioid or serous. Simultaneous primaries of other histotypes are exceedingly rare. Endometrial and Ovarian Endometrioid Tumors Ovarian endometrioid carcinomas occur in tandem with endometrial endometrioid carcinomas in as many as 20% of cases, and the majority of these tumors represent dual primaries. Most synchronous primaries are due to somatic causes and tied to an estrogenic field effect, which is evidenced by the high body mass index, frequent nulliparity, and young age of many women with synchronous ovarian and endometrial endometrioid primaries (295-297). Lynch syndrome underlies only a small subset (