Pathology: Historical and Contemporary Aspects 3031395530, 9783031395536

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Ricardo V. Lloyd

Pathology: Historical and Contemporary Aspects

Pathology: Historical and Contemporary Aspects

Ricardo V. Lloyd

Pathology: Historical and Contemporary Aspects

Ricardo V. Lloyd Department of Pathology & Lab Medicine University of Wisconsin–Madison Madison, WI, USA

ISBN 978-3-031-39553-6    ISBN 978-3-031-39554-3 (eBook) https://doi.org/10.1007/978-3-031-39554-3 © The Editor(s) (if applicable) and The Author(s), under exclusive license to Springer Nature Switzerland AG 2023, Corrected Publication 2023 This work is subject to copyright. All rights are solely and exclusively licensed by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. The publisher, the authors, and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, expressed or implied, with respect to the material contained herein or for any errors or omissions that may have been made. The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. This Springer imprint is published by the registered company Springer Nature Switzerland AG The registered company address is: Gewerbestrasse 11, 6330 Cham, Switzerland Paper in this product is recyclable.

Prologue

Pathology is a branch of medical science that has evolved extensively over the past few centuries. The traditional study of human diseases was done by the pathologist at the time of autopsy. This approach has been practiced for many centuries and has led to major advances in our knowledge about disease processes. However, for the past 60–70 years, new knowledge in pathology, as in many other areas of medicine, has been rapidly advancing. These advances including developments in diagnostic surgical pathology and clinical pathology or laboratory medicine have led to further specialization by practitioners of pathology into specific areas of the field. Today most surgical pathologists in academic institutions specialize in one to three areas of surgical pathology so that they can maintain their expertise in these areas of the field. Forensic pathologists concentrate mainly on forensic autopsy cases, and some of these doctors also perform general autopsies. In surgical pathology, expertise in a limited number of subspecialty fields is partially motivated by the explosion of knowledge in surgical pathology and the increase in the number of limited biopsies in which only a few cells are available for diagnosis. The pathologist needs to use many immunohistochemical and other special stains and molecular diagnostic techniques to narrow the differential diagnosis and to arrive at a specific diagnosis whenever possible. Surgical pathology in the twenty-first century has been made more challenging by clinical colleagues who desire more specific diagnoses even as the number of tissues provided from biopsies specimens continue to decrease in size. Today many biopsies of cancers require a minimum of a few special stains, especially immunohistochemical stains, for diagnosis. In addition, molecular diagnosis may be needed to support a precise diagnosis or for targeted therapy of a disease process as well as for guiding patient treatment. The surgical pathologist has to be familiar with the hundreds of antibodies available in the diagnostic laboratory and the specificity and limitations of these antibodies in assisting to render a precise diagnosis which will determine the type of treatment for individual patients. Rapid developments in diagnostic molecular pathology and digital pathology have provided new challenges for the practicing pathologist who has to develop and maintain expertise in their subspecialty areas.

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Prologue

These new developments have provided a great deal of challenges and excitement in the field for general pathologist. In small community practices in the USA, general pathologists make diagnosis in most areas of surgical pathology (including more than 15 subspecialty) areas. However, these practitioners of diagnostic surgical pathology often wisely share their difficult cases with their colleagues in the same institution or send the case out for consultation to an expert pathologist in another part of the country. These experts are usually widely recognized for their knowledge in a particular field. Advances and continued developments in subspecialty areas of anatomic and clinical pathology, especially in diagnostic surgical pathology, guided by frequent publications by the World Health Organization (WHO) and other influential organizations about tumor classification, have served as a guide for pathologists to remain on top of their field. In addition, attendance at one or more national or international meetings a year provides the background knowledge that is needed to remain current in specific areas of diagnostic pathology. This book presents an overview of specific subspecialty areas of diagnostic pathology including clinical pathology. It is not intended to be used as a comprehensive textbook of diagnostic pathology, but to provide a flavor of past achievements, current practices, and anticipated future developments. The topics discussed are more general in anatomic and clinical pathology and cover only a small fraction of the current knowledge used by experts in these various areas of pathology. This approach provides a flavor of the history and dynamic developments and recent changes in the field. A selected group of illustrations is included in some of the chapters. These illustrations are mainly microscopic examples of histological diagnoses that enable the pathologist to diagnose some specific disease entities. Histology is the basic language of pathology, so a reader without this background may have some difficulties recognizing the pathologic changes. However, even the student who has not had a course in histology should be able to appreciate the changes in the photomicrographs that provide a basis for making specific diagnoses, especially when an illustration of the normal histology is included. A few immunohistochemical images using specific antibodies and historical histochemical stains are also included to demonstrate how these special techniques assist in making a diagnosis. Diagnostic surgical pathology has changed greatly during the past few decades. More than half a century ago, surgical pathology was part of surgery departments. This field was subsequently merged with general pathology laboratories which combined autopsy pathology with clinical laboratory medicine and surgical pathology. This latter subspecialty has continued to make diagnoses on resected organs and other tissues but has been greatly expanded to include small tissue core biopsies as well as cytological examination using a few aspirated cells in order to arrive at specific diagnoses. The explosion of knowledge which began around the 1950s started slowly as electron microscopy became a useful adjunct diagnostic tool that was very helpful in diagnostic surgical pathology. Diagnostic immunohistochemistry became very useful in the 1970s with the development of monoclonal antibodies

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that worked well in formalin-fixed paraffin-embedded tissue, the main laboratory currency of diagnostic anatomic pathology. The advances in diagnostic pathology have occurred in concert with advances in other areas of medicine such as radiology and oncology in which sophisticated techniques for diagnosing and treating various diseases have continued to advance at a rapid pace. The field of pathology attracts many types of medical students to specialize in this area of medicine. Although a pathologist is usually depicted as an individual who is introverted with a reserved personality, many other personality types, including extroverts, decide to pursue a career in this field. Surveys show that practicing pathologist retain a great deal of personal satisfaction with their chosen specialty field after many decades of practice. Professional burnout is less common in this field of practice compared to many other areas of medical subspecialty. This book is written with several groups in mind. Many chapters will be of interest to the general pathologist in anatomic and clinical pathology, especially the chapters dealing with the historical aspects of the profession. Other groups including medical students who are examining various possible specialty areas may be attracted by the intellectual stimulation from the vastness of pathology and the possibility of practicing medicine or doing research in many areas of this field. Undergraduate students interested in biology and in the allied health fields will find many aspects of the book of interest for acquiring a broad overview of the discipline. Graduate students in pathology and closely related fields will be interested in some chapters of the book, such as basic concepts in disease diagnosis as well as the discussions about Nobel laureates who have contributed significantly to progress in medical sciences and pathology.

Acknowledgments

I would like to thank many of my colleagues who generously provided glass slides that I used to make some of the figures for the book. They include: Drs. Rashmi Agni, Shahriar Salamat, Daniel Matson, Jefree Schulte, Jin Xu, and Weixiong Zhong. This book is dedicated to my wife Debbie Lloyd for her encouragement and assistance in creating the book. The encouragement of our son and his family (Vincent, Dana, Anya and Leroy) is greatly appreciated.

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Contents

1

Introduction��������������������������������������������������������������������������������������������     1 Pathology and Subspecialty Areas ����������������������������������������������������������     1 References������������������������������������������������������������������������������������������������     4

2

Historical Aspects of Pathology������������������������������������������������������������     5 Brief History of Islamic Pathology����������������������������������������������������������     9 Brief History of Eastern Pathology����������������������������������������������������������    10 References������������������������������������������������������������������������������������������������    12

3

Surgical Pathology ��������������������������������������������������������������������������������    15 James Ewing (1866–1943)����������������������������������������������������������������������    19 Arthur Purdy Stout (1885–1967) ������������������������������������������������������������    21 Juan Rosai (1940–2020)��������������������������������������������������������������������������    22 References������������������������������������������������������������������������������������������������    24

4

Cytopathology����������������������������������������������������������������������������������������    27 George Papanicolaou (1883–1962)����������������������������������������������������������    29 Leopold G. Koss (1920–2012)����������������������������������������������������������������    30 References������������������������������������������������������������������������������������������������    32

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 Autopsy and Forensic Pathology����������������������������������������������������������    35 DiMaio, Vincent JM (1941–2022) ����������������������������������������������������������    40 Bennet I. Omalu (Born 1968)������������������������������������������������������������������    41 References������������������������������������������������������������������������������������������������    42

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Gynecologic Pathology��������������������������������������������������������������������������    45 Clear Cell Adenocarcinoma of the Vagina and Cervix����������������������������    45 Carcinoma of the Cervix��������������������������������������������������������������������������    46 Endometrial Adenocarcinoma������������������������������������������������������������������    47 Serous Ovarian Cancers ��������������������������������������������������������������������������    48 Endometriosis������������������������������������������������������������������������������������������    51

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Robert E. Scully (1921–2012) ����������������������������������������������������������������    52 Robert Kurman (Born 1943)��������������������������������������������������������������������    54 References������������������������������������������������������������������������������������������������    55 7

Breast Pathology������������������������������������������������������������������������������������    57 Benign Breast Lesions ����������������������������������������������������������������������������    57 Breast Carcinomas ����������������������������������������������������������������������������������    58 Ductal Carcinoma In Situ������������������������������������������������������������������������    58 Lobular Carcinoma In Situ����������������������������������������������������������������������    60 Invasive Ductal Carcinoma����������������������������������������������������������������������    61 Invasive Lobular Carcinoma��������������������������������������������������������������������    62 Molecular Classification of Breast Cancer����������������������������������������������    63 John G Azzopardi (1924–2013) ��������������������������������������������������������������    64 References������������������������������������������������������������������������������������������������    65

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Genitourinary Pathology����������������������������������������������������������������������    67 Prostate Adenocarcinoma������������������������������������������������������������������������    67 Incidental Prostate Cancers����������������������������������������������������������������������    69 Bladder and Urothelial Tract Tumors������������������������������������������������������    70 Renal Cell Carcinomas����������������������������������������������������������������������������    71 Clear Cell Papillary Renal Cell Carcinoma ��������������������������������������������    72 Donald F. Gleason (1920–2008)��������������������������������������������������������������    73 Fathollah Keshvar Mostofi (1911–2003) ������������������������������������������������    74 References������������������������������������������������������������������������������������������������    76

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Medical Kidney Diseases ����������������������������������������������������������������������    77 Examples of Well-Characterized Renal Glomerular Diseases����������������    78 Minimal Change Disease ��������������������������������������������������������������������    78 Acute Proliferative (Post-Streptococcal) Glomerulonephritis ����������������    79 Membranoproliferative Disease��������������������������������������������������������������    80 Diabetic Nephropathy������������������������������������������������������������������������������    81 Dr. Robert Hodgson Heptinstall (1920–2021) ����������������������������������������    82 References������������������������������������������������������������������������������������������������    84

10 Gastrointestinal  Tract, Liver, and Pancreas����������������������������������������    85 Helicobacter Pylori Infection������������������������������������������������������������������    85 Large Intestinal Polyps����������������������������������������������������������������������������    86 Colonic Adenocarcinomas ����������������������������������������������������������������������    89 Liver Diseases������������������������������������������������������������������������������������������    90 Alcoholic Liver Disease��������������������������������������������������������������������������    90 Viral Hepatitis������������������������������������������������������������������������������������������    92 Hepatocellular Carcinoma ����������������������������������������������������������������������    92 Pancreatic Neoplasms������������������������������������������������������������������������������    93 Neuroendocrine Tumors of the Pancreas ������������������������������������������������    94 Morison, Basil Clifford (1921–2016)������������������������������������������������������    96 Popper, Hans (1903–1988)����������������������������������������������������������������������    96 References������������������������������������������������������������������������������������������������    97

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11 Lung Pathology��������������������������������������������������������������������������������������   101 Acute Pneumonia ������������������������������������������������������������������������������������   101 Viral Pneumonia��������������������������������������������������������������������������������������   102 Influenza Types A, B, and C Pneumonia ������������������������������������������������   102 Sars-CoV-2 Pneumonia����������������������������������������������������������������������������   103 Chronic Pneumonia����������������������������������������������������������������������������������   103 Interstitial Pulmonary Fibrosis (IPF) ������������������������������������������������������   103 Lung Neoplasms��������������������������������������������������������������������������������������   105 Squamous Cell Carcinoma����������������������������������������������������������������������   105 Adenocarcinomas������������������������������������������������������������������������������������   106 Lung Neuroendocrine Tumors ����������������������������������������������������������������   107 Mesotheliomas ����������������������������������������������������������������������������������������   110 Outstanding Pulmonary Pathologist��������������������������������������������������������   110 Herbert Spencer (1915–1993)��������������������������������������������������������������   110 Avrill / Abraham Liebow (1911–1978)������������������������������������������������   112 References������������������������������������������������������������������������������������������������   113 12 Neuropathology��������������������������������������������������������������������������������������   115 Alzheimer’s Disease��������������������������������������������������������������������������������   115 Chronic Traumatic Encephalopathy��������������������������������������������������������   116 Brain Tumors ������������������������������������������������������������������������������������������   117 Meningiomas��������������������������������������������������������������������������������������������   119 Pituitary Tumors��������������������������������������������������������������������������������������   120 Prion Diseases������������������������������������������������������������������������������������������   122 Lucien J Rubinstein (1924–1990)��������������������������������������������������������   123 Bernd W. Scheithauer (1946–2011)����������������������������������������������������   124 References������������������������������������������������������������������������������������������������   125 13 Endocrine Pathology������������������������������������������������������������������������������   127 Hashimoto’s Thyroiditis��������������������������������������������������������������������������   128 Follicular Neoplasms of Thyroid ������������������������������������������������������������   129 Follicular Adenoma������������������������������������������������������������������������������   129 Follicular Carcinomas��������������������������������������������������������������������������   130 Papillary Thyroid Carcinoma��������������������������������������������������������������   131 Anaplastic Thyroid Carcinoma������������������������������������������������������������   132 Medullary Thyroid Carcinoma������������������������������������������������������������   133 Adrenals ��������������������������������������������������������������������������������������������������   134 Adrenal Cortex����������������������������������������������������������������������������������������   134 Adrenal Cortical Hyperplasia��������������������������������������������������������������   134 Adrenal Cortical Adenomas����������������������������������������������������������������   135 Adrenal Cortical Carcinoma����������������������������������������������������������������   136 Pheochromocytomas��������������������������������������������������������������������������������   137 Paragangliomas����������������������������������������������������������������������������������������   138 Anthony GE Pearse (1916–2003)������������������������������������������������������������   139 J. Aidan Carney (Born 1934) ������������������������������������������������������������������   140 References������������������������������������������������������������������������������������������������   143

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14 Hematopathology ����������������������������������������������������������������������������������   145 Karl Lennert (1921–2012) ����������������������������������������������������������������������   150 Robert Lukes (1922–1999)����������������������������������������������������������������������   152 References������������������������������������������������������������������������������������������������   153 15 Introduction to Microbiology����������������������������������������������������������������   155 Vaccinations ��������������������������������������������������������������������������������������������   156 Louis Pasteur (1822–1895)����������������������������������������������������������������������   157 References������������������������������������������������������������������������������������������������   159 16 Virology��������������������������������������������������������������������������������������������������   161 Some Common and Uncommon Viral Infections������������������������������������   162 Orthomyxovirus ����������������������������������������������������������������������������������   162 Paramyxovirus ������������������������������������������������������������������������������������   163 Measles Virus (Rubeola)����������������������������������������������������������������������   163 Respiratory Syncytial Virus ����������������������������������������������������������������   163 Picornavirus ����������������������������������������������������������������������������������������   163 Gastroenteritis Viruses ������������������������������������������������������������������������   163 Uncommon Viral Infections��������������������������������������������������������������������   164 Rhabdovirus ����������������������������������������������������������������������������������������   164 Retrovirus��������������������������������������������������������������������������������������������   164 Coronavirus������������������������������������������������������������������������������������������   165 SARS-CoV-2 (COV-19)����������������������������������������������������������������������   165 The 1918 Influenza Pandemic and Comparison with the 2019 COVID-19 Pandemic��������������������������������������������������������������������   166 Comparison of 1918 and 2019 Pandemics����������������������������������������������   168 Viruses and Cancer����������������������������������������������������������������������������������   169 References������������������������������������������������������������������������������������������������   170 17 Bacteriology��������������������������������������������������������������������������������������������   173 Tuberculosis ��������������������������������������������������������������������������������������������   174 Infection with Spirochetes ����������������������������������������������������������������������   175 Lyme Disease ������������������������������������������������������������������������������������������   176 Yersinia- and Vibrio Cholerae-Associated Historical Pandemics��������   177 References������������������������������������������������������������������������������������������������   179 18 Mycology and Parasitology ������������������������������������������������������������������   181 Fungal Infections ������������������������������������������������������������������������������������   181 Candida Species ��������������������������������������������������������������������������������������   182 Histoplasma Species��������������������������������������������������������������������������������   182 Aspergillus Species����������������������������������������������������������������������������������   183 Blastomyces ��������������������������������������������������������������������������������������������   184 Parasitology����������������������������������������������������������������������������������������������   184 Tapeworms ����������������������������������������������������������������������������������������������   185 Malaria ����������������������������������������������������������������������������������������������������   185 References������������������������������������������������������������������������������������������������   186

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19 Clinical Chemistry ��������������������������������������������������������������������������������   189 A Few Specific Areas of Analyses in Clinical Chemistry������������������������   191 Cardiac Troponin ��������������������������������������������������������������������������������   191 Glycosylated Hemoglobin ������������������������������������������������������������������   192 Evaluation of Liver Function in Clinical Chemistry����������������������������   192 Alcoholic Liver Disease����������������������������������������������������������������������   193 Intraoperative Parathyroid Hormone Assessment��������������������������������   193 References������������������������������������������������������������������������������������������������   195 20 Transfusion  Medicine and Hemostasis������������������������������������������������   197 Preparation of Blood Components����������������������������������������������������������   198 Therapeutic Uses of Blood Components ������������������������������������������������   198 Therapeutic Plasma Exchange (Apheresis) ��������������������������������������������   198 Examples of Specific Diseases Requiring Transfusion Medical Intervention ����������������������������������������������������������������������������������   199 Hemophilia A and B����������������������������������������������������������������������������   199 Sickle-Cell Disease������������������������������������������������������������������������������   200 von Willebrand Disease ����������������������������������������������������������������������   201 References������������������������������������������������������������������������������������������������   202 21 Outstanding Women in Pathology��������������������������������������������������������   205 Maude Elizabeth Seymour Abbott (1869–1940) ������������������������������������   206 Dorothy Reed (1874–1964) ��������������������������������������������������������������������   207 Dorothy Russell (1895–1983)������������������������������������������������������������������   208 Emma Sadler Moss (1898–1970)������������������������������������������������������������   209 Sophie Spitz (1910–1956)������������������������������������������������������������������������   210 Dr. Alexandra Piringer-Kuchinka (1912–2004)��������������������������������������   211 Lotte Strauss (1913–1985)����������������������������������������������������������������������   211 Enid Gilbert-Barness (1927–2022)����������������������������������������������������������   212 Dr. Margaret Evelyn Billingham (1939–2009)����������������������������������������   213 Julia Margaret Polak (1934–2014)����������������������������������������������������������   214 Elaine Sarkin Jaffe (Born 1943)��������������������������������������������������������������   215 Virginia LiVolsi (Born 1943) ������������������������������������������������������������������   216 Sharon Ann Whelan Weiss (Born 1945)��������������������������������������������������   217 Fattaneh Tavassoli (Born 1949) ��������������������������������������������������������������   218 References������������������������������������������������������������������������������������������������   219 22 Nobel  Laureates in Pathology ��������������������������������������������������������������   223 Karl Landsteiner (1868–1943)����������������������������������������������������������������   224 Johannes Fibiger (1867–1928)����������������������������������������������������������������   225 George Hoyt Whipple (1878–1976)��������������������������������������������������������   226 Renato Dulbecco (1914–2012)����������������������������������������������������������������   228 Baruj Benacerraf (1920–2011)����������������������������������������������������������������   229 John Robin Warren (Born 1937)��������������������������������������������������������������   230 References������������������������������������������������������������������������������������������������   232

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23 Future  Directions in Pathology ������������������������������������������������������������   235 Molecular Immunohistochemistry ����������������������������������������������������������   235 Multiplex Immunohistochemistry and in Situ Hybridization������������������   237 Molecular Diagnostics ����������������������������������������������������������������������������   238 Digital Pathology ������������������������������������������������������������������������������������   240 References������������������������������������������������������������������������������������������������   242 Correction to: Breast Pathology ������������������������������������������������������������������   C1 Appendices������������������������������������������������������������������������������������������������������   245 Index����������������������������������������������������������������������������������������������������������������   251

Chapter 1

Introduction

Pathology and Subspecialty Areas Pathologists study cells, tissues, and body fluids and make diagnoses based on the findings in these tissues and fluids. This makes pathology a unique branch of medicine. Because anatomic diagnostic pathologists do not see patients, they use all of the available information including patient history, physical examination results, family history, and radiological findings to arrive at the most definitive diagnosis based on the tissue biopsy or resected specimen. The diagnosis established by the surgical pathologist serves as guides for the treatment and prognosis of many human diseases. Training to be a pathologist in the USA involves a very long and rigorous road. After 4 years of college and another 4 years of medical school, the graduating doctor who decides to go into pathology usually does 4 years of residency training in anatomic and clinical pathology (a few pathologists may decide to train in only one of these areas, especially if they plan to go into academic pathology or to do research). Some individuals may decide to spend a few years getting formal training in experimental pathology. After completing residency training, the young pathologist takes an exam given by the American Board of Pathology to become certified in anatomic and/or clinical pathology. Most pathologists then go on to spend between 1 and 3 years getting further training in one or more subspecialty areas of pathology such as neuropathology, hematopathology, gastrointestinal, renal, genitourinary, gynecological, breast, pulmonary, soft tissue, head and neck, or molecular pathology. They may then take another board examination given by the American Board of Pathology in some of these subspecialty areas, such as neuropathology, hematopathology, microbiology, or cytopathology, to become board-certified in a subspecialty area. (Many subspecialty areas in anatomic pathology do not have board-certification as yet.) The pathologist is then prepared to get a job in a

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subspecialty area of pathology or to be a general pathologist who is board-certified in anatomic and clinical pathology. The two main areas of diagnostic pathology include anatomic and clinical pathology. However, there are many subspecialty areas in pathology. The anatomic pathologist analyzes cells and tissues from biopsies or resected organs to determine the diagnosis based on the histological interpretation of the specimen using the light microscope. Autopsy pathology, which examines tissues and body fluids after death, is important in determining the cause of death and for making diagnoses that were not made antemortem. Such diagnoses can be helpful in treating living patients. An example of this application is when infectious diseases are first uncovered in the postmortem period if the deceased patient is found to have undiagnosed tuberculosis at the time of autopsy, all individuals that came in contact with the patient while he/she was alive in the hospital should be tested for the possibility of infection with tuberculosis. Another important aspect of the autopsy would be to diagnose specific genetic diseases and this information can be used to work up, treat, or follow relatives of the deceased person that did not know that they had inherited these mutated genes and had the potential to develop specific diseases before the autopsy was performed. An example of a rare familial disease that may be discovered at autopsy is a thyroid cancer known as medullary thyroid carcinoma [1]. Between 5% and 20% of these cancers are inherited in an autosomal-dominant manner. If one of these cancers is discovered at autopsy, one can examine living relatives by a blood test to determine if they have inherited the genetic mutation that may lead to the development of this particular cancer. Another important part of autopsy pathology is forensic pathology which examines the deceased patient to determine the cause and manner of death. The forensic pathologist investigates causes of unexpected death, sudden death, and violent deaths to determine the manner and cause of death such as homicide or suicide. Forensic pathologists may spend a great deal of time in court testifying in medicolegal cases. Another major area of anatomic pathology is pediatric pathology. Since children are more than simply smaller adults, many of the diseases they develop may be relatively unique for their age group. Pediatric surgical pathologists examine tumors and other conditions that are relatively unique to pediatric patients. Examples of these conditions would be some cancers such as Wilms tumors of the kidney, neuroblastomas of the adrenal glands, and Ewing sarcoma of bone that occurs predominantly in pediatric patients. Pediatric autopsy pathology concentrates on perinatal and intrauterine disorders affecting neonates and children. Heart defects are not uncommonly seen in pediatric patients and a great many advances have been made in pediatric cardiac surgery by historical studies done on these defects at the time of autopsy in previous years. A condition known as tetralogy of Fallot is an example of a complex cardiac malformation that was studied extensively by pediatric pathologists, but is now treated surgically by cardiovascular surgeons in most children born today in developed countries with this defect [2].

Pathology and Subspecialty Areas

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Cytopathology is another subspecialty area of anatomic pathology. This field examines single cells or clusters of cells to make specific diagnoses about a disease process. Because a biopsy is usually done in a noninvasive manner, only a few cells are obtained for the analysis. And in most cases, the side effects from the biopsy are minimal. Cytopathology specimens are more difficult and more challenging to interpret, but are often less accurate than other areas of anatomic pathology such as surgical pathology, because of the small number of cells available for analysis in the typical specimen. In addition, the presence of tissue architecture in the small group of cells that can serve as a guide in formulating a diagnosis as done with surgical specimens or tissue biopsies is not available to the cytopathologist. Clinical pathology (also known as laboratory medicine) consists of multiple subspecialty areas including microbiology, clinical chemistry, transfusion medicine, molecular pathology, and hematopathology. Microbiology includes virology, the study of viruses involved in disease causation; bacteriology, the study of bacteria in disease causation; mycology, the study of fungi involved in disease causation; and parasitology, the study of parasites involved in human diseases. Another area of clinical pathology, clinical chemistry, includes the analysis of the amounts of specific chemicals in the blood and other body fluids such as the blood glucose levels in blood or uric acid and creatine in urine. These measurements provide critical information about the function of specific body organs in health and in disease states such as measurements of specific liver enzymes to indicate liver cell function in health and in disease. Transfusion medicine includes analysis of blood and blood products which can be used for transfusion in sick patients. Hematopathology studies abnormal cells in the blood as well as analysis of lymph nodes, spleen, and bone marrow specimens to diagnose abnormal cell growth or the presence of abnormal cells and tissues in these organs and in other parts of the body. Other relatively new areas of pathology include molecular pathology and digital pathology. Molecular pathology analyzes DNA and RNA in cells or tissues from a patient to make diagnoses about disease causation or methods to treat specific diseases such as cancers that are not cured by surgery or conventional radiation therapy and chemotherapy. Digital pathology includes artificial intelligence and machine learning. Digital pathology uses a combination of imaging technologies and artificial intelligence to assist in diagnoses in different areas of anatomic and clinical pathology. Experimental pathology dates back from before the time of Virchow when physicians and other scientist used experimental systems along with studies in animals to learn about the biology of diseases. The study of pathobiology has been a major reason for many of the outstanding advances in pathology and in the biological sciences. Many pathologists at the time of Virchow worked in both diagnostic and experimental pathology and this transfer of knowledge from one area to another served to accelerate the development of knowledge about the pathological basis of diseases. Although many diagnostic pathologists were well trained in experimental pathology in the nineteenth and twentieth centuries, today most experimental pathologists have a doctorate degree (PhD) with training in basic biological and/or physical sciences and do not usually engage in the practice of diagnostic pathology.

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Most experimental pathologists are biomedical scientists who investigate the mechanisms of diseases with highly competitive nationally funded research grants in the USA. Some pathologists go through the traditional training in anatomic and clinical pathology and then go on to do postdoctoral training in more basic sciences; but this is an exception today rather than the more common pathway to a research career. Many heads of major academic pathology departments in the USA are basic researchers who currently or previously ran a research laboratory with grant funding from the National Institutes of Health or some other national funding agency and work closely with graduate and postgraduate students in the research laboratory. Experimental pathology has contributed immensely to advances in pathology and medical sciences during the past century. Many experimental pathologists in the USA today are members of The American Society of Investigative Pathology ([3, 4], and Appendix 1). The broad goals of experimental pathology and diagnostic pathology are overlapping; both fields aim to make scientific discoveries or diagnoses to help patients and/or to help people directly by diagnosing the disease process and providing a scientific means of treating diseases and predicting patient prognosis for specific type of neoplasms or other disease processes.

References 1. Ceolin L, da Silveira Duval MA, Benini AF, et al. Medullary thyroid carcinoma beyond surgery: advances, challenges, and perspectives. Endocr Relat Cancer. 2019;26:R499–518. 2. Pinsky WW, Arciniegas E. Tetralogy of Fallot. Pediatr Clin N Am. 1990;37(1):179–92. 3. Fausto N. What’s in a name? (editorial). The American Society for Investigative Pathology. Am J Pathol. 1993;142(1):1. 4. Rosai J. Pathology: a historical opportunity. Am J Pathol. 1997;151(1):3–6.

Chapter 2

Historical Aspects of Pathology

Pathology, which includes the study of the structural and functional changes produced in tissues by disease processes, developed many centuries ago. Some of the earliest accounts of pathology in recorded history included data from the seventeenth-­ century BC in the Edwin Smith Egyptian Papyrus from 1600 BC [1, 2]. The Smith Papyrus showed that there was recognition of specific entities such as deafness after fracture of the temporal bone and paralysis from dislocation of the cervical vertebrae [2]. Recorded studies of ulcerating lungs which may have been neoplastic or inflammatory, bone injuries, and other diseases have been documented [1]. Unfortunately, only a small amount of information on pathological anatomy over many centuries of Egyptian dynasties have been uncovered in recent times. A few diseases such as spinal tuberculosis and bone tumors had been well documented. But a systematic analysis about the possible causes of these conditions have not been uncovered. It was the early Greeks such as Hippocrates during the period of 400–300 BC who had a great deal of influence on the development of studies in human pathology. Hippocrates of Kos (460–370  BC) and his disciples exerted a great deal of influence on Greek and Roman medicine. Hippocrates’ influence was so vast that it lasted until the seventeenth-century AD. For example, his “Humoral Theory” suggested that the body was made up of four humors or substances including blood, phlegm, black bile, and yellow bile and that for ideal health the humors had to be in perfect balance, while loss of this balance would lead to sickness, was widely adopted by brilliant physicians such as Galen many centuries later. Hippocrates’ observational skills and writings on topics such as tuberculosis and tumors were very insightful and influential. Other gifted Greek writers and thinkers such as Aristotle (384–322 BC) and his successors contributed to our knowledge of human pathological processes, but a great deal of their writings have been lost [1]. Herophilus (335–280  BC) was reportedly one of the first Greeks to dissect human bodies [1, 2]. The influence of the Greeks on the development of pathology was extensive. They used the word pathology to mean suffering or a diseased state. The Romans later under the influence of Galen would designate pathology to mean © The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 R. V. Lloyd, Pathology: Historical and Contemporary Aspects, https://doi.org/10.1007/978-3-031-39554-3_2

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a disturbance of vital processes [1, 2]. The influence of the Romans on medicine and on pathology included contributions from outstanding scholars such as Celsius and Galen. Cornelius Celsius (25 BC–50 AD), although not a physician by training, was a well-educated scholar. He described the cardinal signs of inflammation which included “rubor, tumor, calore and dolore—redness, swelling, heat and pain” which remains familiar to medical students studying pathology even in the twenty-first century. Galen (129–201 AD) exerted a great deal of influence on the development and thinking in medicine and pathology way beyond his time. His influence was widely felt for many centuries after his demise. His dissection of animals and his emphasis on the importance of organ systems added scientific rigor to pathology and medicine. His writings in such books as “Seats of Diseases” and “Abnormal Tumors” influenced medicine for many centuries and even into the Middle Ages. Unfortunately, Galen adopted the Hippocratic theory of the four humors which impeded the development of rational theories about disease causation for many centuries through the Middle Ages [1]. During the Middle Ages, Byzantine and Arab physicians made more contributions to the advancement of medical sciences, but Galen’s influences persevered. During the late Middle Ages and the early Renaissance period advances were made in further understanding human diseases. This was stimulated by renewed interest in medicine, anatomy, and pathological anatomy especially in Italy. In Bologna, Italy, human dissection was performed as early as 1270 AD [1]. Dissections were done routinely for teaching purposes such as studies in anatomy and for studies of disease processes. Antonio Benivieni (1443–1502) advanced the field of pathology by recording case histories and performing autopsies on some of his patients and he initiated the clinical-pathological correlation for the first time. This approach is widely used in the education of medical students today. Vesalius (1514–1564), who was not a follower of Galen, did many autopsies and published his correlative studies. Jean Fernel (1497–1558) classified diseases as general or special and distinguished between these in his main publication (Medicina). He was one of the first physicians to suggest that some aneurysms could be secondary to syphilitic infections, a disease common in the sixteenth century. William Harvey (1578–1657) made great inroads in studies of the etiology of disease. His observations about blood circulation and the role of the heart in the circulatory process helped to decrease the influence of the Hippocratic humoral theory [2]. Many new ideas were advanced by performing autopsies and linking the findings to patient history. Giovanni Batista Morgagni (1682–1771) was one of the early giants of pathology and his observations contributed to the decline of some of Galen’s erroneous theories [2, 3]. Morgagni is sometimes considered as the father of modern pathology. He studied medicine in Bologna, Italy, and became an outstanding teacher. His many contributions include the description of angina pectoris, description of aneurysms of the aorta, mitral stenosis, syphilitic lesions of the skin, tuberculous peritonitis, and many other lesions. Antonie van Leeuwenhoek (1632–1723) made critical discoveries, leading to the development of the microscope [4]. His discoveries made microscopes a basic tool in pathology. Although the compound microscope was built

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before van Leeuwenhoek, his use of the single lens microscope made his discoveries easier to be independently reproduced and confirmed. Van Leeuwenhoek kept his lens grinding technique a secret, so his achievements remained unmatched until the nineteenth century [1, 4]. Morgagni was a student of the outstanding anatomist Anton Valsalva (1666–1723). Morgagni’s book on anatomy (Adversaria Anatomica Prima) provided him with a great deal of recognition by his peers at the relatively young age of 24 [3]. His productivity continued for many decades and at the age of 79 he published a book on the seats and causes of diseases through anatomical examination. This outstanding work described the anatomic findings and correlated these with the antemortem symptoms and helped to support his observations that disease processes were organ-based. A major step forward in pathological anatomy was made by the increasing usage of the light microscope. John Hunter (1728–1793) contributed to experimental pathology with his studies on inflammation including excellent microscopic descriptions. One of his relatives, Matthew Baillie (1761–1823), published the first systematic book of pathology. Thomas Hodgkin (1708–1866) was a British physician with many interests including studies of pathological changes in tissues [5]. His range of studies included characterization of tuberculosis at autopsy to studies of lymphatic tissues (absorbent glands) and spleen to characterize a disease that was later better characterized and named in his honor (Hodgkin’s disease). Interestingly, Hodgkin’s disease was studied at a gross level and he did not characterize the microscopic features of the disease bearing his name. Subsequent investigators performed microscopic studies to complete the circle. Hodgkin reported on seven cases describing what he considered as a new entity [6] and it was later shown by microscopic studies that three or four of the described lesions were true cases of Hodgkin’s lymphoma with diagnostic Reed–Sternberg cells [5, 6]. Interestingly, after it was published, Hodgkin’s 1832 paper was not noticed by most of his contemporaries until it was recognized by Baronet Sir Samuel Wilks in 1856. Wilks generously applied the term “Hodgkin’s disease” to the lesion that he described in his study. Hodgkin was a prolific scholar and described other conditions such as aortic insufficiency and acute appendicitis and its sequalae [5]. The microscope was a major driving force in moving medicine and pathology forward in the 1800s. The first half of the 1800s also saw an increasing interest in the basic sciences, in physiology, and in medicine. Carl von Rokitansky (1804–1878) made many contributions to pathological anatomy. He was born in what is now considered the Czech Republic and studied in Prague and Vienna. He oversaw more than 50,000 autopsies during his career performed either by himself and/or his assistants. He maintained a systematic and comprehensive approach to the study of human diseases. Unfortunately, on rare occasions, when there were no clear-cut autopsy findings to explain a disease process, he would rely on the ancient humoral pathology [7]. Rokitansky did not master the microscope as readily as Virchow, but he was a strong supporter of histological and pathological studies [7]. It was his extensive pathological studies and clinicopathological studies of autopsies and patient history that helped to establish pathology as a rapidly growing specialty. Rokitansky had a great deal of other interests outside of pathology including

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evolutionary biology and philosophy. He had reportedly interacted with the evolutionary biologist Charles Darwin and the German philosopher Arthur Schopenhauer [7]. He died of a myocardial infarction at the relatively advanced age of 74. Rudolf Virchow (1821–1902) is probably the most outstanding figure in the history of pathology. He was one of the first pathologist to use the microscope routinely in his work [1, 8]. The continuity of cellular life was recognized by his famous aphorism, “Omnis cellula e cellula” or “All cells come from cells.” His extensive studies involved both cellular and experimental pathology [8]. His studies moved pathology from an organ-based system to a cell-based system. This created a lot of new insights and excitement about the “new pathology” in his days [9]. Some of his most significant observations have ranged from describing metastatic carcinoma in supraclavicular lymph node usually metastasizing from the stomach (Virchow’s node) to his description of the mechanisms of thromboembolism [10]. He also performed experiments to validate his theory about the origin of thromboembolism [10]. In addition to all of his scholarly publications, he started a pathology journal (Virchows Archiv) and was the first lifetime editor of this famous pathology journal that still remains a major pathology journal today. Robert Koch was born in Prussia in 1843 [11]. As a medical student, he won a prize for studies on the neural innvervation of the uterus. After graduation, he moved to Berlin and was able to meet Virchow. For a while he did general practice in Langenhagen. He received a microscope from his wife for one of his birthdays and this stimulated him to begin studying infectious diseases. He studied microbiology at Germany’s major scientific centers. In his studies of the anthrax bacillus, he was able to trace the route of the infection and the life cycle of the organism including the spores which were capable of surviving in the soil for years. This was the first infectious agent of a specific disease and its life cycle was demonstrated under controlled in  vitro conditions [11]. He later created solid culture media for growing bacteria in the 1870s and 1880s. One of his major contributions was the use of a scientific approach to bacteriology that led to the establishment of Koch’s postulate which included the following tenets: (a) one had to establish that a specific organism was present in every case of an infectious disease, (b) be able to cultivate the organism, (c) to inoculate an experimental animal such as a guinea pig with the culture to reproduce the disease, and (d) recover the organism from the inoculated animal and grow it in pure culture. These rigorous criteria established microbiology on a sound scientific footing. Koch won the Nobel Prize in Physiology or Medicine in 1905 for his studies about tuberculosis. One downfall later in his career was the suggestion that he could cure tuberculosis with tuberculin. Although this turned out not to be true, tuberculin is used today as a marker for tuberculosis infection. Koch also considered human and bovine tuberculosis to be the same disease. The start of the twentieth century saw rapid progress in various areas of pathology. The neoplastic Reed–Sternberg cell in Hodgkin’s disease was described by Sternberg in 1898 and by Reed in 1902. Major advances in anatomic pathology including autopsy studies by Anitschkov (1885–1964) describing the changes in the heart in rheumatic fever and his studies also suggested that cholesterol could contribute to atherosclerosis. Karl Landsteiner performed more than 3000 autopsies

Brief History of Islamic Pathology

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during his training and later discovered various antigens on red blood cells which established the field of blood typing and transfusion medicine. In addition to histochemical stains established by Pierre Masson (1880–1959) [12], Santiago Ramon Cajal (1852–1934) [13], AG Everson Pierce (1916–2003), and many others, advances in tissue fixation, embedding, microtome sectioning, and other techniques provided the foundation for the advances in basic research and in diagnostic pathology. The electron microscope was introduced in the 1930s by Ruska and collaborators and this greatly advanced the field of pathology [14], since it was now possible to visualize viruses and to observe subcellular organelles directly [14]. The development of immunohistochemistry was also another great leap forward in pathology. Albert Coons (1912–1978) [15], Ludwig Sternberger (Born 1921) [16], Paul Nakane (Born 1935) [17], and Barry Pierce (1925–2015) [17] all contributed to immunohistochemical advances. Immunohistochemistry became a dominant technique in experimental and diagnostic pathology after the development of monoclonal antibodies by Kohler and Milstein leading to their Nobel prize in 1970 [18]. The histopathological classification of neoplasms and the discovery of many new types of neoplasms as well as major changes in the classification of cancers were linked to advances in immunohistochemistry. Molecular diagnostics is currently the most recent technical diagnostic armamentarium in pathological diagnoses in fields as disparate as hematopathology and neuropathology [19, 20]. Most fields of diagnostic surgical pathology currently rely on molecular diagnostics and in situ hybridization for daily accurate diagnoses in the laboratory.

Brief History of Islamic Pathology The Islamic civilization once had a major influential impact on medicine and pathology as well as on the traditional sciences [21]. During the Islamic Golden Age between the eighth and fourteenth centuries, Arab scholars had major influences on science, medicine, and philosophy. They translated works from Greek, Syriac, Pahlavi, and Sanskrit into Arabic [1]. Pathology in the period between Galen and the late Middle Ages was largely influenced by Arab physicians [1]. One of the outstanding physicians was Aetius of Amida (502–575), who was the physician to the emperor Justinian. He described uterine cancer, hemorrhoids, and condyloma among other disease entities [1]. Avicenna (980–1037) was another outstanding Arab physician who was influenced by Galen and Aristotle. His major work was “Canon Medicinae.” He remained influential until the fifteenth century [1]. The physician Ibn Zuhr (Avenzoar) (1091–1165  AD) was one of the most influential Arabic physicians. He described cancer of the esophagus and stomach [1]. He also described serous pericarditis and the pathological findings in fibrous pericarditis [21, 22]. In his textbook “Al-Taisir,” Ibn Zuhr used the approach of classifying diseases according to the organs affected which is still used today in many pathology textbooks [22]. Avenzoar was reportedly the first physician to have done a postmortem dissection [22, 23]. Another outstanding physician, Ibn Al-Nafis, who lived in

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the thirteenth century described the pulmonary circulation in1242 which was several centuries before the description of William Harvey [24]. The Medieval Islamic physicians were influential during the Middle Ages and this influence lasted until the 1400s after the decline of the Arab influence in Spain and other places in Europe [25]. After that time, there was the decline of the Byzantine and Arabic schools of influence toward the end of the crusades. Granada was the last Islamic state in Spain and this was taken over by the Spanish in 1492 when Columbus set sail to “discover” the new world.

Brief History of Eastern Pathology While pathology was developing in the West, there were also ongoing activities in the East, especially in China and Japan. Since China has had a continuous written history for over 5000 years, some of the development and contributions to medicine and pathology have been well documented [26]. The history of pathology in China has been embedded in the literature of Traditional Chinese Medicine (TCM). TCM is based on experience rather than experimentation, so the diagnoses of TCM is based on “observation, auscultation, olfaction, interrogation and feeling the pulse” [26]. The feature of observation is closest to results produced in anatomic pathology. The first description of autopsy in Chinese history was in the first-century BC [26]. Another ancient Chinese medical record described the anatomy of the heart, lungs, liver, gallbladder, spleen, stomach, intestine, and bladder and also described their functions. These findings reportedly established a foundation for TCM. Between 202 BC and 24 AD, Zhongjing Zhang wrote about his extensive experience in diagnosing and treating surgical infections and described a wide range of pathological changes [26]. Royal physicians and skilled butchers performed autopsies on executed criminals during the first century to learn about medicine and pathology for medical practice. Between 581 and 907 AD during the Sui and Tang Dynasties, a book by Yuanfung Chao described wound infections, parasitic diseases, and endocrine diseases [27]. For example, it was pointed out that some parasitic diseases could be caused by undercooked pork, an early appreciation about the etiology of trichinosis. Between 907 and 960 AD, the first book of human anatomy was published by the Taoist priest Luozi Yan which was a major contribution to anatomy [28]. During the Ming Dynasty (1368–1644), there was little progress in pathology and anatomy. In contrast, this was the great period of advancement in European anatomy and pathology. Around 1644 AD at the end of the Ming Dynasty, sea travel to China increased and medical and pathological concepts from Western medicine were brought in by Roman Catholics. This increased the spread of medicine and pathology in China. After the Opium War in 1840, China was a semifeudal society, but with the subsequent invasion of Western culture, things began to change. The first Western style medical school (Boji Medical School) was started in 1866  in Guangzhou by the church. Some of the subjects taught included anatomy and pathology [29]. The first Department of Pathology was started in 1921 by Xiecheng

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at Union Hospital which was supported by the Rockefeller Foundation in Peking (Beijing) [30]. Pathologist such as Dr. Zhenxiang Hu reported on his studies of visceral leishmaniasis and identified Phlebotomus as being responsible for transmitting the disease [31]. From 1949 AD, pathology started to develop rapidly with the opening of China and the rapid development of technologies such as immunohistochemistry, electron microscopy, and more recently molecular pathology. The Japanese adopted Chinese medicine when they conquered Southern Korea in the fourth-century AD. In Japan, as in China, laws against dissection were based on reverence for the human body and this slowed progress in pathological anatomy. Some Japanese doctors were performing dissections in the eighteenth century under the influence of Western medicine. There were many interactions between Japanese and German physicians in the late nineteenth century [32, 33]. Virchow in 1901 talked about the friendship and new contacts between Germany and Japan. In the late 1800s and early 1900s, Japanese physicians visited Germany and made important contributions to medicine, pathology, and bacteriology. Dr. Kitisato worked with Robert Koch around 1885. He was able to isolate Clostridia tetani in the laboratory and, in 1894, he discovered the pathogenic organism responsible for the plague. Mikito Takayasu, an ophthalmologist in Tokyo, described Takayasu’s arteritis, a granulomatous inflammation of the medium and large arteries in 1908 [34]. Tomisaku Kawasaki, who worked at the Red Cross Hospital in Tokyo for over 40 years, observed two children with high fever, conjunctival hyperemia, and cervical lymphadenopathy. He collected another 50 patients with similar signs and symptoms and reported for the first time about acute febrile mucocutaneous lymph node syndrome or Kawasaki’s disease [35]. Hashimoto’s publication on an inflammatory disease of the thyroid that was named in his honor (struma lymphomatosa or Hashimoto’s thyroiditis) in 1912 was another major milestone. It illustrated the close correlation between surgery, medicine, and pathology in Japanese medicine. Hashimoto did his studies of four female patients with chronic thyroid disorder as a student [36]. He published his observations in a German journal, since this was one of the most influential journals and German was the dominant language in medicine at the time. He later visited Germany and the Pathologist Professor Kaufmann years after his original discovery. World War I interrupted the close contact of Japan with Western medicine, but this contact resumed in the postwar years. In the 1930s, Masugi described experimental glomerulonephritis (Masugi nephritis) as an inflammatory disease of the kidney illustrating the importance of experimental pathology in Japan and its influence in Western medicine. Aschoff visited Japan in 1924. After World War II, the relationship between Japanese and German physicians improved and there were increased interactions of Western and Japanese medicine. Since these early days, biology and pathology research have increased greatly in Japan and many original discoveries have led to the awarding of Nobel Prizes to Japanese scientists working in Japan as well as in other countries such as the USA.

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References 1. Van den Tweel JG, Taylor CR. A brief history of pathology. Preface to a forthcoming series that highlights milestones in the evolution of pathology as a discipline. Virchows Arch. 2010;457:3–10. 2. Krumbhaar EB. Pathology. New York: Hafner Publishing Company; 1962. 3. da Silva VD, Prolla JC.  Morgagni Giovanni B 1682–1771. In: van den Tweel JG, editor. Pioneers in pathology. In: van Krieken JHJM, series editor. Encyclopedia of pathology. Switzerland: Springer; 2017. p. 385–92. 4. da Silva VD. Van Leeuwenhoek, Antoine (1632–1723). In: van den Tweel JG, editor. Pioneers in pathology. In: van Krieken JHJM, series editor. Encyclopedia of pathology. Switzerland: Springer; 2017. p. 505–10. 5. Geller SA, Taylor CR. Hodgins Thomas (1798–1866). In: van den Tweel JG, editor. Pioneers in pathology. In: van Krieken JHJM, series editor. Encyclopedia of pathology. Switzerland: Springer; 2017. p 251–7. 6. Hodgkin T.  On some morbid appearances of the absorbent glands and spleen. Medico-­ Chirurgical Trans. 1832;17:68–114. 7. Sedivy R. Von Rokitansky Carl (1804–1878). In: van den Tweel JG, editor. Pioneers in pathology. In: van Krieken JHJM, series editor. Encyclopedia of pathology. Switzerland: Springer; 2017. p. 527–32. 8. Canzonieri V, Italia F. Virchow Rudolph (1821–1902). In: van den Tweel JG, editor. Pioneers in pathology. In: van Krieken JHJM, series editor. Encyclopedia of pathology. Switzerland: Springer; 2017. p 514–7. 9. Byen JM 3rd. Rudolph Virchow-Father of cellular pathology. Am J Clin Pathol. 1989;92(4 Suppl 1):S2–8. 10. Kumar D, Hamlin E, Glurich E, et al. Virchow’s contribution to the understanding of thrombosis and cellular biology. Clin Med Res. 2010;8(3/4):168–72. 11. da Silva VD, Tonietto RG Koch Robert (1843–1910). In: van den Tweel JG, editor. Pioneers in pathology. In: van Krieken JHJM, series editor. Encyclopedia of pathology. Switzerland: Springer; 2017. p. 294–300. 12. Canzonieri V. Masson, Pierre (1880–1959). In: van den Tweel JG, editor. Pioneers in pathology. In: van Krieken JHJM, series editor. Encyclopedia of pathology. Switzerland: Springer; 2017. p. 366–9. 13. Wick MR.  Ramon y Cajal, Santiago (1852–1934). In: van den Tweel JG, editor. Pioneers in pathology. In: van Krieken JHJM, series editor. Encyclopedia of pathology. Switzerland:Springer International Publishing; 2017. p. 451–2. 14. Kruyer DH, Schneck P, Gelderblom HR.  Helmut Ruska and the visualization of viruses. Lancet. 2000;355(9216):1713–7. 15. Greer A.  Albert coons: harnessing the power of the antibody. Lancet Respir Med. 2016;4(3):181–2. 16. Sternberger LA, Hardy PH Jr, Cuculis JJ, et al. The unlabeled antibody enzyme method of immunohistochemistry: preparation and properties of soluble antigen-antibody complex (horseradish peroxidase-anti horseradish peroxidase) and its use in identification of spirochetes. J Histochem Cytochem. 1970;18:315–33. 17. Nakane PK, Pierce GB.  Enzyme-labeled antibodies for the light and electron microscopic localization of tissue antigens. J Cell Biol. 1967;33:307–18. 18. Kohler G, Milstein C.  Continuous culture of fused cells secreting antibodies of predefined specificity. Nature. 1975;256:495–7. 19. Mullauer L. Molecular pathology of cancer. The past, the present and the future. J Pers Med. 2021;11(7):676–9. 20. Posey JE, Rosenfeld JA, James RA, et al. Molecular diagnostic experience of whole-exome sequencing in adult patients. Genetics. 2016;18(7):678–85.

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21. Majeed A. How Islam changed medicine. Arab physicians and scholars laid the base for medical practice in Europe. Br Med J. 2005;331(7531):1486–7. 22. Abdel-Halim RE, Elfaqih SR.  Pericardial pathology 900 years ago. Saudi Med J. 2007;28(3):323–5. 23. Abdel-Halim RE. Contribution of ibn-Zuhr (Avenzoar) to the progress of surgery. A study of translations from his book Al-Taisir. Saudi Med J. 2005;26:1333–9. 24. Soubani AO, Kahn FA. The discovery of the pulmonary circulation revisited. Ann Saudi Med. 1995;15(2):185–6. 25. Conrad LI. The western medical tradition 800 to 1800 AD. Cambridge: Cambridge University Press; 1995. p. 93–130. 26. Jiang C, Gu J.  History and current state of pathology in China. Virchow Arch. 2013;463(4):599–608. 27. Maciocia G. The foundation of Chinese medicine. Amsterdam: Elsevier; 2005. 28. Hong FF.  History of medicine in China: when medicine took an alternative path. McGill J Med. 2004;8(1):79–84. 29. Rockefeller Foundation China Medical Board. Medicine in China. Chicago: University of Chicago Press; 1914. 30. Bullock MB. An American transplant: the Rockefeller Foundation and Peking union medical college. Berkeley: University of California Press; 1980. 31. Ackerknecht EH.  A short history of medicine. Baltimore: Johns Hopkins University Press; 1982. 32. Schmiedebach H-P. German-Japanese relationship in pathology and forensic medicine during the late 19th and early 20th centuries. Rechtsmedizin. 2006;16:213–8. 33. Veith I.  On the mutual indebtedness of Japanese and Western medicine. Bull Hist Med. 1978;52:383–409. 34. Takayasu M. A case with peculiar changes of the central retinal vessels. Acta Soc Opthalmol Japonica. 1908;12:554. 35. Kawasaki MM. Tomisaku (1925–). In: van den Tweel JG, editor. Pioneers in pathology. In: van Krieken JHJM, series editor. Encyclopedia of pathology. Switzerland: Springer; 2017. p. 281–3. 36. Sawin CT. The heritage of Dr. Hakaru Hashimoto (1881–1934). Endocr J. 2002;49(4):399–403.

Chapter 3

Surgical Pathology

Surgical pathologists examine gross specimens with diseases excised by surgeons as well as small biopsy specimens and process them for microscopic examination in order to make a diagnosis and to quantify the amount of neoplasms in the resected specimens. Surgical pathologists also study and diagnose many nonneoplastic lesions such as inflammatory and degenerative diseases. When examining neoplasms, the surgical pathologist documents the size of the neoplasm; for cancers, they determine the depth of invasion and the quality of the margins, and decide if there is invasive growth of the cancer into lymphatic and vascular channels with spread into lymph nodes. If the patient was treated before surgery, they document the response of the cancer to prior radiation therapy, chemotherapy, and immunotherapy. Today, the immunohistochemical and molecular features of neoplasms and other lesions are also assessed by the pathologist and this assists clinicians in deciding about further therapy. In addition to larger resected gross specimens, the surgical pathologist also examines biopsy specimens performed for diagnostic purposes. Another major role of the surgical pathologists is in the rapid evaluation of frozen sections of tissues to provide rapid feedback to the surgeon about the presence of tumor and type of tumors that the patient has and in some case about the adequacy of the tumor margins. This has been summarized well in a book by the late Dr. Juan Rosai, an outstanding surgical pathologist, who edited the book, ”Guiding the Surgeon’s Hand: The History of American Surgical Pathology.” Surgical pathology as a subspecialty in American pathology developed during the last decade of the 1800s. In the USA, surgeons often served as their own pathologist in most institutions at this time [1]. One major exception in the history of pathology was at the Mayo Clinic, founded by the Dr. Mayo in the 1880s in Rochester Minnesota and further developed by his two sons William and Charles Mayo, who were both surgeons. The Mayo brothers recognized early on the importance of a fulltime pathologist as well as of the necessity of intraoperative frozen sections for patient care [2]. Around the year 1900, surgeons in most medical centers in the USA operated only on palpable masses such as breast lesions and decide if they were benign or © The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 R. V. Lloyd, Pathology: Historical and Contemporary Aspects, https://doi.org/10.1007/978-3-031-39554-3_3

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malignant based on gross inspection. The surgery would end after excision of the lesion if the tumor was thought to be benign or after a radical mastectomy for malignant lesions. The standard of practice at the time was to discard the excised specimen without detailed pathological examination [1]. Frozen sections at Johns Hopkins started in 1891 with Dr. Welch as the pathologist and Dr. Halsted as the surgeon. In one of their first cases working as a team, the final diagnosis by the pathologist was reported correctly. However, the turnaround time was so long that the surgeon had to make his own diagnosis and completed the operation [3]. Frozen section techniques similar to those used at Johns Hopkins were reported by the Germans, British, and others in the late 1890s. At the Mayo Clinic, Dr. Wilson was the first pathologist to publish about the frozen section in 1905 [4]. One difference between the frozen sections done at the Mayo Clinic and other places in the USA was that hematoxylin and eosin dyes were used at most other institutions today, while the Mayo Clinic used methylene blue or other dyes that stained more quickly. This allowed completion of the frozen section slide more quickly which was always helpful to the surgeon. Dr. Bloodgood, a famous surgeon working at Johns Hopkins recommended the use of the frozen section at his presentation to the American Association of Pathologist and Bacteriologist in 1906, especially when it was impossible to make a clinical diagnosis of a malignant tumor [1, 5]. However, there were many challenges leading to poor-quality frozen sections including artifacts such as ice crystal formation during freezing and other issues that made rapid interpretation of the specimens very challenging. In the 1920s, Bloodgood wrote editorials supporting the use of frozen sections [1]. Around this time, the use of the microscope became more important in surgical pathology and by 1911 pathologists such as Dr. James McCarthy at the Mayo Clinic emphasized the necessity of microscopic examination of tissues to make the correct pathological diagnosis [6]. The American Society of Clinical Pathology (ASCP), which was founded in 1922, developed as pathology searched for a unified voice (Appendix 1). The American Board of Pathology, which was founded in 1935 established the formal examination and certification of pathologists with the first examination for official certification in 1936. Clinical pathology was not limited to the laboratory, but was intended to emphasize the clinical relevance of surgical pathology, which was also a laboratory practice [1]. Much of surgical pathology continued to be practiced in surgery departments until around the 1950s. This was especially true in some specialties such as gynecology. The ASCP emphasized in the 1930s and 1940s that tumor pathologists did not only have to make a tissue diagnosis, but should also be familiar with the cellular elements making up the tumor, and the natural course of tumors as they developed in different parts of the body [7]. To educate pathologist in the broad area of surgical pathology, the ASCP held its first seminar in anatomic pathology in 1934 at the Mayo Clinic with Dr. McCarthy as the presenter. Dr. Will Mayo delivered the welcoming address. In his talk, Dr. Mayo stressed the importance of the pathologist in patient care [1]. During the past half century in addition to histochemical stains advancing diagnostic pathology, the development of immunohistochemistry helped to advance the field of diagnostic pathology extensively. The development of polyclonal and monoclonal antibodies accelerated advances in

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diagnostic surgical pathology including the description and classification of new tumors. These achievements including biopsy interpretation helped to make advances in surgical pathology more prominent with the development of small needle core biopsies by clinicians and radiologists for the diagnosis of primary and metastatic lesions [8]. Needle biopsies of tumors and other lesions are frequently interpreted by the surgical pathologist. The handling and interpretation of needle biopsies depends on the interactions between the pathologist, radiologist, clinicians, and surgeons. The type of needle and other equipment used to perform the biopsy can determine if adequate biopsy material was taken for proper interpretation of the lesion. For example, when a radiologically guided suspicious breast biopsy is done, the type of equipment used can make a great deal of difference in what the pathologist has to examine and diagnose under the microscope. Comparison of the 14-gauge needle core biopsy with the vacuum-assisted biopsy has shown that vacuum-assisted biopsies consistently outperformed 14-gauge needle core biopsies. In one study, needle core biopsies had a false negative rate of 8% using the 14-gauge core biopsy compared to 0.7% for vacuum-assisted biopsies [8]. In another study, the core biopsy of in situ carcinoma (noninvasive) was upgraded to invasive carcinoma in 15–36% of patients with core biopsies compared to only about 10% in patients with vacuum assisted biopsies [8]. Evaluation of some of the landmarks seen on the mammogram can vary with the procedure used. When calcification is seen on the mammogram and is not seen in the biopsy material, the difference between using a core biopsy (14%) and a vacuum-­ assisted biopsy (1%) is also significant in the event that the biopsy shows only benign breast tissue with or without calcification [8]. A false negative rate of up to 74% for the biopsy occurs when calcification is not present in the specimen X-ray after the biopsy is completed [9]. To overcome some of the problems between differences in biopsy techniques, pathologist often make an X-ray of the resected breast biopsybiopsy specimen to search for calcification in the specimen radiograph after the biopsy is received in the pathology laboratory. Another serious problem that can occur in the surgical pathology laboratory when the biopsy specimen is being examined microscopically is the question of whether an additional piece of tissue present in the biopsy that is being examined under the microscope is really from the patient with the larger biopsy specimen on the same slide or if it represents a contaminant that was acquired during the processing of the tissues (sometimes referred to as a contaminant or floater) [10]. The incorrect interpretation of the extra piece of tissue could lead to a false positive or false negative result in the diagnosis and an erroneous decision on whether further surgery or other treatment is needed for the patient. For example, if a biopsy from patient A shows mainly normal breast tissue in most of the biopsy and a small cluster of malignant cells as a separate focus on the same slide, does patient A have breast cancer as revealed by the biopsy? If it is a false positive, the malignant cells could have been acquired from another patient during the processing of the biopsies and picked up with patient A’s biopsy from the water bath during cutting or during the staining of patient A’s biopsy in the pathology laboratory. Such questions arise

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occasionally in the pathology laboratory. In spite of meticulous and careful techniques used in the laboratory, these problems do occur on occasion even in the most carefully run labs. Fortunately, molecular techniques are available to answer such questions when they arise [11, 12]. Molecular analyses such as short tandem repeat analysis or microsatellite analysis can be performed. In these procedures, the polymerase chain reaction (PCR) in which DNA from the cells in question are extracted and amplified many times in a test tube with 10 or more polymorphic microsatellite markers from various chromosomes and alleles, the samples are analyzed semiquantitatively by fluorescent capillary electrophoresis [10]. Other samples from the patient who provided the current biopsy such as previous biopsy, or the use of a buccal swab or peripheral blood lymphocytes from the patient are analyzed at the same time for comparison. Such techniques are usually successful 92–100% of the time in resolving the question about possible specimen mix up and ensures highquality patient care. In a well-run pathology laboratory, all of the laboratory procedures involved in patient tissue processing are reexamined on a regular basis to avoid these potential problems. Differences between the appearances of tissues, including benign and malignant tissues, at the time of frozen section on even optimally processed specimens can be very subtle and a great deal of expertise is required to make a correct diagnosis either on frozen section or permanent section. In some institutions, different pathologists may be covering several hospitals on a given day, so the expert in bone tumors or liver tumors may not be available on site to assist with a frozen section or permanent section. The practice of telepathology has assisted in the past few years to assist with such problems. Telepathology uses telecommunications lines for electronic transmission of digital pathology images that can assist in making a rapid diagnosis [13]. It is most helpful for frozen tissue sections, but can also be used for permanent section of tissues when an expert subspecialist is not available at an institution at the time that the specimen is being examined. Telepathology can also be used for teaching, research, and for quality assurance to check on the accuracy of a rendered diagnosis [13]. Although telepathology is relatively expensive and there are no universal standards, it is rapidly becoming a widely used tool in pathology [14, 15]. It is especially useful for intraoperative frozen section diagnosis when an expert pathologist in, for example, neuropathology or thyroid pathology is not available to directly examine the frozen section. When digital pathology is performed by frozen sections, diagnostic accuracy greater than 92% has been reported [14]. This approach is widely popular and is being used with increasing frequency in the USA, Europe, China, Japan, and in many developing countries. In Japan, some of the larger hospitals provide smaller hospitals with expertise in frozen section analysis using digital pathology. Over the past 170 years, surgical pathology has been evolving from areas dealing only with autopsy and experimental pathology into a major discipline in diagnostic pathology. A great deal of progress in the diagnostic criteria for new disease entities seen under the microscope have led to more specific treatments and disease prognosis. Although many outstanding surgical pathologists have contributed to this progress,

James Ewing (1866–1943)

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a few individuals have stood out from the others. The following section will summarize the lives of three outstanding pathologists who have contributed greatly to progress in surgical pathology.

James Ewing (1866–1943) James Ewing was born on Christmas day in 1866, a little more than 1 year after the end of the American Civil War when the country was still healing. He grew up in Pittsburgh. Around age 14 he had an accident, developed osteomyelitis, and remained bedridden for a few months [16]. Since this was in the pre-antibiotic era, the doctors considered amputating his limb; but a conservative course of treatment was successful and he recovered without an amputation. However, he walked with a limp that was present for the rest of his life. In spite of his physical handicap, he was able to play tennis, a sport that he enjoyed immensely. He even tried the sport of boxing for a while [17]. He attended Amherst College in Massachusetts. He received his medical training at Columbia College of Physicians and Surgeons in New York. He learned about pathology as a specialty while doing his internship at Roosevelt Hospital in New York. His interest in pathology increased and he even assisted in performing autopsies during his clinical training. He was later offered a position as a tutor in histology at Physicians and Surgeons Medical Center based on his excellent skills in this field. He later visited Austria in 1894 and studied under Professor Kalesky, one of Rokitansky’s successors. After returning to the USA, he became as assistant in Clinical Pathology at Columbia. Around that time, physicians were not reimbursed much for academic positions, so Ewing worked in private practice with a colleague to earn a living wage. At the relatively young age of 33, he was appointed as the first Professor of Pathology at Cormell University Medical School [16, 17]. He was married in 1900 and the young couple had a son. Unfortunately, his wife developed eclampsia during her second pregnancy and died. Ewing never remarried and dedicated most of his energy to his profession. Around 1902 Collis P. Huntington Fund for Cancer Research was established at the Alfred L Loomis Library at Belleview Hospital in New  York. At this time, Belleview was associated with Cornell University Medical College where Ewing worked. The laboratory under his leadership was very productive and he had written several research papers by 1910. His scientific work covered several areas and included studies on venereal lymphosarcoma in dogs and studies of immune serum in tumor transplantation. On revisiting Europe in 1910 Ewing was impressed with the great potential use of radium for tumor treatment by radiotherapy. Madam Curie had worn the Nobel Prize a few years earlier for her discovery of radium and its vast potential therapeutic utility. Around this time, Ewing proposed that Cornell establish a commission for clinical cancer research. It was also around this time that he met Dr. James Douglas. Dr. Douglas had previously trained in medicine and was the founder of the Phelps Dodge Corporation. His friendship with Douglas would mean great deal for

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Ewing’s future career. Douglas’s daughter had developed breast cancer and had died in spite of treatment with radium purchased in France. Douglas became focused on building a center dedicated to the study and treatment of cancer. Ewing and Douglas discussed with Memorial Hospital about the possibility of focusing mainly on cancer patients and they negotiated a permanent affiliation with Cornell Medical Center. The hospital would receive a gift of $100,000 and several grams of radium for cancer treatment as well as equipment to start an X-ray treatment facility. Douglas later donated more funds to the renamed Memorial Hospital for the Treatment of Cancer and Allied Diseases for the establishment of Douglas Deeds of Trust. Ewing became the Director of the hospital and maintained this position for the next 24 years. Ewing published the first edition of his book, “Neoplastic Diseases” in 1919. It was very successful and was updated in four editions over several years. “Neoplastic Diseases” brought Ewing a great deal of recognition in the USA and Europe [18]. A large part of “Neoplastic Diseases” was based on a review of the literature in addition to work based on Ewing’s own work and experience with various cancers. Ewing devoted most of his efforts to the study of oncologic pathology for many years. He emphasized the origin of cancers, the natural disease courses and the histology of the tumor. He published a description of the disease that was named in his honor in 1921. He designated this lesion as diffuse endothelioma of bone and emphasized that it was different from the widely recognized osteogenic sarcoma of bone and from myeloma [19, 20]. He had reported on a 14-year-old girl who had had congenital syphilis, but also developed a pathological fracture of her ulna in 1918. He noted that the radiological studies showed features that were inconsistent with osteogenic sarcoma. The tumor appeared to have regressed after radium treatment, a finding that was also different from the response of osteosarcoma to radium therapy. Unfortunately, the cancer recurred 2 years later and the tumor was biopsied for the first time. The biopsy showed small round cells. The patient developed metastases and died of cancer. Ewing remembered that he had seen another six cases with similar histological features as well as radiological appearance. One feature that was noted with all of the tumors was that they all responded to radium therapy. He mistakenly suggested that the tumors were of endothelial origin and this was later disproven with more sophisticated studies using newer techniques. Ewing emphasized that the primary role of the pathologist in making a diagnosis of cancer was to provide guidance for therapy. He stressed that the pathological findings should guide treatment design and that the study of the tumor should help to predict the clinical course of the disease. Interestingly, Ewing had some reservation about the use of frozen sections. He felt that while they were occasionally useful for their decisive value, they usually encouraged hasty conclusions and often led to errors in diagnosis. The publication of “Neoplastic Diseases” in multiple editions firmly established Ewing as a leading expert in oncologic surgical pathology. His leadership skills at Memorial Sloan Kettering Hospital established him as an outstanding leader in the newly emerging field of surgical pathology. Dr. Ewing died in 1943 when the USA was in the middle of World War II.

Arthur Purdy Stout (1885–1967)

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Arthur Purdy Stout (1885–1967) Arthur Purdy Stout was born in New York City in 1885. One of his grandparents was a physician. His family was well off financially and he spent about 15 months in Europe visiting Switzerland, Florence, and Paris when he was around age 11 [21–23]. Stout attended boarding school in Connecticut where he was a good student. He attended college at Yale University. Sadly, his father died when he started college. After graduation from Yale, he and three classmates made a 15-month trip around the world. They spent a great deal of time in China exploring parts of the country in depth. Arthur developed a lifelong interest in China from his travels. He collected many books about that part of the world. Later in his life he would donate several hundred books and pamphlets of the Yun-Nan region of China to the New York Public Library. While they were on a trip to Burma and Ceylon, one of his traveling companions from Yale died of smallpox. After his trip around the world, he enrolled at the College of Physicians and Surgeons of Columbia University. During his surgical training Stout spent 6 months on the pathology service and participated in performing frozen sections. Around this time, it was customary for surgery residents to spend a few months in pathology as part of their surgery training. After Stout completed his surgery residency, the College of Physicians and Surgeons offered him a position in the Department of Surgery. Before starting this job, he and his fiancé went to Europe and were married in Paris in 1914.The couple was in Scandinavia when World War I started and they traveled to England and stayed a few months before returning to the USA in September of that year. They had no significant financial problems at this time, since Stout’s father had left him an endowment [23]. At the New York Presbyterian Hospital Stout worked as an instructor in surgery and as an assistant attending surgical pathologist. Stout and his wife had a daughter born in 1916 and he became a dedicated father in spite of his very busy academic schedule. Stout’s interest in diagnostic pathology continued to expand. He reported on the clinicopathological studies on a tumorlike lesion of the epididymis in 1917 to the New York Pathologic Society. He also reported on the first case ever of a ganglioneuroma in 1918 as his interest in soft tissue and nerve lesions increased. When the USA was fighting in World War I, Stout served in Liverpool, England and in France. He was impressed by Ewing’s “Neoplastic Diseases” book, since he found many tumors in the book that he was not familiar with. Although Stout had to perform many autopsies, he was more attracted to biopsy diagnoses and in the diagnoses from resected tumors in living patients compared to the autopsy findings. With the resected specimens he could evaluate the adequacy of the resection margins, the tumor characteristics, and the metastasizing ability of the tumors. He also had the ability to follow the patient to learn more about the behavior of the tumors. He began preparing to write a book on the pathology of cancer around 1928. When he went to Paris with his family in1928, he would spend every morning in the medical school library at the Rue des Ecoles [22, 23]. In the fall, the family moved to the Riviera in Cannes and Stout continued his daily work on the book in the mornings.

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When they returned to New York, he became the head of surgical pathology and the daily demands of his duties delayed completion of the book for a few years. When the book was finally published in 1932, it was well received by clinicians as well as pathologists [24]. It provided information not only about pathological diagnoses, but correlated these with the origin of specific tumors, symptoms reported by the patients, and the different modalities of treatment. One of Stout’s future collaborators, Margaret Murray, was an expert in culturing cells and she had studied absorption of calcium in normal tissues. Stout and Murray became collaborators and they performed extensive studies on the histogenesis of tumors based partly on her cell culture systems. His interest in tumors of the peripheral nerves continued to increase and it extended to other soft tissue tumors including glomus tumors and subcutaneous leiomyomas. Together with data from their cell culture studies, he and Murray described hemangiopericytomas and mesenchymal tumors with pericytes [25]. With their rigorous in vitro studies of many tumors, Stout and Murray were able to show that some tumors arose from the lining of the pleura or mesothelium and these were designated as mesotheliomas. As his fame increased, Stout continued receiving many consultation cases from the USA and many other countries. He dedicated a great deal of time to processing these cases and responding to the submitters. He would often spend Saturday and Sunday mornings working on his consultation cases. He retired from the Department of Surgery in 1951 and from the Physician and Surgeons Department of Pathology in 1954, becoming an Emeritus Professor. After retirement he continued to maintain his interest in soft tissue tumors ranging from fibromatosis, a term that he had introduced, to fibrosarcomas. He used to comment that he has saved the limbs of many children with fibromatosis by making this diagnosis instead of a more malignant lesion. Stout died on December 20,1967. One of the many honors that he enjoyed was the Arthur Purdy Stout Pathology Club, which expanded to the Arthur Purdy Stout Society of Pathology in subsequent years. This society continues to attract many members at the annual meeting with the US and Canadian Academy of Pathology. This is one of the largest groups in attendance with more than 1000 pathologists attending the Arthur Purdy Stout Society presentations.

Juan Rosai (1940–2020) Juan Rosai was born in Poppi, Italy, a small town close to Florence, during World War II in 1940 [26–28]. At the end of World War II, his family moved to Argentina because of the poor economic conditions in Italy at that time. Juan was 8 years old at that time. His younger brother was born in Argentina and went on to become a lawyer. Juan fractured his femur while riding a bicycle when he was around age 13. It took a long time to recover and this was probably one of the influences that led him into a career in medicine. He was an outstanding student and enrolled in medical school at age 15. At the University of Buenos Aires, the freshman class in medical school was very large with over 4000 enrolled students. It was difficult to interact

Juan Rosai (1940–2020)

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with the professors in the first few years. Juan came under the influence of Professor Eduardo Lascano, who taught pathology in his third year of medical school. His interactions with Professor Lascano were a major influence in his decision to pursue pathology as a medical specialty. He completed medical school at age 21 and started his residency training in pathology under Professor Lascano’s mentorship. After a few years of residency training at the regional Hospital of Mar del Plata, Rosai was considering immigrating to the USA.  Coincidentally, Dr. Lauren Ackerman, a famous American Pathologist who was at Washington University in Missouri, was visiting Argentina around this time and Juan interviewed with him and expressed his interest in studying pathology in the USA. Dr. Ackerman agreed and Juan immigrated to the USA and started another residency training program in pathology at Washington University, St Louis. After completing his residency and fellowship in pathology, he was persuaded by the Chairman of the Department, Dr. Lacy, to acquire some additional training in the basic sciences. Rosai spent a year at the National Institutes of Health in Dr. Vincent Marchesi’s laboratory. However, Juan decided that he enjoyed diagnostic surgical pathology a lot more than basic research, so he returned to Washington University and joined the pathology faculty in surgical pathology. He moved to the University of Minnesota as the Director of Anatomic Pathology a few years later. He increased the quality of the faculty in diagnostic pathology at the University of Minnesota with outstanding pathologists such as Dr. Louis Dehner and Dr. Mark Wick [28, 29]. Rosai took a 1-year sabbatical to Italy at the University of Florence and the University of Bologna from 1982 to 1983. He did a major study of poorly differentiated thyroid carcinomas [30] and several other projects during that time. He worked with Dr. Carcangiu on the thyroid project and they fell in love and married. After returning to Minnesota for a few years, he moved to New Haven to become the Director of Anatomic Pathology at Yale University Medical Center in 1985. His diagnostic and teaching skills continued to increase. In 1991, he was recruited to be the Chairman of Pathology and the James Ewing Alumni Professor at Memorial Sloan Kettering Cancer Center in New York. His reputation as a brilliant diagnostic oncologic pathologist and teacher continued to grow. His photographic memory about cases that he had seen previously and about publications that he had read was legendary. He had many young pathologists seeking to spend some time with him as a Fellow at Memorial Sloan Kettering Cancer Center. In 2000, he returned to Italy as Chairman of the Department of Anatomic Pathology at the Instituto Nazionale del Tumori in Milan. In 2005, he created the Centro Diagnostico Italiano in Milan and continued his consultation and teaching activities in oncologic surgical pathology. Dr. Rosai was an outstanding surgical pathologist who described various new entities in surgical pathology including sinus histiocytosis with massive lymphadenopathy (Rosai–Dorfman disease) [31]. This benign condition, which affects people of color more commonly, was not known before he and Dr. Dorfman described the first four cases. Some cases had been misdiagnosed and the patients were treated as if they had a malignant neoplasm because of the enlarged lymph nodes in the thorax and other parts of the body [31]. He and his colleagues also described another new

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entity—desmoplastic small cell tumor which occurred mainly in the abdomen of young men [32]. The team led by Dr. Rosai was able to elucidate the immunohistochemical and molecular features of this new disease. (These features are also used to assist in the diagnosis.) The list of other conditions and diseases that he described is extensive and included spindle and epithelial cell tumors with thymus-like differentiation, another new entity [33]. Rosai also had a major impact on pathology as the appointed successor to Dr. Ackerman to update his textbook which was later named Rosai and Ackerman’s Surgical Pathology. Dr. Ackerman had created the book and wrote the first five editions. Dr. Rosai continued from the sixth to the tenth editions and expanded the book greatly over many years as knowledge in surgical pathology increased exponentially. The current two-volume book remains the main reference book for residents training in pathology. Most of the textbook is written and illustrated by the author, although a few chapters are written by other experts in their areas of expertise. Based on the many seminars and consultations that he performed over many years, Dr. Rosai collected, organized, and then donated the Juan Rosai Collection of Surgical Pathology Seminars to the US and Canadian Academy of Pathology. Most of the slide seminars that he presented over many decades are available as digital pathology files which can be accessioned by pathologists in many countries (“The Juan Rosai Collection” http:/www.rosaicollection.net/). He made other major contributions to pathology including serving as editor-in-chief of the third series of the Atlas of Tumor Pathology of the Armed Forces Institute of Pathology. He was the lead author for the “Fascicles on Tumors of the Thymus and Tumors of the Thyroid Gland.” He was also the editor of a major book on the history of American surgical pathology, “Guiding the Surgeons Hands.” It is safe to say that Dr. Juan Rosai’s contributions to surgical pathology have had the greatest effects on the teaching and practice of pathology in the past half a century. Dr. Rosai died of Parkinson’s disease on July 7, 2020.

References 1. Fechner RE.  The birth and evolution of American surgical pathology. In: Rosai J, editor. Guiding the Surgeon’s Hand: the history of American surgical pathology. Washington, DC: American Registry of Pathology; 1997. p. 7–21. 2. Woolner LB. Surgical pathology at the Mayo Clinic. In: Rosai J, editor. Guiding the Surgeon’s Hand: the history of American surgical pathology. Washington, DC: American Registry of Pathology; 1997. p. 145–79. 3. Carter D.  Surgical pathology at Johns Hopkins. In: Rosai J, editor. Guiding the Surgeon’s Hand: the history of American surgical pathology. Washington, DC: American Registry of Pathology; 1997. p. 23–9. 4. Wilson LB. A method for the rapid preparation of fresh tissues for the microscope. J Am Med Assoc. 1905;45:1737.

References

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5. Bloodgood JC. Senile parenchymatous hypertrophy of female breast. Its relationship to cyst formation and carcinoma. Surg Gynecol Obstet. 1906;3:721–30. 6. MacCarty WC. Carcinoma of the breast. Trans S Surg Gynecol Assoc. 1911;23:262–70. 7. Crowell BC. The relationship of clinical pathologists to the cancer problem (Editorial). Am J Clin Pathol. 1931;1:477–81. 8. Ames V, Britton PD.  Stereotactically guided breast biopsy. A review. Insights Imag. 2011;2(2):171–6. 9. Bagnall MJ, Evans AJ, Wilson AR, et al. When has mammographic calcification been adequately sampled at needle core biopsy? Clin Radiol. 2000;55(7):548–53. 10. Hunt JL, Swalsky P, Sasatomi E, et al. A microdissection and molecular genotyping assay to confer in the identity of tissue floaters in paraffin-embedded tissue blocks. Arch Pathol Lab Med. 2003;127(2):213–7. 11. Hunt JL.  Identifying cross contaminants and specimen mix-ups in surgical pathology. Adv Anat Pathol. 2008;15(4):211–7. 12. Pfeifer JD, Zehnbauer B, Payton J.  The changing spectrum of DNA-based specimen provenance testing in surgical pathology. Am J Clin Pathol. 2011;135(1):132–8. 13. Farahani N, Pantanowitz L. Overview of telepathology. Surg Pathol Clin. 2015;8(2):223–31. 14. Laurent-Bellue A, Poullier E, Pomorel J-F, Adret E, Reduri M-J, Possere K, et al. Four-year experience of digital slide telepathology for intraoperative frozen section consultations in a two-site French Academic Department of Pathology. Am J Clin Pathol. 2020;154(3):414–23. 15. Kayper K, Beyer M, Blum S, et al. Recent developments and present status of telepathology. Anal Cell Pathol. 2000;21(31):101–6. 16. Coetzee A. Ewing, James (1866–1943). In: van den Tweel JG, editor. Pioneers in pathology. van Krieken JHJM, series editor. Encyclopedia of pathology. Cham: Springer; 2017. p. 164–7. 17. Koss LG, Lieberman PH.  Surgical pathology at Memorial Sloan Kettering Cancer Center. In: Rosai J, editor. Guiding the Surgeon’s Hand: the history of American surgical pathology. Washington. DC: American Registry of Pathology; 1997. p. 61–85. 18. Del Regato JA.  James Ewing “Oncology…The most complex and fascinating field of Pathology”. Int J Radiol Oncol Biol Phys. 1977;2:185–98. 19. Ewing J. Diffuse endothelioma of bone. Proc N Y Pathol Soc. 1921;21:17–21. 20. Huvos AE.  James Ewing MD.  Contemporary oncologist exemplar. Arch Surg. 1998;122:1240–3. 21. Nguyen DP, Farre X. Stout Arthur Purdy (1885–1967). In: van den Tweel JG, editor. Pioneers in pathology. van Krieken JHJM, series editor. Encyclopedia of pathology. Cham: Springer; 2017. p. 496–9. 22. Stout AP. Notes on the education of an oncological surgical pathologist: the autobiography of Arthur Purdy Stout. In: Rosai J, editor. Guiding the Surgeon’s Hand: the history of American surgical pathology. Washington, DC: American Registry of Pathology; 1997. p. 197–274. 23. Del Regato JA. Arthur Purdy Stout. Int J Radiat Oncol Biol Phys. 1989;14:799–812. 24. Stout AP.  Human Cancer. Etiological factors, precancerous lesions, growth; spread; symptoms, prognosis; principles of treatment. Philadelphia: Lea and Febiger; 1932. 25. Stout AP, Murray MR. Hemangiopericytoma. A vascular tumor featuring Zimmerman’s pericytes. Ann Surg. 1942;116:26–33. 26. Armstrong S. A matter of life and death. Conversations with pathologists. Wiltshire: Dundee University Press; 2008. p. 94–112. 27. Conversations with Pathologists. Interview with Juan Rosai: full transcript. March 12, 2008. Available from: https://conversations.pathsoc.org/index.php?option=com_ content&view=article&id=86:juan-­rosai-­full-­transcript&catid=17&Itemid=200. Accessed 3 Nov 2021. 28. Klimstra DS, Young RH.  Juan Rosai, MD (1940–2020) A tribute. Am J Surg Pathol. 2021;45(12):e24–34. 29. Dehner LP. Juan Rosai, MD. Arch Pathol Lab Med. 2021;145(2):135–6.

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30. Carcangui ML, Zampi G, Rosai J. Poorly differentiated (“insular”) thyroid carcinoma. A reinterpretation of Langerhans “Wochenende struma”. Am J Surg Pathol. 1984;8(9):665–8. 31. Rosai J, Dorfman RF. Sinus histiocytosis with massive lymphadenopathy. A newly recognized benign clinicopathological entity. Arch Pathol. 1969;87(1):63–70. 32. Gerald WL, Rosai J.  Desmoplastic small cell tumor with divergent differentiation. Pediatr Pathol. 1989;9(2):177–83. 33. Chan JK, Rosai J. Tumors of the neck showing thymic or related branchial pouch differentiation: a unifying concept. Hum Pathol. 1991;22(4):349–67.

Chapter 4

Cytopathology

Cytopathologists make diagnoses by examining exfoliated or aspirated cells, especially cancer cells. This field was established many decades ago and Dr. Papanicolaou was one of the pioneers in cytopathology in the 1940s. Exfoliated cells consist of cells from body fluids, brushings, washings, lavages, and scrape cytology. Aspiration cytology which analyzes deep-seated cells are sampled from targets which are located and sampled through modern imaging techniques such as ultrasound or computerized axial tomography scans. These procedures are usually done by radiologist, but cytopathologist in many centers perform some of the superficial biopsies. It is the cytopathologist that interprets the results of the aspiration biopsy to decide if the lesion is reactive, inflammatory, infectious, or neoplastic. Study of cancer cells for diagnostic purposes had been described in many types of specimens by the end of the nineteenth century [1, 2], but it was not until the work of Papanicolaou and Trout in the early 1940s that cytopathology became more influential in medicine and in pathology [2]. Other advances included the development and uses of aspiration cytology in the 1980s, the automated screening systems in the 1990s, and the application of immunohistochemistry, molecular diagnosis, and telecytology to cytopathology in the last few decades. Basic cytological staining relies on the Papanicolaou and the Romanowsky stains. The Romanowsky stain is also used for studies of bone marrow cells and blood cells as well as cytology, and there are some issues that may still develop in experienced laboratories such as problems in standardization and problems with instability of acidic dyes-basic dye mixtures [3]. One area of cytopathology that has seen a great deal of progress in the last few decades has been in thyroid cytopathology. Thyroid nodules are fairly common and more than 50% of a given population have at least one thyroid nodule. Thyroid fine-­ needle aspiration (FNA) has become one of the most commonly practiced areas in non-gynecologic cytopathology. Although thyroid nodules are relatively common, most thyroid nodules are benign, so it is important to have a simple and reliable © The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 R. V. Lloyd, Pathology: Historical and Contemporary Aspects, https://doi.org/10.1007/978-3-031-39554-3_4

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method to distinguish benign from malignant nodules. Today, the combination of cytological diagnosis along with radiologic imaging, serology, and molecular studies can all be used to separate benign and malignant thyroid nodules. With this combined approach the incidence of finding thyroid cancer in an FNA has increased from around 14% to greater than 50% in the last two decades [4, 5]. Thyroid cytopathology has included a multidisciplinary approach with the assistance of the pathologist, radiologist, endocrinologist, and head and neck surgeon. The different diagnostic categories used in evaluating the thyroid cytology FNA with the Bethesda system which was officially adopted around 2009 includes (1) nondiagnostic, (2) benign, (3) atypia of undetermined significance, (4) suspicious for follicular neoplasm, (5) suspicious for malignancy, and (6) malignant [4, 5]. The most challenging category in the Bethesda system is the atypia of undetermined significance and this is where recent developments in molecular diagnostic have been very helpful in resolving cases with this diagnosis as will be discussed later. The development of ancillary diagnostic techniques adopted from the basic sciences has helped to establish cytopathology on a firmer footing. For example, flow cytometry, immunohistochemistry, image analysis, fluorescence in situ hybridization (FISH), karyotyping, and molecular diagnostics have all contributed to advances in diagnostic cytopathology [6]. The use of flow cytometric immunophenotyping of cancer cells in effusions specimens has facilitated the diagnosis of metastatic adenocarcinomas, malignant mesotheliomas, and lymphomas especially when using a broad panel of antibodies [7, 8]. Research cytopathologists have used EGFR mutation testing by next-generation sequencing and anaplastic lymphoma kinase (ALK), gene rearrangement testing by FISH in non-small-cell lung carcinoma with liquid-­ based cytology specimens to assist in the diagnosis of difficult cases. Thyroid cytology tests for malignancy in thyroid nodules have used BRAF V600E point mutations along with several mutational panels such as RAS genes. In addition, RET/PTC and PAX8/PPAR gamma rearrangements have been used for analysis of thyroid specimens. Comprehensive multigene next-generational sequencing with Thyroseq or markers of prognosis such as TERT promoter mutations and other gene mutations have been used to assist in the workup of thyroid aspiration specimens [9, 10]. Another area that has assisted in the advancement of cytopathology as a diagnostic tool is telecytology which uses digital transfer of images from distant sites to assist in cytological diagnoses [11]. This approach makes possible the electronic transmission of microscopic images using static dynamic and whole slide imaging systems. It has also been used to evaluate fine-needle aspiration (FNA) biopsies for diagnosis. Telecytology has also been used for rendering second opinions about specific cases, quality assurance, proficiency testing, slide archiving, and distance-­ based evaluations when individuals are at two different hospitals or other locations [12]. In one study, telecytology with rapid on-site evaluation reduced the unsatisfactory rate in examining FNA specimens from 8.8% to 1.6% after on-site evaluation and to 3.8% after telecytology evaluation. This was much better than ultrasound-­ guided evaluation in which the unsatisfactory rate was not significantly reduced in the same institution [13].

George Papanicolaou (1883–1962)

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Although many outstanding cytopathologists have contributed progress in this field, a few individuals have stood out from the others. The next section will summarize the lives of two of these outstanding individuals who have contributed greatly to progress in cytopathology.

George Papanicolaou (1883–1962) George Papanicolaou was a pioneer in cytopathology. His interest in this field began with his early studies of the physiology and cytology of the female reproductive tract in guinea pigs and in humans. Papanicolaou was born in Kymi, Greece on the island of Euboca in 1883. His father was a medical doctor and also served as the mayor of the town [14]. George was one of four siblings. He and his siblings attended school in Athens. He was a good student and started at the University of Athens at age 15. He subsequently graduated with honors at age 21 [14–16]. He enjoyed music especially playing the violin. After college, he served in the Greek military for 2 years as an assistant surgeon. His father tried to get him to remain in the military as a physician, but George had dreamed of a career in medicine as a scientific researcher. In 1907, he studied in Germany under Professor Haecke. He continued his studies under Dr. Weismann in genetics. He also obtained graduate school training with Dr. Goldschmidt in Munich, Germany and became a skilled microscopist. He completed his doctorate thesis around this time. Around this time, he married Andromache. They spent many years together as scientific collaborators. His wife would also become one of his research subjects and contribute to his cytological studies of female reproduction. He and his wife emigrated to the USA in 1913 [14, 15]. This was a bold move, since they did not have any financial savings and they did not speak English at the time. However, they were both intellectually gifted, worked very hard, and were eventually successful. Dr. Papanicolaou was recommended for a position at New York Hospital as a technician by someone who knew him based on his research work for his doctoral dissertation. He started out as a technician in the Pathology Department at New York Hospital, but was soon made an assistant professor of anatomy at Cornell Medical School based on his outstanding performance in the laboratory. He continued his research studies using guinea pigs as a research model as he had previously done for his doctoral degree. His research questions required knowledge about the ovulation of guinea pigs. However, not very much information was available about the ovulatory cycle of guinea pigs so Papanicolaou devised a method to examine the vaginal cells of guinea pigs to learn about their ovulatory cycle. He approached this by collecting vaginal cells and examining them under the microscope. His careful and meticulous studies revealed a sequence of distinct cell types and he saw cytological changes with time. Because he was also interested in these changes in humans, his wife, Andromache, reportedly served as his human guinea pig and provided vaginal smears and this was presumably the first Pap smear [4, 5]. His studies were extensive and he was able to

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organize the cytological changes and correlate them with endometrial changes in the female genital tract in both guinea pigs and humans [17, 18]. He expanded his studies in humans to include smears from female workers at the New York Women’s Hospital. Fortuitously his studies in females included some patients that had cancer or who subsequently developed cancer. The findings were first presented at a conference in Battle Creek, Michigan [19, 20]. However, it was not well received. There was skepticism among pathologists who thought that a biopsy with tissue in which there was distinct architectural features and not just single cells or cell clusters without architectural orientation was required to make a diagnosis of malignancy. Extensive studies were done by Papanicolaou and his collaborators before his findings were finally accepted as capable of diagnosing cancer after they were published in 1941 [6]. Dr. Papanicolaou published the definitive atlas more than a decade later when he was 71 years old and was at that time an Emeritus Professor at Cornell University Medical School. In the early 1960s, his work was nominated for the Nobel Prize at least twice, but he did not receive this prestigious award in spite of the revolutionary changes in pathology that resulted from his work. One possible reason for not receiving the Nobel Prize was suggested below. However, he received many other awards from multiple countries including the United Nations Award in 1962. Many original discoveries or observations are sometimes made by different research groups around the same time. In the case of Dr. Papanicolaou, it had been noted that observations by a British Physician Walter Hayle Walshe had observed malignant cytological specimens as summarized in a book about lung diseases many years earlier [16]. More importantly, a Rumanian doctor, Dr. Aurel Babes, had used a loop to collect cells from the cervix for cytological examination and to diagnose malignant cells. However, this was not mentioned by Papanicolaou in any of his publications, although it is not known if Dr. Papanicolaou had read any of the publications of Dr. Babes. According to at least one expert, even though Papanicolaou’s experiments and interpretations may have been better than Dr. Babes, the failure of Papanicolaou to cite the work of Babes may have been one of the reasons that he did not receive the Nobel Prize for his work. However, the truth may never be known. Interestingly, the Pap technique in Rumania is referred to as the Methode BabesPapanicolaou [16] which gives credit to Babes for his research and observations. Papanicolaou was described as a hardworking scientist who worked 7  days a week and did not take vacations. After working 50 years at Cornell, he moved to Miami to be in charge of the Cancer Institute of Miami. Unfortunately, he died a few months after arriving in Miami. The name of the Cancer Institute at this institution was renamed as the Papanicolaou Cancer Research Institute [15].

Leopold G. Koss (1920–2012) Leopold Koss was born in Langfuhr, Poland in 1920. He attended middle school (gymnasium) in Lodz, Poland, and graduated in 1938. He then spent the following year at the University of Vienna [21–23]. As a first-year medical student, he observed

Leopold G. Koss (1920–2012)

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the annexation of Austria by Germany as World War II approached. Since Austria was under the influence of Germany, Koss transferred to the Free University of Brussels in Belgium. He returned home the following July and was present when the Germans attacked Poland on September 1, 1939. Koss was able to escape from Poland to Belgium and witnessed the fall of the Polish Army in France. When France surrendered to Hitler in June 1940, Koss travelled to Montpellier to continue medical school. He spent most of that academic year as a farm hand and learned very little medicine [24]. He was able to transfer to the University of Bern and was fortunate to have completed medical school in 1946. His parents and sister were all killed during the Holocaust. Koss’ interest in pathology began when he was at the University of Bern in the Department of Anatomy under the influence of Professor Hans Bluntschli, a comparative anatomist. Koss was assigned to study the ovaries of a primitive mammal, a small insectivore, from Madagascar. His job was to review hundreds of sections of ovaries stained with a special stain. At the end of the study, his findings were submitted for his doctoral thesis. He noted that the extensive studies of the ovaries increased his interest in microscopy and pathology. Koss migrated to the USA in 1947. He did his residency training in pathology at Kings County Hospital in Brooklyn, New York, and then spent several years in the Department of Pathology before moving to Memorial Sloan Kettering Cancer Center in the Department of Pathology. At Memorial, he became heavily involved in cytopathology which was an evolving new area of pathology and he came under the influence of Dr. Papanicolaou who worked across the street at Cornell Medical Center. While working with Papanicolaou, the young Dr. Koss noted that Papanicolaou’s techniques were accepted by the gynecologists, but not by the pathologists who were the experts in interpreting tissue abnormalities. Dr. Papanicolaou was an excellent and creative physician and researcher, but he had not had any formal training in pathology. Dr. Koss wrote that Dr. Papanicolaou was an insecure diagnostician who did not use clear terminology to describe his findings [24]. He divided his diagnoses into classes from I to V. On the other hand, the underlying pathology was not appreciated by individuals interpreting the smears and the pathologists had only a vague idea about the cytological appearance, so there were difficulties in communication between pathologists and cytopathologists [24, 25]. Koss saw these difficulties and decided to write a book in which he hoped to clarify the relationship of the two groups [26]. With his book on Diagnostic Cytology and Its Histopathologic Basis [26], he was able to combine cytopathology and histopathology into a single entity combining cells with tissues. His book also helped to clarify the origin of many human cancers. His main subspecialty interests were in cancers on the uterine cervix and bladder. He published the first comprehensive textbook of cytology in 1961 which covered all branches of cytology. The textbook rapidly became the standard textbook and went through several editions. After leaving Memorial Hospital, Dr. Koss spent some time at the University of Maryland and at Thomas Jefferson Hospital before going to Montefiore Hospital in New  York in 1973 as the chairman of pathology with a professorship at Albert Einstein College of Medicine where he remained until retiring in 2002.

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Dr. Koss worked with Grace Durfee and they were the first persons to recognize a cervical abnormality that they designated as koilocytic atypia. They designated the corresponding changes in tissue sections as warty atypia, because of the similarity in appearance to skin warts with halo cells which were considered as precancerous. A colleague of Koss had used the term halo cells to describe these unique cells. It was later suggested that these cells were pathognomonic of human papilloma virus infection [24]. Dr. Koss received many honors including an honorary fellowship from the Royal College of Pathologists and the Medal of Officer of the Order of Merit from the Republic of Poland. Dr. Koss died in 2012.

References 1. Wang H, Jhala N.  The evolving field of cytopathology and its expanding role in pathology practice. Arch Pathol Lab Med. 2019;143(6):662–3. 2. Naylor B. The century for cytopathology. Acta Cytol. 2000;44(5):709–25. 3. Krafts KP, Pambuccian SE. Romanowsky staining in cytopathology: history, advantages and limitations. Biotech Histochem. 2011;86(2):82–93. 4. Cibas ES, Ali SZ.  The Bethesda System for reporting cytopathology. Am J Clin Pathol. 2009;132:658–65. 5. Ali SZ. Thyroid cytopathology: Bethesda and Beyond. Acta Cytol. 2011;55:4–12. 6. Baloch ZW, Gupta PK. Cytopathology comes of age. Acta Cytol. 2020;64(1-2):5–6. 7. Davidson B, Dong HP, Holth A, et  al. Flow cytometric immunophenotyping of cancer cells in effusion specimens: diagnostic and research applications. Diagn Cytopathol. 2007;35(9):568–78. 8. Davidson B, Dong HP, Berner A, et al. The diagnostic and research applications of flow cytometry in cytopathology. Diagn Cytopathol. 2012;40(6):525–35. 9. Ohori NP, Nikiforova MN, Schoedel KE, et al. Contribution of molecular testing to thyroid fine-needle aspiration cytology of follicular lesions of undetermined significance/atypia of undetermined significance. Cancer Cytopathol. 2010;118:17–23. 10. Poller DN, Glaysher S.  Molecular pathology and thyroid FNA.  Cytopathology. 2017;28(6):475–81. 11. Khurana KK.  Telecytology and its evolving role in cytopathology. Diagn Cytopathol. 2012;40(6):498–502. 12. Lin DM, Tracht J, Rosenblum F, et  al. Rapid-on-site evaluation with telecytology significantly reduced unsatisfactory role of thyroid fine-needle aspiration. Am J Clin Pathol. 2020;153(3):342–5. 13. Witt BL, Schmidt RL. Rapid onsite evaluation improves the adequacy of the fine- needle aspiration for thyroid lesions: a systematic review and meta-analysis. Thyroid. 2013;23:428–35. 14. Chantziantoniou N, Al-Abbadi MA. Papanicolaou, Georgios (1883–1962). In: van den Tweel JG, editor. Pioneers in pathology. van Krieken JHJM, series editor. Encyclopedia of pathology. Springer; 2017, p. 424–7. 15. Vilos GA.  Dr. Georgios Papanicoleau and the birth of the Pap test. Obstet Gynecol Surg. 1999;54(8):481–3. 16. Ton SY, Tatsumma Y.  Georgios Papanicolaou (1883–1962): discoverer of the Pap smear. Singap Med J. 2015;56(10):586–7. 17. Stockard CR, Papanicolaou GN. The existence of a typical oestrous cycle in the guinea pig with a study of its histological and physiological change. Science. 1917;46:42–4.

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18. Papanicolaou GN. The sexual cycle in the human female as revealed by vaginal smear. Am J Anat. 1933;52(Suppl):519–637. 19. Papanicolaou GN, Traut HF.  The diagnostic value of vaginal smears in carcinoma of the uterus. Am J Obstet Gynecol. 1941;42:193–206. 20. Papanicolaou GN, Traut HF. The demonstration of malignant cells in vaginal smears and its relation to the diagnosis of carcinoma of the uterus. N Y State J Med. 1943;43:767–8. 21. Al-Abbadi M.  History of cytopathology. In: van den Tweel JG, editor. Pioneers in pathology. van Krieken JHJM, series editor. Encyclopedia of pathology. Cham: Springer; 2017. p. 228–36. 22. Waisman J. Koss Leopold G (1920–2012). In: van den Tweel JG, editor. Pioneers in pathology. van Krieken JHJM, series editor. Encyclopedia of pathology. Cham: Springer; 2017. p. 300–1. 23. Nelson B. A career with “No regrets” Leopold Koss, MD, reminisces about his role in the history of cytopathology. Cancer Cytopathol. 2010;118(5):227–8. 24. Koss LG.  On being a pathologist. Of tissues, cells and molecules: reminiscences of an old pathologist. Hum Pathol. 2007;38(10):1447–53. 25. Sanchez M. A personal memory of Leo Koss. Cancer Cytopathol. 2012;120(6):419–26. 26. Koss LG. Diagnostic cytology and its histopathologic basis. Philadelphia: Lippincott; 1961.

Chapter 5

Autopsy and Forensic Pathology

The development of the autopsy as a technique to directly observe abnormalities or disease processes while dissecting the human body was dependent on the appreciation of normal human anatomy [1–3]. The Alexandrian scientist Herophilus (395–280 BC) was one of the first scientists to dissect the human body. Knowledge of normal human anatomy increased slowly in ancient times and it was not until the Middle Ages that this knowledge had a major impact on medical theory [3]. Egyptian embalmers, who were thought to be from the lower class, probably saw a range of abnormalities as they were removing human organs during the embalming process. However, their observations were probably not communicated to the priests and other upper-class individuals who would have been the individuals that would have been able to evaluate these findings and write about them in a scientific manner [3]. During the Middle Ages, Antonio Benivieni (1443–1502) from Florence, Italy, wrote several medical manuscripts including cases with autopsies that were focused on causes of diseases [1, 2]. Theophile Bonet (1620–1689) from Genoa, Italy, wrote about anatomical investigations on dead bodies impaired by diseases in 1679 [2]. Giovanni Batista Morgagni (1682–1710) who studied medicine as well as philosophy in Bologna was an assistant to Valsalva and he succeeded him as a demonstrator in anatomy, and published a book on The Seats and Causes of Diseases Through Anatomical Investigation in 1761. He described a series of autopsies in which he was able to correlate the patient’s symptoms with pathological findings during the autopsy. This novel approach was different from the traditional approach of Hippocrates’ and Galen’s humoral theory and emphasized the role of pathological anatomy in explaining diseases observed at the time of performing the autopsy. Bichat Marie-F-Xav (1771–1802) was a brilliant student who unfortunately died at a very young age. He carefully examined various tissues at autopsy and saw the effects of physical changes such as boiling, freezing, and putrefaction the tissues. Using this approach, he was able to categorize 21 different tissue types. His findings emphasized that diseases were tissue-based [1]. Matthew Baillie, who was the nephew of John and William Hunter, observed many autopsies performed by his © The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 R. V. Lloyd, Pathology: Historical and Contemporary Aspects, https://doi.org/10.1007/978-3-031-39554-3_5

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uncles and he also performed many autopsies. He subsequently wrote a book The Morbid Anatomy of Some of the Most Important Parts of the Human Body. This text provided new insights into pathological anatomy [1]. Carl Rokitansky (1804–1878), considered by many to be the father of the autopsy, used a very systematic approach in performing thousands of autopsies to advance many concepts in gross pathological anatomy. His findings and teachings led to the acceptance of the autopsy as a standard procedure in medicine. Even today, the Rokitansky method of performing autopsies remains a standard protocol. Rokitansky was not as conversant with microscopic findings during autopsy studies as his successor, Rudolf Virchow (1821–1902), who combined gross and microscopic autopsy findings to further advance the field of the autopsy study of diseases. In the twentieth century, the autopsy continued to contribute to progress in medicine. Observations at the autopsy table were closely linked to discoveries in infectious diseases in microbiology and to laboratory research in experimental pathology such as studies in atherosclerosis or hardening of the arteries. The Flexner report in 1910 [4] helped to raise the standards of medical practice which included the capacity to do pathological examination of specimens and autopsy practices. Starting around 1970 there was a gradual and then steep decline in performance of autopsies—first in private practice hospitals and then in teaching hospitals in the USA and then in many other countries. The changes occurred over many decades. In the 1960s, some hospitals reported performing autopsies in around 70% of patients dying in hospitals where as a few decades later autopsy rates had declined to between 0% and 10% of patients dying in hospitals. Some of the reasons given for this decline have included the following: (1) the increased sophistication in clinical medicine with more advanced instruments such as computerized tomographic (CT) scans, magnetic resonance imaging (MRI), and related techniques such as positive emission tomography (PET) that could detect very small lesions; (2) more sophisticated serum biochemical and molecular testing leading to more accurate and (3) sophisticated diagnoses while the patient was still alive, obviating the need to perform autopsies after the patient died. However, many studies of antemortem and postmortem diagnoses for the past few decades have continued to show that autopsies still find 20–25% of disease processes contributing to the patient’s death that were not diagnosed antemortem [5–9]. In one study, up to 10–13% of the diagnoses made at autopsy could have changed patient management before the patient died [9]. In another study, there was a discrepancy rate of 26% between premortem clinical diagnoses and postmortem findings in cancer patients in a medical-surgical intensive care unit in a tertiary-care cancer center [6]. Thus, the autopsy remains a very efficient tool for quality control in the workup and care of patients. Another reason given for the decline in the autopsy rate in the USA is that the cost of the autopsy is not reimbursed by a third party, so the expense must be covered by the hospital or by a relative. In addition, the Joint Commission for the Accreditation of Hospitals abandoned the requirement for a minimum number of autopsies needed for hospitals to maintain accreditation in the 1970s. This was another reason for the decline in the autopsy rate in the USA.

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In China, the first officially permitted autopsy in modern times was in 1913 [10]. Anatomy and pathology were introduced in the medical curriculum in China around 1840, but the autopsy was prohibited until 1912. The highest autopsy rate was between 1950 and 1970, similar to the USA. Since that time the autopsy rate has declined and ranges from 0% to 2% in major medical centers. The reasons given for the decline in China have included a lack of motivation by clinicians to get permission for autopsies, probably related to some of the same issues in the USA, and a lack of interest by hospital administrators. In China, the hospital autopsy rate in large teaching hospitals is less than 1% [10]. The autopsy has been a source of discovery in cardiovascular and other areas of medicine for a long time [11]. William Harvey, in addition to describing the role of the heart in circulation in the body, observed valves in veins around 1602. His studies of the heart and blood vessels led to the first description of circulation in humans as well as in the deer that he studied in the forest surrounding Windsor Castle [8]. Morgagni reported his studies of over 700 autopsies. In his book On Seats and Causes of Diseases Investigated by Anatomic Dissecting, in which he correlated autopsy findings with the clinical history and studies of autopsies. This led him to recognize several major cardiovascular abnormalities such as atrioventricular block, aortic dissection, cardiac rupture, and atrial septal defects by clinicopathologic correlation studies [8]. This combination of autopsy findings and clinical findings led to the birth of the Clinicopathological Conference first at Harvard Medical School in the USA in 1924 and then this approach spread to most hospitals in the USA and, subsequently, to other countries. The observations about cardiovascular abnormalities at autopsy were all grounded in basic anatomic principles established at the time of the autopsy [11, 12]. For example, Monroe Schlesinger, a pathologist at Beth Israel Hospital in Boston, worked with Herrman Blumgart, who was Chief of Medicine and the father of nuclear cardiology, and in 1938 they investigated the coronary system by X-ray of autopsied hearts injected with lead-based solutions. They described some of the fundamental mechanisms of angina pectoris and acute myocardial infarction. They also described collateral circulation of the coronary arteries and the vascular changes of congestive heart failure and cardiogenic shock [12]. Autopsies in cardiovascular medicine have also been very useful in studies of sudden death both in adults and in pediatric patients. Many of the causes of sudden death in pediatric patients are secondary to genetic heart disorders, resulting in arrythmias such as familial long PT syndrome or structural abnormalities such as in hypertrophic cardiomyopathy [13]. In some cases, these conditions may present as an underlying cardiac problem and the family may be at a loss about why a seemingly healthy youngster died so suddenly and unexpectedly [14]. These developments also present a dilemma to the clinician managing the patient and to the surviving family members. A multidisciplinary approach that includes a cardiologist, clinical geneticist, genetic counselor, and pathologist involved with the autopsy is usually needed for evaluation of the data and management of the surviving family members [13, 14]. The number of patients affected in the USA is quite large with estimates of over 450,000 people each year. Many of the cases may be caused by

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genetic abnormalities ranging from long QT interval as one of the most common findings to Brugada syndrome with over 40 genetic heart diseases described [13]. Management usually involves first identifying the genetic basis of sudden cardiac death [13]. Molecular genetics and molecular diagnostics have added to the diagnostic strengths of many areas of pathology including clinical and surgical pathology. Although some aspects of molecular genetics have been used in forensic pathology, especially DNA identification, many of these techniques have not been incorporated into the routine aspects of autopsy pathology. An expert in molecular genetics, Dr. Jeffrey Sklar at Yale University, has advocated that certain aspects of molecular genetics be incorporated into the routine autopsy to improve patient care and for pathology resident education [15]. He recommends that next-generation sequencing (massive parallel sequencing) and sequencing of large portions of the genomic DNA be done routinely at autopsy [15]. He indicated that genomic DNA remains intact in dead tissues for long periods of time and could be readily extracted or used from formalin-fixed paraffin-embedded tissues. Such an approach would advance personalized or precision medicine for the individual patient [15]. One advantage of broad-based DNA sequencing as part of the routine autopsy is that it may detect inherited mutations and DNA-sequencing polymorphisms that may contribute to disease processes and may provide input about the etiology of specific diseases [15]. One disadvantage of genomic sequencing is that it remains expensive even today (about $3000–4000); however, exome sequencing would be a lot less expensive (around $300–400). Thus, the cost of genomic sequencing would have to decrease significantly to make it a routine procedure as part of the general autopsy [15]. Another new approach in autopsy procedures includes use of the minimally invasive autopsy. The decline in autopsies during the past few decades and the increasing sophistication of imaging techniques have combined to make minimally invasive autopsies more attractive [16, 17]. MRI has been used for fetal, infant, and adult autopsies for the past two decades [13]. Studies with fetuses have been useful because of the distinct pathological changes usually associated with congenital malformations. Imaging and cytogenetics may be more useful than histopathological examination with these malformations. Some disadvantages include the requirement for multiple specialized radiologists to interpret the images for each case and the time that it takes to acquire the images, which is usually at least a few hours [16]. Postmortem MRIs are also more difficult to interpret because of postmortem blood clots and autolysis. Virtopsy, which is a combination of postmortem examination with imaging methods including computed tomography (CT), magnetic resonance imaging (MRI), and three-dimensional imaging, to find the cause of death has been studied as a substitute for the standard autopsy with examination of the whole body. Although it is very time consuming, it is a good substitute when religious issues make the surviving relatives upset about conventional autopsies. Interest in virtopsy has been increasing in some medicolegal cases especially when there could be toxic exposure during the autopsy with an increased risk of contamination. In one study

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using virtopsy with minimally invasive autopsies, the cause of death was found in 77% of cases compared to the conventional autopsy. However, significant disadvantages of minimally invasive autopsies include the failure to detect myocardial infarctions and endocarditis as the causes of death in most patient with these conditions [17, 18]. Forensic pathologists perform autopsies on people who die unexpectantly, suddenly, or violently to determine the cause and manner of death [19, 20]. The cause of death is usually the immediate reason for the cessation of life. While the manner of death determines if the person was killed by homicide, if the death was accidental, if the person committed suicide, or if the death was of natural causes. In a few cases, the forensic pathologist may not be able to determine the cause of death, and the manner of death may remain unknown. The forensic pathologist uses all available information to arrive at their conclusions. This usually includes examination of the person’s history (which in some cases is very brief or nonexistent), analysis of evidence at the crime scene including speaking with witnesses, examination of the body methodically by performance of an autopsy, and collecting and analyzing available evidence including patient history and trace evidence from the patient’s body using rigorous scientific analyses. The analyses may include some or all of the following: firearms/ballistics, toxicology, blood analysis, trace evidence, and DNA analyses. In the USA, forensic autopsies may be performed by the medical examiner who is often a forensic pathologist or by a coroner who is usually an elected or appointed person. Many coroners do not have a medical degree. Forensic pathologists in the USA are educated first by earning a bachelor’s degree, then graduation from medical school after 4 years, performance of residency training in anatomic and clinical pathology for 4  years, followed by fellowship training in forensic pathology for 1 or 2 years. The forensic pathologist in the USA is usually board-­ certified in general pathology and forensic pathology by the American Board of Pathology (Appendix 2). One of the many challenging areas of forensic pathology is to analyze cases of natural and unexpected death occurring within 1  h of new symptoms. The most common causes of these sudden death cases usually involved the cardiovascular system such as coronary heart disease [21]. However, other cardiac diseases such as left ventricular hypertrophy, left ventricular function impairment, and genetic mutations affecting the heart may be involved [21]. Major factors such as past medical history and symptoms before death are important points to investigate. A history of other sudden deaths in the family may suggest a familial cardiac mutation. Whether the person was involved in physical activities is an important part of the history, since catecholamines released from the adrenal glands (epinephrine or norepinephrine) during stressful overactivity may lead to arrythmias and sudden death [21]. Sudden death in younger individuals less than 35 years of age may be related to arrythmias or right ventricular cardiomyopathy [21]. In some cases of sudden death without morphological changes, the pathologist should also consider functional cardiac pathology such as long QT syndrome, Brugada syndrome, or idiopathic ventricular fibrillation. In many of these cases, molecular diagnostic studies may be very useful. During the past two decades, molecular genetic studies have helped to

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solve some of these problems [22, 23]. More recent molecular diagnostic studies such as analysis of microRNA have been useful in forensic molecular analysis. For example, microRNA concentration in the vitreous humor of the eye may help to establish the time of death [22]. Historically, short tandem repeats have been used (and continue to be used) to compare different tissue types to determine if various tissues or fluids are identical (from the same source) by comparing allele repeats at specific DNA loci [23]. Molecular analyses may involve a range of problems ranging from genetic mutations, defects in ion channels, or viral infections that may lead to or contribute to the patient’s death. Although many outstanding pathologists have contributed to the progress in autopsy pathology since Rokitansky and Virchow, two of the most prominent autopsy pathologists in history, many individuals have contributed to the development of forensic pathology. The careers of two of these individuals who have contributed to progress in forensic pathology are summarized in the next section.

DiMaio, Vincent JM (1941–2022) Vincent DiMaio was born in Brooklyn, New York in 1941 during World War II. His father was a forensic pathologist and he and his three sisters all became doctors [24]. He attended St. John’s University and the State University of New York. He received postgraduate training at Duke University in North Carolina and the State University of New York. He then completed a fellowship in forensic pathology at the Office of the Chief Medical Examiner of Maryland [24, 25]. He was board-­ certified in anatomic and clinical pathology as well as in forensic pathology. His expertise in forensic sciences was in gunshot wounds and he wrote many articles and testified in court about forensic cases in his area of expertise. He served in the US Army Medical Corps and was the Chief Medical Examiner of San Antonio, Texas, until he retired in 2006. He was also a professor in the Department of Pathology at the University of Texas Health Science Center in San Antonio, Texas. Dr. DiMaio was the editor-in-chief of The American Journal of Forensic Medicine and Pathology from 1991 to 2017 [26, 27]. He had a productive academic career having authored 4 books and more than 75 original papers on forensic pathology and gunshot wounds [26]. In 2016, he wrote his memoir with Ron Franscell entitled “Morgue: A Life in Death” which was written during his retirement. The book was well received and won an award. He also received numerous awards for his work including the Outstanding Service Award from the National Association of Medical Examiners. He has been involved with many legal cases over the years including high-profile cases such as the exhumation and identification of Lee Harvey Oswald, the individual involved in the assassination of President John Kennedy. Using dental records, they confirmed that Lee Harvey Oswald was the person buried in his grave. He was also involved in the investigation of the death of Vincent van Gough who was thought to have committed suicide. Dr. DiMaio and his team concluded that van Gough was murdered and that his death could have been accidental. He later worked

Bennet I. Omalu (Born 1968)

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with the George Zimmerman murder trial for the killing of 17-year-old Trayvon Martin in 2013. Zimmerman was acquitted for the second-degree murder of Treyvon Martin. Dr. DiMaio died on September 18, 2022. The cause of his demise was attributed to COVID 19.

Bennet I. Omalu (Born 1968) Bennet Omalu was born in southeastern Nigeria, Africa, in 1968. His family left the Igbo region in southeastern Nigeria during the 1968 Civil War in Nigeria and then returned 2 years later. He started school at age 3 and then attended secondary school at the Federal Government College Enugu. He started medical school at the young age of 16 at the University of Nigeria and completed his medical studies in 1990. He then completed an internship and served for 3 years in the medical service in Jos, Nigeria [28]. He subsequently emigrated to the USA in 1993 first studying at the University of Washington in Seattle with an epidemiology fellowship. He subsequently moved to New York City and received residency training at Harlem City Center which was part of Columbia University Medical Center. He then moved to Allegheny County Coroner’s Office in Pittsburgh for training in forensic pathology. In 2002, he also received fellowship training in neuropathology. In 2008, he received a master’s degree in business administration from Carnegie Mellon University. He and his family moved to California in 2007 and he became the Chief Medical Examiner of San Joaquin County and served in this position until 2017. During his training in pathology in 2002, he performed an autopsy on a former football player, Mike Webster, who had died suddenly. His medical record included playing professional football for the Pittsburgh Steelers and a long history of mood disorders, depression, drug abuse, suicide attempts, and cognitive impairment. Omalu observed large amounts of tau protein in the patient’s brain as shown by immunohistochemistry. He and his colleagues published a paper in 2005 as a report of the first documented case of chronic traumatic encephalopathy (CTE) in football players. CTE had been well described in boxers and other athletes after repetitive head injury many years earlier in the 1920s [29]. Omalu and his collaborators documented other cases of CTE in former football players over the next few years [29– 34]. CTE remains a very controversial topic [30, 34] since it is usually diagnosed by neuropathological examination at the time of autopsy and it is difficult to diagnose antemortem. There were conflicts with the National Football League (NFL) as to whether trauma sustained during football playing would lead to CTE. A book by the journalist Jeanne Marie Laskas, Concussion, was later made into a movie with the same title and starred the actor Will Smith, which popularized the controversies. Dr. Omalu is married to Prema Mutiso and they have two children. He is currently a professor of Pathology and Laboratory Medicine at the University of California Davis and also works as an independent pathology consultant [28].

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References 1. van den Tweel JG. History of autopsy. In: van den Tweel JG, editor. Pioneers in pathology. van Krieken JHJM, series editor. Encyclopedia of pathology. Cham: Springer; 2017. p. 224–6. 2. van den Tweel JG, Taylor CR. The rise and fall of the autopsy. Virchows Arch. 2013;462:371–89. 3. King LS, Mechan HC. The history of the autopsy: a review. Am J Pathol. 2013;73(2):514–44. 4. Flexner A.  Medical education in the United States and Canada: a report of the Carnegie Foundation for the Advancement of Teaching, Bulletin 4. New York: The Carnegie Foundation for the Advancement of Teaching; 1911. Reproduced in 1950 5. Burgesser MV, Camps D, Calafat P, Diller A. Discrepancies between clinical diagnosis and autopsy findings. Medicina (B Aires). 2011;71:135–8. 6. Pastores DM, Dulu A, Voigt L, et al. Postmortem clinical diagnoses and postmortem autopsy findings: discrepancies in critically ill cancer patients. Crit Care. 2007;11(2):R48. 7. Lundberg GD. Low-tech autopsies in the era of high-tech medicine: continued value for quality assurance and patient safety. JAMA. 1998;280(14):1273–4. 8. Shojania KG, Burton EC.  The vanishing non-forensic autopsy. N Engl J Med. 2008;358(9):873–5. 9. Kajiwara JK, Zucoloto S, Manco AR, et al. Accuracy of clinical diagnoses in a teaching hospital: a review of 997 autopsies. J Intern Med. 1993;234(2):181–7. 10. Zhu M-H, Yu D-H.  Fluctuations in the rate of autopsy in China. Chin Med J. 2011;124(20):3403–7. 11. Thiene G, Saffitz JE. Autopsy as a source of discovery in cardiovascular medicine. Then and Now. Circulation. 2018;137:2683–5. 12. Schlesinger MJ. An injection plus dissection study of coronary artery occlusion and anastomoses. Am Heart J. 1938;15:528–68. 13. Ingles J, Semasarian C. Sudden cardiac death in the young: a clinical genetic approach. Int Med J. 2007;37(1):32–7. 14. Zheng ZJ, Croft JB, Giles WH, et al. Sudden cardiac death in the United States, 1989–1998. Circulation. 2001;104:2158–63. 15. Sklar J. The clinical autopsy and genomic testing. Am J Pathol. 2019;189(9):1682–5. 16. Sudhin T, O’Cleary JO, Rosemary JS, et al. Post-mortem examination of human fetuses and a comparison of whole-body high-field MRI at 9.4T with conventional MRI and invasive autopsy. Lancet. 2009;374(9688):467–75. 17. Tejaswii KB, Periya EA. Virtopsy (virtual autopsy): a new phase in forensic investigation. J Forensic Dent Sci. 2013;5(2):146–8. 18. Weustink AC, MGM H, van Dikje CF, et  al. Minimally invasive autopsy: an alternative to conventional autopsy? Radiology. 2009;250(3):897–904. 19. DiMaio DJ, DiMaio D. Forensic pathology. 2nd ed. Boca Raton: CRC Press LLC; 2001. 20. Payne-James J, Byard R, Corey TS, Henderson C. Encyclopedia of forensic and legal medicine. London: Elsevier; 2005. 21. De la Grandmaison GL. Is there progress in the autopsy diagnosis of sudden unexpected death in adults? Forensic Sci Int. 2006;156(2-3):138–44. 22. DeMatteis A, DelFante Z, Santoro P. Forensic pathology: past, present, and future. Clin Ter. 2020;171(4):e302–3. 23. Maeda B, Saukko P. Molecular pathology in forensic medicine – introduction. Forensic Sci Int. 2010;20(1-3):3–14. 24. Roberts, S. Dr. Vincent DiMaio, pathologist in notorious murder cases, dies at 81. New York: The New York Times; 2022, October 14. https://www.nytimes.com/2022/10/14/us/dr-­vincent-­ dimaio-­dead.html. Accessed 15 May 2023. 25. Suzannna D. “The scalpel is passed” a conversation with Dr. Vincent JM DiMaio. Am J Forens Med Pathol. 2019;40(3):199–209. 26. DiMiao VJM, Molina K. DiMaio’s forensic pathology. 3rd ed. Boca Raton: CRC Press; 2021.

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27. Parnell WT. My interview with Dr. Vincent JM DiMiao. 2001. https://www.jfk-­assassination. net/parnell/dimaio.htm. Accessed 19 Dec 2021. 28. Bennet O. Curriculum vitae. UC Davis. 2015. https://health.ucdavis.edu/pathology/our_team/ faculty/OmaluB/BennetIOmaluMD,MBA,MPH,CPE,CurriculumVitaeAndTestimonies,Aug ust2015.pdf. Accessed 15 May 2023. 29. Omalu BI, Dekosky ST, Minster RL, et al. Chronic traumatic encephalopathy in a National Football League player. Neurosurgery. 2005;57(1):128–34. 30. McKee AC, Cantu RC, Nowinsk CJ, et al. Chronic traumatic encephalopathy in athletes: progressive tauopathy after repetitive head injury. J Neuropathol Exp Neurol. 2009;68(7):709–35. 31. Omalu BI, DeKosly ST, Hamilton RL, et al. Chronic traumatic encephalopathy in a national football league player. Part II. Neurosurgery. 2006;59(5):1086–92. 32. Omalu BI, Small GW, Bailes J, et al. Postmortem autopsy-confirmation of antemortem (F-18) FDDNP-PET scans in a football player with chronic traumatic encephalopathy. Neurosurgery. 2017;82(2):237–46. 33. Omalu BI, Bailey J, Hammers JL, Fitzsimmoms RP. Chronic traumatic encephalopathy, suicides and parasuicides in professional American athletes: the role of the forensic pathologist. Am J Forensic Med Pathol. 2010;31(2):130–2. 34. Cherry JD, Babcock KJ, Goldstein LE.  Repetitive head trauma induces chronic traumatic encephalopathy by multiple mechanisms. Semin Neurol. 2020;40(4):430–8.

Chapter 6

Gynecologic Pathology

Gynecologic pathology is the subspecialty of surgical pathology that studies and makes diagnoses of diseases of the female genital tract. Some gynecologic pathologists include breast pathology in their subspecialty practice while others focus only on the organs that form part of the female genital tract from the vulva to the ovaries and fallopian tubes. Gynecologic pathologists work closely with gynecologists and radiologists in their subspecialty areas to make diagnoses of difficult and unusual cases. Many studies have shown that sex hormones such as estrogens and specific subtypes of viruses are implicated in the pathogenesis of vaginal and cervical cancers. Studies on the pathogenesis and manifestation of these conditions by pathologists, gynecologists, and other researches have supported these observations.

Clear Cell Adenocarcinoma of the Vagina and Cervix This uncommon disease was associated with intrauterine exposure to diethylstilbestrol (DES) and related drugs. Mothers of the patients with these lesions had been treated with these drugs for threatened abortions and related high-risk pregnancies between 1938 and 1971. Clear cell carcinomas made up about 10% of vaginal malignancies during the peak periods of these diseases. The studies of pathologist Dr. Robert Scully and the gynecologist Dr. Arthur Herbst showed that around two thirds of the patients with these lesions had a history of maternal exposure to DES or related drugs during pregnancy. This was one of the first demonstrations of the epidemiologic relationship between maternal hormone use and cancer developing in female offsprings (Fig. 6.1). Patients with these malignancies had a 5-year survival rate of around 78% [1–3] However, there was a recurrence rate of 23% after 5  years [1]. Recurrences occurred in the pelvis and lungs. The association was established by careful epidemiologic and pathologic studies by Scully and Herbst and their collaborators with the first few cases reported in 1970–1971 [2–4]. Some © The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 R. V. Lloyd, Pathology: Historical and Contemporary Aspects, https://doi.org/10.1007/978-3-031-39554-3_6

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Fig. 6.1  Clear cell adenocarcinoma of cervix. The tumor cells in the biopsy are composed of predominantly eosinophilic cells with occasional clear cells (arrow). The tumor cells have prominent cell borders, oval nuclei, and eosinophilic cytoplasm. Many of these tumors have been associated with maternal diethylstilbesterol exposure during early pregnancy. The premalignant analog for this lesion has not been identified

patients with diethylstilbestrol exposure also developed vaginal adenosis which reflected residual Mullerian ducts from accessory mesonephric ducts in the wall and stroma of the vagina after complete vaginal development. When a series of radical hysterectomies and vaginectomies cases were studied, atypical vaginal adenosis was frequently associated with clear cell adenocarcinoma cases implicating them as precursors of clear cell adenocarcinomas [4]. Clear cell adenocarcinomas of the vagina and cervix have become very uncommon in the past few decades, since diethylstilbestrol and related drugs are no longer used for high-risk pregnancies. However, rare cases of these malignancies are still reported.

Carcinoma of the Cervix Multiple studies have implicated specific viruses in the pathogenesis of some cervical and vulvar cancers in women. Cervical cancer is the second most common cancer in women worldwide. In the USA, the incidence and mortality have decreased in recent decades mainly secondary to cytological screening with PAP smears. Squamous cell carcinoma of the cervix is preceded by varying degrees of dysplasia or precancerous lesions including mild to severe dysplasia (Fig. 6.2). These degrees of dysplasia are also known as cervical intraepithelial neoplasia grades 1, 2, and 3. Dysplasia is often followed by noninvasive carcinoma in situ and then invasive cancer. Some of the subtypes of human papilloma viruses (HPV) have been linked to cervical squamous dysplasia and squamous cell carcinoma of the cervix as well as other types of cervical carcinomas. Morphological recognition of the early changes of HPV infection is termed koilocytosis and is often seen by cytopathologist looking at cervical smears. Many studies have

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Fig. 6.2  Cervical biopsy showing cervical intraepithelial neoplasia I (mild squamous dysplasia). The cells in the lower third of the epithelium are enlarged with an increased nuclear-cytoplasmic ratio. Focal koilocytotic changes (clearing with perinuclear halo) are present in the upper portion of the epithelium

established a relationship between koilocytosis, different degrees of dysplasia, and squamous cell carcinoma of the cervix, especially with HPV types 16,18, 31, 33, 45, and other less common subtypes of HPV [5–7]. HPV has been associated with both squamous cell and adenocarcinomas of the cervix. Although cytological smears can readily detect HPV changes in the cervix infected with human papilloma viruses, a combination of cytological examination and HPV DNA testing usually increases the sensitivity of detecting squamous cell carcinoma to more than 97% [8]. Other viruses may interact with HPV leading to cervical cancer. For example, cervical cancer is the most frequently detected cancer in women having human immunodeficiency virus infections and it is linked to AIDS [9]. Thus, women with human immunodeficiency virus infection have a sixfold increased incidence of developing cervical cancer [9]. HPV vaccines (Gardasil) have been shown to be effective in reducing the incidence of cervical intraepithelial neoplasia 2 and higher degrees of dysplasia and squamous cell carcinomas of the cervix and from other sites [10]. HPV 16 and 18 infections cause about 70% of cervical cancers, while HPV 6 and 11 cause around 90% of genital warts [10]. Thus, the use of HPV vaccines can decrease both cervical cancer development and genital warts [10]. With the approval of the HPV vaccines since 2006 by the US Food and Drug Administration, these vaccines have been very effective in reducing the risk of cervical intraepithelial neoplasia types 2 and 3 as well as invasive squamous cell carcinomas of the cervix [10].

Endometrial Adenocarcinoma Endometrial adenocarcinoma is the most common gynecologic malignancy in the USA and in other developed countries. It is the second most common gynecologic malignancy worldwide. Endometrial adenocarcinoma (Fig. 6.3) is often preceded by varying degrees

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Fig. 6.3  Uterine well-differentiated (grade I) adenocarcinoma composed of increased numbers of back-to-back gland-forming cells with enlarged nuclei and decreased amounts of cytoplasm. Focal infiltration into the underlying stroma is present. This is the most common gynecologic malignancy in developed countries

of endometrial hyperplasia. Risk factors for carcinoma development include endometrial hyperplasia, diabetes, obesity, oral contraceptives, hypertension, exogenous estrogens, diet, and prior pelvic irradiation [11]. Endometrial hyperplasia has been studied extensively by pathologists, since it is associated with an increased risk of progression to carcinoma. Atypical forms of endometrial hyperplasia are considered as premalignant lesions, that is, they may progress to carcinoma [12]. The most common presentation in patients is that of endometrial bleeding. Hyperplasia usually develops from chronic stimulation of estrogens when it is unopposed by progestins. The concept of endometrial intraepithelial neoplasia (EIN) has been adopted by some pathologists as a mutationally activated monoclonal premalignant condition that differs from the unopposed estrogen hypothesis in that EIN represents a direct precursor lesion for the development of endometrial carcinoma and avoids the traditional heterogeneity of the unopposed estrogen stimulation in the usual endometrial hyperplasia model [12]. Molecular analysis of the development of endometrial hyperplasia has included analysis of phosphate and tensin homologue (PTEN), TP53, paired box gene 2 (PAX2), DNA mismatch repair pathways, beta-catenin, and E-cadherin [12]. However, these molecules do not have a well-defined role in the progression from hyperplasia to carcinoma [12]. In addition to estrogen and progesterone, other sex steroid hormones such as androgens and the receptor for all of these hormones may have important roles in the progression from hyperplasia to neoplastic tumor development in endometrial cancer [13].

Serous Ovarian Cancers Ovarian surface epithelial cancers (Figs. 6.4 and 6.5) have traditionally been thought to arise from the ovarian surface epithelium and more specifically from the portion of the ovarian epithelium that has invaginated to produce surface epithelial glands

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Fig. 6.4  Low power view of an ovarian serous high-grade adenocarcinoma with prominent papillary features

Fig. 6.5  High-power view of ovarian papillary serous adenocarcinoma. The tumor cells have a high nuclear-cytoplasmic ratio in a background of complex papillary architecture. These carcinomas are associated with a poor prognosis because they are usually diagnosed late in the course of the disease

and cysts [14]. Another more recent hypothesis for the origin of serous tumors is that they arise from the fimbriated ends of the fallopian tubes and related Mullerian epithelium of the fallopian tubes (Figs. 6.6 and 6.7) [15, 16]. Serous tumors constitute about one fourth of ovarian tumors. About 30–50% of these carcinomas are bilateral. High-grade serous cancers may show papillary, cribriform, and glandular structures with solid growths in areas of necrosis. The carcinomas are usually cystic and solid when examined grossly and under the microscope. The higher-grade cancers are usually mostly solid. Immunohistochemical markers that can be useful in establishing the diagnosis when only a small biopsy is available include CK7, ARIDIA, PAX8, estrogen and progesterone receptors, WT1, and mutant TP53. Calcifications with concentric lamellated calcified structures are usually present microscopically and radiologically. Ovarian cancers are some of the most lethal cancers. The mortality rate for ovarian cancers has not changed very much since the 1980s [17]. Because there are no specific signs and symptoms early on in the development of these cancers, they are usually diagnosed at an advanced stage when they first come to medical attention.

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Fig. 6.6  Low-power view of epithelial lining cells of the fallopian tube showing carcinoma in situ. The carcinoma in situ cells in the tube are hyperchromatic with high nuclear cytoplasmic ratios. This lesion is a precursor for high-grade ovarian papillary serous adenocarcinoma and are sometimes designated as serous tubal intraepithelial carcinoma (STIC)

Fig. 6.7  Higher magnification of fallopian tube with STIC (arrow). These carcinoma in situ cells have BRCA 1 and BRCA 2 mutations and are characterized by p53 positive staining and TP53 mutations

The discovery of serous tubular intraepithelial carcinomas (STICS) in the fallopian tubes of patients who were BRCA mutation carriers was a significant advance in understanding ovarian serous high-grade cancers (Fig. 6.6 and Fig. 6.7). The fallopian tubes in these patients stained for p53 immunohistochemically, suggesting that they had TP53 mutations and this was a significant finding [17]. The distal fallopian tube cells were postulated the be the origin of high-grade serous carcinomas in women with BRACA1 or BRACA2 mutations. Other genes with low penetrance

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such as BARD1, CHECK2, RAD50, and ATM have also been associated with increased risk of developing serous high-grade ovarian cancers. One study has shown that STICS were also found in 52% of patients with sporadic advanced-stage high-grade serous ovarian cancers [18]. Molecular studies have shown that the genomic landscape of high-grade serous ovarian cancers show extensive genomic instability with only a few recurrent genetic mutations other thanTP53. TP53 mutations appear to be ubiquitous in these cancers [19]. TP53 is also mutated in STICs, so it is probably an early event in the development of high-grade ovarian serous cancers.

Endometriosis Endometriosis indicates the presence of endometrial glands and stroma outside of the uterine cavity (Fig. 6.8 and Fig. 6.9) [20]. This occurs in about 10% of reproductive age women and is often associated with pelvic pain and infertility. Adenomyosis refers to the presence of endometrial glands and stroma within the myometrium or smooth muscle of the uterus. The etiology of endometriosis is uncertain, but possible origins include retrograde menstruation, coelomic metaplasia, and Mullerian remnants. The retrograde menstruation hypothesis suggests that endometrial fragments reach the pelvis by trans tubal retrograde flow and are implanted onto the peritoneum and abdominal organs. Proliferation of these tissues, which occurs with the normal menstrual cycles, leads to chronic inflammation and adhesion formation at the sites of implantation. Endometriosis has been associated with various types of

Fig. 6.8  Endometriosis of fallopian tube. The endometrial glands and stroma (arow) are embedded in the fallopian tube. The histological appearance of the glands is similar to normal endometrial glands

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Fig. 6.9  Endometriosis with endometrial glands and stroma in the rectal wall. The endometrial glands are surrounded by smooth muscle from the rectum and there is adjacent stromal fibrosis

ovarian cancers including clear cell, endometroid, and low-grade serous carcinomas [20], but they are not thought to be related to high-grade serous cancers. Molecular analyses of deeply infiltrating endometriosis, which is usually associated with minimal risks of malignant transformation into cancer [21], using exome-wide sequencing or cancer-driver targeted sequencing has found somatic cancer-driver mutations in 79% of cases in one study [21]. Mutations included ARIDIA, PIK3CA, KRAS, and PPP2R1A. Interestingly, these mutations were found in the epithelial cells only and not in the stromal cells. Since the estimated rate of malignant transformation for endometriosis is close to 1% and because the mutations were only in epithelial cells and not in stromal cells of the same lesion, it has been suggested that the mutations occur by chance and that the mutations may provide some selective advantages to the endometriotic epithelial glands [21, 22]. The finding of these mutations in benign endometrial glands is a startling and surprising finding and reminds us that much remains to be learned about molecular markers that are simply thought to be somatic cancer drivers. Although many outstanding gynecologic pathologists have contributed to progress in this field, a few individuals have stood out from the others. The next section will summarize the lives of two of these outstanding individuals who have contributed greatly to progress in gynecologic pathology.

Robert E. Scully (1921–2012) Robert Scully was born in Pittsfield, Massachusetts in 1921. His father died when he was only 3 months old and he was raised by his mother who was a schoolteacher. He graduated from the College of Holy Cross with honors in 1941 [23–27]. He then

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attended Harvard Medical School and graduated in 1944 [23–27]. Although he wanted to do an internship at the Harvard teaching hospitals, he was not successful in gaining admission. He also applied for a pathology internship at Peter Brent Brigham Hospital and Massachusetts General Hospital and was accepted at the former institution. During his first year of pathology training, he contracted conjunctival tuberculosis while doing an autopsy and had to spend 9 months at a sanitarium in New York. He then continued with his internship and residency training in pathology at the Brigham and Women’s Hospital. An early indication of his scholarly academic interest and productivity was when he encountered an unusual case as a resident and then undertook a review of all of the testicular tumors in the files at the Brigham. He subsequently published two papers with Dr. Asa Parham based on his reviews on germ cell tumors and sex cord stromal and related tumors. After he completed his training at the Brigham and at Children’s Hospital, he spent a year as a resident at the Free Hospital for Women in Brookline, Massachusetts and Boston Lying-In Hospital with Dr. Arthur Hertig which stimulated his interest in gynecologic pathology. He then completed a residency at Pondville Cancer Hospital and served as the director of cytology and anatomic pathology. He then spent a year as an instructor of pathology at Massachusetts General Hospital where he taught anatomic pathology to second-year medical students. He joined the staff at Massachusetts General Hospital and worked there for 54 years. His early service at Mass Gen was interrupted when he was in the army during the Korean War between 1952 and 1954. While he rose through the academic ranks at Massachusetts General Hospital, one of the challenging duties that he took on was to serve as editor of the Case Records of the MGH in the New England Journal of Medicine. He served as editor of case reports for 52 weeks each year for 27 years. During his illustrious career, Dr. Scully published over 400 original papers and probably described more new entities in surgical pathology than any other pathologist in recent times. Some of these included small-cell carcinoma of the ovary with hypercalcemia [28], gonadoblastoma [29], juvenile granulosa cell tumor [30], and many others including clear cell carcinoma of the vagina and cervix after intrauterine exposure to diethylstilbestrol [1, 2], and large-cell calcifying Sertoli cell tumor [24]. He wrote two books on ovarian pathology and one on the endocrine pathology of the ovary. His busy external consultation practice included over 27,000 cases that he worked on 7 days a week. He was also sought after for his consultation advice about difficult cases from his colleagues at Mass Gen, by residents and fellows, because of his encyclopedic knowledge of many areas of general surgical pathology. He was the principal leader of the group that developed the ovarian tumor classification for the World Health Organization in 1973 and was also the leader of the group responsible for the classification of all genital tract tumors in 1998. Dr. Scully died of a stroke in 2012. Six months after his death he was given the lifetime achievement award by the Massachusetts Medical Society, the only pathologist to receive this award in the long history of that society [25]. Colleagues, residents, fellows, and most others that he worked with over many decades considered him to be “a modest, friendly person who treated all from the most eminent to the most junior with the same warmth and amiability” [23].

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Robert Kurman (Born 1943) Dr. Kurman was born in Harlem, New York in 1943. His parents moved to Jackson Heights, Queens, New York, when he was 8 years old. He was considering a career in medicine even when he was in the elementary school. This was influenced in part by an uncle who was a general practitioner and a cousin who was a psychoanalyst and professor of psychiatry at the University of Rochester [31, 32]. He attended Stuyvesant High School and went on to Queens College where he graduated with a major in chemistry in 1964. Dr. Kurman attended Upstate Medical Center in Syracuse New York, and graduated in 1968. He went on to do an internship in medicine and pathology at Beth Israel Hospital in New  York from 1968 to 1969. Following the advice of one of his mentors, Robert Scully, he completed a residency in pathology at Peter Brent Brigham Hospital in Boston from 1969 to 1971. After completing his residency, he became a fellow in gynecologic pathology with Dr. Scully. He learned a great deal from reviewing the consultation files of Dr. Scully and noted that the consultation letters were like reading a textbook in gynecologic pathology [32]. Because of his interest in clinical medicine from his internship training, he then decided to do a residency in obstetrics and gynecology. He was also influenced by outstanding gynecologists such as Dr. Donald Woodruff and Dr. Emil Novak who were outstanding gynecologists and experts in gynecologic pathology. He became a resident in obstetrics and gynecology from 1972 to 1973 at the Los Angeles County Hospital at the University of Southern California Hospital for Women. He was then enrolled in the service as assistant chief in the division of gynecologic and breast pathology at the Armed Forces Institute of Pathology from 1973 to 1976. He subsequently completed his training in obstetrics and gynecology at the Los Angeles County Hospital at the University of Southern California. Dr. Kurman joined the faculty of Georgetown University School of Medicine in 1978 and remained at this medical center until 1988. In 1989, he joined Johns Hopkins Department of Pathology as distinguished professor of gynecologic pathology and worked there until he retired in June 2017. Dr. Kurman’s research covered many areas of gynecologic pathology. As a fellow with Dr. Scully, he participated in the studies of diethylstilbestrol-associated clear cell adenocarcinoma of the vagina and cervix. Some of his early research was focused on gestational trophoblastic disease including studies of the intermediate trophoblastic lesions including placental-site epithelial trophoblastic tumors, endometrial hyperplasia, germ cell tumors of the ovary, and the relationship of human papilloma virus to cervical neoplasia [31–35]. Later, his research shifted to ovarian epithelial tumors. Dr. Kurman was also an early pioneer in applying immunohistochemical methods to addressing important pathological questions using formalin-­ fixed paraffin-embedded tissue sections. His studies in this area showed how this approach could be applied to diagnostic surgical pathology [31, 32]. In his studies of human ovarian epithelial tumors, he and his collaborators were able to combine morphological, immunohistochemical, and molecular diagnostic studies of low-grade, borderline, and high-grade ovarian tumors. They proposed that these tumors developed along different pathways [33, 34]. Type I tumors were lowgrade neoplasms and included low-grade serous, low-grade endometroid, clear cell

References

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and mucinous carcinomas, and Brenner tumors. These tumors are generally indolent, present at a low stage when they are confined to the ovary and have specific molecular changes with mutations including BRAF, KRAS, PTEN, ARIDIA, and a few others. They usually do not have mutations of TP53. Type II tumors include high-grade serous, high-grade endometroid, malignant-mixed mesodermal tumors, and undifferentiated carcinomas. These are aggressive tumors and patients present at an advanced stage have a high frequency of TP53 mutations and rarely have the mutations detectable in Type I tumors. BRCA mutations are present in Type II tumors or they may have promoter methylation of the BRCA gene [35]. It is thought that both Types I and II tumors develop outside of the ovary and involves this organ secondarily. His work has had a great impact on ongoing studies by many other investigators who study ovarian cancer pathogenesis and development, and Dr. Kurman’s findings may 1 day play a role in reducing the ravages of ovarian cancers. Dr. Kurman has been an author and editor of several major textbooks in gynecologic pathology including Blaustein’s “Pathology of the Female Genital Tract, Diagnosis of Endometrial Biopsies and Curettings: A Practical Approach.” He has been involved in several Armed Forces Institute of Pathology Fascicles including the third and fourth series of tumors of the cervix, vagina, and vulva, and the third series of tumors of the uterine corpus and gestational trophoblastic disease. Dr. Kurman has been actively involved in major leadership activities in gynecologic pathology and has served as president of the International Gynecologic Pathologists, membership on numerous editorial boards, and international committees. He recently became an honorary fellow of the Royal College of Pathologists. Dr. Kurman has been married for more than 34 years and he and his wife have one daughter. His hobbies include traveling, reading, and photography.

References 1. Herbst AL, Cole P, Norusis MJ, et al. Epidemiologic aspects and factors related to survival in 384 registry cases of clear cell adenocarcinoma of the vagina and cervix. AJ Obstet Gynecol. 1979;135(7):876–86. 2. Herbst AL, Scully RE. Adenocarcinoma of the vagina in young women. A report of 7 cases including clear cell carcinomas (so-call mesonephromas). Cancer. 1970;25:745–57. 3. Herbst AL, Ulfelder H, Poskanzer DC.  Adenocarcinoma of the vagina. Association of maternal stilbestrol therapy with tumor appearance in young women. New Engl J Med. 1971;284(15):878–81. 4. Robboy SJ, Young RH, Welch NR, et  al. Atypical vaginal adenosis and cervical ectropion. Association with clear cell adenocarcinoma in diethylstilbestrol-exposed offspring. Cancer. 1984;54(5):869–75. 5. Stoler MH. Human papilloma virus and cervical neoplasia. Human papilloma virus and cervical neoplasia: a model for carcinogenesis. Int J Gynecol Pathol. 2006;19:16–28. 6. Thompson J 3rd, Thomas LK, Shroyer KP. Human papilloma virus: molecular and cytologic/ histologic aspects related to cervical intraepithelial neoplasia and carcinoma. Hum Pathol. 2008;39:54–166. 7. Nuovo CJ.  Human papilloma virus DNA in genital tract lesions histologically negative for condyloma. Analysis by in situ, southern blot hybridization and the polymerase chain reaction. Am J Surg Pathol. 1990;14:643–51.

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8. Miller RA, Waters LL, Mody DR, et al. Squamous cell carcinoma of the cervix: a cytology-­ histology-­ human papillomavirus correlation in Chinese practice. Arch Pathol Lab Med. 2015;139(6):776–81. 9. Stelzle D, Tanaka LF, Lee KK, et al. Estimates of the global burden of cervical cancer associated with HIV. Lancet Glob Health. 2021;9(2):e161–9. 10. Castle PE, Xie X, Xue X, et al. Impact of human papillomavirus vaccination on the clinical meaning of cervical screening results. Prev Med. 2019;118:44–50. 11. Noumoff JS, Faruqi S. Endometrial adenocarcinoma. Microsc Res Tech. 1993;25(3):246–54. 12. Sanderson PA, Critchley HOD, Williams ARW. New concepts of an old problem: the diagnosis of endometrial hyperplasia. Hum Reprod Update. 2017;23(2):232–54. 13. Gibson DA, Simitsidellis I, Collins F, Sanders PTK, Androgens, oestrogens and endometrium. A fine balance between perfection and pathology. J Endocrinol. 2020;246(3):R75–93. 14. Feeley KM, Wells M.  Precursor lesions of ovarian epithelial malignancy. Histopathology. 2001;38:87–95. 15. Crum CP, Drapkin R, Kindelberger D, et al. Lessons from BRCA: the tubal fimbria emerges as an origin for pelvic serous cancer. Clin Med Res. 2007;5:35–44. 16. Salvador S, Gilks B, Kobel M, et al. The fallopian tube: primary site of most pelvic high-grade serous carcinomas. Int J Gynecol Cancer. 2009;19:58–64. 17. Lisio M-A, Fu L, Goyereche A, et al. High-grade serous ovarian cancer: basic sciences, clinical and therapeutic standpoints. Int J Mol Sci. 2019;20(4):952–6. 18. Kindelberger DW, Lee Y, Miron A, et al. Intraepithelial carcinoma of the fimbria and pelvic serous carcinoma. Evidence for a causal relationship. Am J Surg Pathol. 2007;31:161–9. 19. Network GGAR. Integrated genomic analysis of ovarian carcinoma. Nature. 2011;474:609–15. 20. Vercellini P, Vigano P, Somigliana E, et al. Endometriosis: pathogenesis and treatment. Nat Rev Endocrinol. 2014;10:261–75. 21. Anglesio MS, Papadopoules N, Aylan A, et al. Cancer-associated mutations in endometriosis without cancer. N Engl J Med. 2017;376(19):1835–48. 22. Wei JJ, William J, Bulun S. Endometriosis and ovarian cancer: a review of clinical, pathologic and molecular aspects. Int J Gynecol Pathol. 2011;30:553–68. 23. Young RH. Scully, Robert E. (1921-2012). In: Louis DN, Young RH, editors. Keen minds to explore the dark continents of disease. A history of the pathology service at the Massachusetts General Hospital. Boston: Mass Gen Hospital & Harvard Medical School; 2011. p. 134–46. 24. Young RH, Scully, Robert E. (1921–2012). In: van den Tweel JG, editor. Pioneers in pathology. In: van Krieken JHJM, series editor. Encyclopedia of pathology. Cham:Springer; 2017. pp. 479–481. 25. Young RH, Mills SE, Wick MR. In memoriam. Robert E. Scully, MD 1921-2012. Am J Clin Pathol. 2013;139:126. 26. Talerman A, Robert Scully MD. A pathologist’s perspective. Hum Pathol. 1991;22(8):747–9. 27. Young RH. Robert Scully MD. Am J Surg Patholog. 2013;37:464–8. 28. Young RH, Oliva E, Scully RE.  Small cell carcinoma of the ovary, hypercalcemic type. A clinicopathological analysis of 150 cases. Am J Surg Pathol. 1994;18:1102–16. 29. Scully RE. Gonadoblastoma. A review of 74 cases. Cancer. 1970;25:1340–56. 30. Young RH, Dickerson GR, Scully RE. Juvenile granulosa cell tumor of the ovary. A clinicopathological study of 125 cases. Am J Surg Pathol. 1984;8:575–96. 31. Young RH, An interview of Robert J Kurman, MD. Learning, teaching, passing the torch. Int J Gynecol. Pathology. 2018;37(1):1–16. 32. Kurman RJ, Young RH, Main CS, et al. Immunocytochemical localization of placental lactogen and chorionic gonadotropin in the normal placenta and trophoblastic tumors with emphasis on intermediate trophoblast and the placental site trophoblastic tumor. Int J Gynecol Pathol. 1984;3:101–21. 33. Shih I-M, Kurman RJ. Ovarian tumorigenesis: a proposed model based on morphological and molecular genetic analysis. Am J Pathol. 2004;164:1511–8. 34. Kurman RJ, Shih I-M. The dualistic model of ovarian carcinogenesis. Revisited, revised and expanded. Am J Pathol. 2016;186:733–47. 35. Kurman RJ, Shih I-M. Molecular pathogenesis and extraovarian origin of epithelial ovarian cancer – shifting the paradigm. Hum Pathol. 2011;42(7):918–31.

Chapter 7

Breast Pathology

Breast pathology is a specialized area of surgical pathology that studies and makes diagnoses of diseases of the female and male breast tissue. Many gynecological pathologists also combine breast pathology in their practice. Breast pathologists work closely with radiologists, breast surgeons, and oncologists to evaluate and diagnose breast lesions. Radiologists have a major role as the first physicians who usually diagnose breast lesions, especially neoplasms in patients with breast diseases. The American College of Radiologist developed the BI-RADS scoring system to standardize mammographic reporting of breast lesions. In this system, negative to benign lesions usually have RI-RADS scores of 1, 2, and 3. A BI-RADS score of 4 is suspicious for malignancy, while a BI-RADS score of 5 is highly suspicious for malignancy. A BI-RADS score of 6 refers to a biopsy-proven malignancy. This classification highlights the importance of the correlation between radiologic and pathologic findings in making a diagnosis of breast malignancies with BI-RADS 4, 5, and 6 [1]. The importance of the type of biopsy instrument and correlations to calcification in breast biopsy evaluations by the pathologist and radiologist is summarized in Chap. 3.

Benign Breast Lesions Fibroadenoma is a benign biphasic lesion of the breast arising from intralobular stroma which is associated with nonneoplastic epithelial cells. Fibroadenomas are the most common benign tumors of the breast in females [2]. Patients may have a palpable mass or it may be a mammographic density, especially in older patients. Fibroadenomas may increase in size during pregnancy secondarily to increased hormonal secretion. Histopathological examination shows a well-circumscribed nodule The original version of this chapter was revised. The correction to this chapter is available at https://doi.org/10.1007/978-3-031-39554-3_24 © The Author(s), under exclusive license to Springer Nature Switzerland AG 2023, Corrected Publication 2023 R. V. Lloyd, Pathology: Historical and Contemporary Aspects, https://doi.org/10.1007/978-3-031-39554-3_7

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Fig. 7.1  Fibroadenoma of the breast consisting of tubules of cuboidal cells with round uniform nuclei and associated myoepithelial cells. The stroma is composed of loose connective tissue. This common breast lesion is benign

that feels rubbery on palpation and microscopically shows slit-like spaces (Fig. 7.1). The stroma is often myxoid. Fibroadenomas are usually benign with minimal risk of becoming malignant. Phyllodes tumors are uncommon fibroepithelial tumors of the breast that may recur locally after excision. Metastatic disease is uncommon, but may occur in phyllodes tumors [3]. Benign phyllodes tumors make up the majority of these lesions and recurrences may occur in around 20% of lesions [3]. These tumors are more variable than fibroadenomas. They are quite variable in size and are more cellular, more pleomorphic, and may have increased numbers of mitoses compared to fibroadenomas. They are usually positive for stromal mesenchymal markers such as vimentin, but are usually negative for cytokeratins and for p63 [3]. Some molecular studies have found TP53, RB1, and NF1 mutations as well as EGFR amplifications in malignant phylloides tumors [4]. MED12 mutations have also been noted in malignant tumors [5].

Breast Carcinomas Breast carcinomas include ductal carcinomas which may be present as ductal carcinoma in situ or invasive ductal carcinoma as well as other subtypes.

Ductal Carcinoma In Situ Ductal carcinoma in situ is the most common breast carcinoma detected mammographically. Histologically, the carcinoma is limited to ducts and lobules by a basement membrane (Fig. 7.2). Once the tumor breaks through the basement membrane, it is no longer in situ, but microinvasive or frankly invasive. Histologically, there is

Ductal Carcinoma In Situ

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Fig. 7.2 Ductal adenocarcinoma in situ of breast with proliferating epithelial cells which have not invaded the adjacent stroma. Focal areas of calcification (arrow) and adjacent necrosis are present

Fig. 7.3  Invasive ductal carcinoma with infiltration of stroma by carcinoma

necrosis and/or fibrosis in ductal carcinoma in situ. Careful histopathological examination is needed to separate ductal carcinoma in situ from ductal hyperplasia and atypical ductal hyperplasia [6]. Ductal carcinoma in situ is more frequently associated with calcifications seen mammographically compared to invasive ductal carcinoma (Fig. 7.3). Ductal carcinoma in situ usually shows strong uniform staining for estrogen receptor unlike atypical ductal hyperplasia [6]. Use of immunohistochemical markers for basal myoepithelial cells can help to identify microinvasion [6]. The size of the lesion and marginal status are important markers that the breast pathologist must evaluate, since they help to predict tumor behavior. A margin of 2 mm or greater is usually considered a negative margin for these types of carcinomas [6]. Molecular analysis with array and genomic studies have shown a low- and high-­ grade pathway to ductal carcinoma in situ [6]. The low-grade pathway is associated with loss of 16q and variable gains of 1q. The high-grade pathway is associated with

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alterations of 8p, 13q,17q, and 20q, but rarely shows alterations in 16q. Fluorescent in situ hybridization studies of ductal carcinoma in situ have found alterations of copy numbers of HER2, C-MYC, CDX2, CCND1, CDH1, and TP53 [7, 8]. Some studies have suggested a multiclonal invasion model with clones assisting during progression to invasive ductal carcinoma [6].

Lobular Carcinoma In Situ Lobular carcinoma in situ is usually a primary diagnosis by the pathologist, since it is not often detected radiographically [9]. Lobular carcinoma in situ is usually not associated with calcification or with stromal desmoplasia. It may be bilateral in about 20–40% of cases compared to bilaterality in about 10–20% of cases of ductal carcinoma in situ. Loss of E-cadherin expression is a common finding in lobular carcinoma in situ and this observation has led to the application of this immunohistochemical stain to separate lobular and ductal carcinomas in situ by the pathologist. Histological findings in lobular carcinoma in situ and atypical lobular hyperplasia are similar with dyscohesive cells with round to oval nuclei and inconspicuous nucleoli (Fig. 7.4). Absence of E-cadherin staining is seen in both lobular carcinoma in situ and invasive lobular carcinoma and helps to separate these subtypes from ductal carcinomas (Fig. 7.5). Histologically, there are several subtypes of lobular carcinoma in situ with classical lobular carcinoma in situ more common in premenopausal women. The tumor cells are usually positive for estrogen and progesterone receptor proteins, while HER2 is frequently negative [9]. Lobular carcinoma in situ usually shows deletion of 16q and gains of 1q by comparative genomic hybridization. There are usually mutations of CDH1, PIK3CA, and CBFB [9].

Fig. 7.4 Lobular carcinoma in situ within a duct showing uniform cells within the duct. There is no invasive growth

Invasive Ductal Carcinoma

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Fig. 7.5 Lobular carcinoma in situ with immunohistochemical staining for E-cadherin. The lobular carcinoma in situ cells are negative for E-cadherin, while the adjacent benign ductal cells at the periphery are positive for E-cadherin

Fig. 7.6  Invasive ductal carcinoma of breast. Nests of cancer cells with high nuclear cytoplasmic ratios are infiltrating the stroma and there is a stromal desmoplastic reaction

Invasive Ductal Carcinoma Most patients with invasive ductal carcinoma present with a palpable mass. The gross appearance of the carcinoma is usually a firm hard lesion with irregular borders. The histopathological pattern varies from well to poorly differentiated (Figs.  7.6, 7.7, and 7.8). The majority of invasive ductal carcinomas include no-­ special type invasive ductal carcinoma and range from 70% to 80% of cases [10]. The other types range from lobular to metaplastic carcinomas [10] with invasive lobular carcinoma as the second most common type and constituting about 10% of invasive carcinomas [10].

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Fig. 7.7  Invasive ductal carcinoma of breast with immunohistochemical staining for estrogen receptor protein. There is strong nuclear immunoreactivity as indicated by the dark brown nuclear staining after immunohistochemistry. The cell cytoplasm and stroma are negative for estrogen receptor

Fig. 7.8  Invasive ductal carcinoma of breast with cells staining positively for HER2/Neu in an invasive ductal carcinoma of the breast. Strong membranous staining (3+) is present

Invasive Lobular Carcinoma Invasive lobular carcinoma is the second most common type of invasive breast carcinoma [10]. It has a distinct morphological appearance and clinical behavior compared to other types of invasive breast carcinomas. Patients usually present with a palpable mass or an irregular mammographic lesion. About 25% of invasive lobular carcinomas may not be reliably detected clinically or radiologically because of the subtle infiltration of the tissue without causing significant desmoplasia. Histopathologic appearance of invasive lobular carcinoma includes dyscohesive tumor cells invading in a single file or in sheets or clusters without tubule formation (Fig. 7.9). Some of the tumor cells may have a signet ring appearance secondary to intracellular mucin. The amount of desmoplastic connective tissue may be quite variable in this subtype of invasive breast carcinoma, since invasive lobular carcinoma does not usually illicit a desmoplastic

Molecular Classification of Breast Cancer

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Fig. 7.9  Invasive lobular carcinoma of breast composed of tumor cells with a high nuclear cytoplasmic ratio infiltrating the desmoplastic stroma. The tumor cells infiltrate in a rectilinear pattern

response. Distinction between invasive lobular carcinoma and the more common invasive ductal carcinoma can be done by E-cadherin immunostaining. E-cadherin is a tumor suppressor gene in invasive lobular carcinoma [10]. This biomarker shows loss of expression in invasive lobular carcinoma as well as lobular carcinoma in situ. However, it is retained in many other ductal carcinomas [11, 12].

Molecular Classification of Breast Cancer Molecular classification of breast carcinomas led to a major leap forward with studies in which high-throughput analyses showed the breast cancer heterogeneity at the molecular level [13, 14]. These cancers were classified into molecular subtypes based on the earlier findings. The subtypes included luminal A, luminal B, HER-2 overexpressing, basal-like breast cancer, and normal-like tumors. Luminal A and luminal B are enriched with estrogen receptor-positive carcinomas while HER2 overexpressing, basal-like and normal-like breast cancer are estrogen receptor negative [13, 14]. Luminal A is the most common subtype comprising up to 55% of the no-special types of carcinomas. Luminal B make up about 20% of no-special types of carcinomas and usually overexpressed HER2. The normal breast-like group made up 10% of no-special types and are well-differentiated, estrogen receptor positive and HER2 negative. The basal-like cancers lacked estrogen receptor, progesterone receptor, and HER2 and have markers associated with myoepithelial differentiation. This latter group included some carcinomas with a high grade and a high proliferative index [13, 14]. Next-generation sequencing has shown that some of the most common mutated genes in breast carcinoma include TP53, PIK3CA, GATA3, MYC, CCND1, PTEN, FGFR1, RB1, EERB2, and MAP3K1 [14].

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Triple-negative breast carcinoma represents a subgroup of the basal-like carcinomas, but they are also heterogeneous. Many patients in this subgroup have BRCA1 mutations. Triple-negative breast cancers may be high grade and associated with an aggressive clinical course, while other subgroups of triple-negative breast carcinomas are low grade such as adenoid cystic carcinoma and fibromatous-like metaplastic carcinomas [14]. Because of the complexity of the molecular classification of breast carcinomas, the traditional classification, which is based on histopathological features and immunohistochemical evaluation of hormonal receptors and HER2 is most frequently used in clinical practice. The relatively inexpensiveness, reliability, and ease of adaptation to clinical practice are some of the reasons that the traditional classification rather than the molecular classification has remained as the standard in clinical practice [14]. There are many pathologists that have contributed to advances in surgical breast pathology, especially in writing a definitive textbook in the field. One of these is Dr. JG Azzopardi.

John G Azzopardi (1924–2013) Dr. John Azzopardi was born in Valletta, Malta on June 25, 1929. He was a precocious student and at the age of 13 started medical training at The Royal University of Malta. In spite of his young age, he graduated first in his class from medical school in 1949 [15, 16]. He moved to England that year and spent the first few years in Sheffield in junior house jobs. During that time, he attended a course at the Royal Postgraduate Medical School at Hammersmith Hospital in London. He was later appointed to the academic staff at Hammersmith where he spent most of his academic life. He rose through the ranks to become professor of oncology. He later did a sabbatical at the Armed Forces Institute of Pathology in the USA from 1960 to 1961. He was also a visiting professor at the University of Bologna in Italy for 2 months in 1972. Dr. Azzopardi was married to his devoted wife, Sally Azzopardi, and they had three children. It was Sally who courageously typed his entire masterpiece book on breast pathology in the days that computers were not yet available to facilitate major typing projects [19]. Dr. Azzopardi was an excellent general surgical pathologist, although he is best remembered for his expertise in breast pathology. He published his masterpiece book Problems in Breast Pathology in 1979 [17]. His broad interest in many areas of surgical pathology is supported by his original observations about the encrustation of DNA in the wall of blood vessels which is still referred to as the Azzopardi phenomenon [18]. His textbook on breast pathology became a classic for excellent morphologic description of lesions of the breast and the lucid descriptions of the various diagnostic breast lesions. Pathologists from many countries came to study breast pathology under Dr. Azzopardi during his many years in academic practice. Some of these pathologists went on to become outstanding pathologists and leaders

References

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at their own countries and institutions such as Dr. V. Eusebi [16] and Dr. C. Capella from Italy [19]. Dr. Azzopardi was noted to be a brilliant but humble surgical pathologist. It was noted that he never compiled his CV [15], a task that is usually critical in major academic centers. He was consulted by pathologists from around the world with difficult diagnostic problems in breast pathology. He gave many lectures in Europe. In 1975, he presented a slide seminar at the California Tumor Registry at Stanford Medical Center. This outstanding presentation prompted many institutions in the USA to try to recruit him to their institutions, but he chose to remain at the Hammersmith for all of his academic career, excepts for his sabbaticals. Toward the end of his academic career, a symposium was held in Malta in his name and was entitled “Problems in Breast Pathology Revisited” in honor of his outstanding textbook. Many of his former students and other outstanding pathologists presented at this symposium [16]. Dr. Azzopardi died in London in 2013 at the age of 84 [16].

References 1. Boyer B, Canale S, Arfi-Rouche J, et al. Variability and errors when applying the BIRADS mammography classification. Eur J Radiol. 2013;82(3):388–97. 2. Houssami N, Cheung MNK, Dixon JM.  Fibroadenoma of the breast. Med J Aust. 2001;174(4):185–8. 3. Zhang Y, Kleer CE. Phyllodes tumor of the breast: histopathologic features, differential diagnosis and molecular/genetic updates. Arch Pathol Lab Med. 2016;140(7):665–71. 4. Kersting C, Kuijper A, Schmidt H, et al. Amplifications of the epidermal growth factor receptor gene (egfr) are common in phyllodes tumors of the breast and are associated with tumor progression. Lab Investig. 2006;86(1):54–61. 5. Cani AK, Hovelson DH, McDonald AS, et  al. Next-gen sequencing exposes frequent MED12 mutations and actionable therapeutic targets in phyllodes tumor. Mol Cancer Res. 2015;13(4):613–9. 6. Badve S, Gokmen PM. Ductal carcinoma in situ of breast: update. Pathology. 2019;5(6):563–9. 7. Salomon V, Lucchesi C, Gruel N, et  al. Integrated genomic and transtriptomic analysis of ductal carcinoma in situ of the breast. Clin Cancer Res. 2008;14:1956–85. 8. Hesselmyer-Haddad K, Berroa Garcia LY, Braley A, et  al. Single cell genetic analysis of ductal carcinoma in situ and invasive ductal carcinoma reveals enormous tumor heterogeneity yet conserved genomic imbalances and gain of MYC during progression. Am J Pathol. 2012;181:1807–22. 9. Sokolova A, Lakhani SR. Lobular carcinoma in situ. Diagnostic criteria and molecular correlates. Mod Pathol. 2021;34:8–14. 10. Lester SC. The breast. In: Kumar V, Abbas AF, Fausto N, Aster TC, Robbins and Cotran, editors. Pathologic basis of disease. 8th ed. Cambridge: Elsevier; 2010. p. 1065–95. 11. Alsaleem M, Toss MS, Joseph C, et  al. The molecular mechanisms underlying reduced E-cadherin expression and invasive ductal carcinoma of the breast: high throughput analysis of large cohorts. Mod Pathol. 2019;32:967–76. 12. McCart Reed AE, Kutasovic JR, Lakhani AR, et al. Invasive lobular carcinoma of the breast: morphology, biomarkers and ‘omics’. Breast Cancer Res. 2015;17(1):12–8. 13. Perou CM, Sorlie T, Eisen MB, et al. Molecular portraits of human breast tumours. Nature. 2000;406:747–52.

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14. Tsang JYS, Tse GM.  Molecular classification of breast cancer. Adv Anat Pathol. 2020;27(1):27–35. 15. Picciotti ER, Ramieri T. Azzopardi JG. (1929–2013). In: van den Tweel JG, editor. Pioneers in pathology. In: van Krieken JHJM, series editor. Encyclopedia of pathology. Cham:Springer; 2017. p. 43–45. 16. Eusebi V, Kraus T. In memoriam of JG Azzopardi. Am J Surg Pathol. 2013;37(7):1120–2. 17. Azzopardi JG. Problems in breast pathology. Major Probl Pathol. 1979;11:1–466. 18. Azzopardi JG. Oat-cell carcinoma of the bronchus. J Pathol Bacteriol. 1964;88:213–8. 19. Capella C, Eusebi V, Mann B, et  al. Endocrine differentiation in mucoid carcinoma of the breast. Histopathology. 1980;4:613–30.

Chapter 8

Genitourinary Pathology

Genitourinary pathology is a branch of surgical pathology that makes diagnoses of lesions of the prostate, bladder, testis, penis, kidney, and other portions of the genitourinary tract. Many genitourinary pathologist include the adrenal glands in their subspecialty area. Lesions involving the prostate, bladder, and kidney are the most common ones seen by genitourinary pathologists. Genitourinary pathologists work closely with urologists and radiologists to make specific diagnoses in urologic pathology. Some of the most common and challenging lesions seen daily by the urologic pathologist will be summarized below.

Prostate Adenocarcinoma Prostate cancer biopsies and resections are some of the most common lesions seen by the genitourinary pathologist. Prostate cancer is the most common non-­cutaneous cancer in men worldwide. Around 1,600,000 cases with 366,0000 deaths are reported annually [1]. The highest incidence and mortality are in Northern Europe and the Caribbean. South Central Asia has one of the lowest incidences [2]. Family history, race, and hereditary syndromes are some of the major risk factors [2]. Most patients are in their sixth decade or older, although a few younger patients may have positive biopsies for prostate cancer which is usually followed by resection of the prostate [1, 2]. Morphological features of prostate adenocarcinoma consist of cells with enlarged nuclei, prominent nucleoli, and loss of the peripheral basal layer or myoepithelial cell layer in the prostatic acinus (Figs. 8.1 and 8.2) [3]. Basal cell loss is not totally specific for prostate carcer, since some benign glands may lack basal cells also. Collagenous micronodules or mucinous metaplasia which consist of microscopic aggregates of hyalinized stroma in response to invasive adenocarcinoma which is usually associated with abundant mucin can be another helpful diagnostic feature of prostate adenocarcinoma [3]. A small percentage of prostate © The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 R. V. Lloyd, Pathology: Historical and Contemporary Aspects, https://doi.org/10.1007/978-3-031-39554-3_8

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Fig. 8.1  Benign prostate tissue showing large glands with epithelial cells. Myoepithelial spindle cells are present at the periphery of the glands adjacent to the epithelial cells

Fig. 8.2  Prostate adenocarcinoma composed of small glands with enlarged nuclei and prominent nucleoli. The myoepithelial cells are lost in the proliferating tumor cells

cancers may have glomeruloid features also known as glomerulations which represent renal-glomerular-like epithelial aggregates with acini of prostatic adenocarcinomas. However, these features are present only in a small percentage of adenocarcinomas. Immunohistochemical analysis has become another powerful tool to confirm a diagnosis of prostate cancer. Increased expression of AMACR or alpha methyl CoA racemase also known as p504S and the loss of the basal cell markers such as p63 and high molecular weight keratin positive which are usually combined in a single immunohistochemical staining procedure (Fig. 8.3) [3]. The grading of prostate cancer includes the traditional Gleason grade which adds the

Incidental Prostate Cancers

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Fig. 8.3  Immunohistochemical staining of a prostatic adenocarcinoma using three combined antibodies. The cancer cells show positive red staining for racemase (arrow). The larger nonneoplastic cells on the upper left are negative for racemase. The brown staining cells in the normal prostatic glands are positive for myoepithelial markers (p63 and keratin), while the malignant prostate cancer cells are negative for brown staining indicating loss of myoepithelial cells

two most prominent grade patterns of carcinoma and the more recently adapted Grade Group which extends from Grade Group1 to Grade Group 5 [4]. Genomic studies of prostate cancer have shown a great deal of genetic and phenotypic heterogeneity in any given tumor [5]. Molecular tumor characteristics show a great deal of diversity between different patients and within a given tumor. Some of the complexity of primary prostate cancer is probably related to the multifocal nature of individual tumors [5]. It has been estimated that 80% of primary prostate cancers have different tumor foci including histomorphological heterogeneity [5]. Subclonal heterogeneity is common in prostate cancer with many tumors showing different subclones. It has been suggested that tumors with a high level of subclonal diversity in the primary tumor may increase the chances of metastasis in a given prostate cancer [5].

Incidental Prostate Cancers Small incidental prostate cancers are often seen at autopsy or in patients undergoing transurethral resection of the prostate secondary to benign prostatic hyperplasia. These small cancers are usually incidental in older patients and usually do not progress to more aggressive cancers. The incidence of prostatic cancer detected at transurethral resection of the prostate is quite variable. In one large study from Utah, the USA, between the years 1980 and 1999, the incidence rates tended to decrease for patients 45 years and older and ranged from 39% in 1980 to 1984 and was only 7.4% in 1995 to 1999. The main reason for these changes was the introduction of

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prostate specific antigen screening in the later years [6]. In another study from Denmark with 1450 men who had radical cystoprostatectomy, 33.2% had an incidental prostate cancer [7]. There was no association with Gleason score, positive surgical margins and locally advanced prostate cancer was not associated with increased mortality. After reviewing 29 studies the authors concluded that patients with incidental prostate cancers following radical cystoprostatectomy were not likely to benefit from additional follow-up. In a recent study that combined published autopsy series in adult men, it was reported that incidental prostate cancers increased with age [8]. Using 29 studies from more than 20 countries over 60 years, the prevalence of prostate cancer was related to age with prevalence doubling every 14 years. Prostate cancer was detected even in some young men with an increasing rate with age [8]. The rate of detection increased with age. These studies all support the concept that it is not possible to distinguish between prostate cancers that are simply incidental in older patients from prostate cancers that will show progression. These studies also highlight the observation that a great deal of research is needed to distinguish incidental prostate cancers associated with increasing age from truly progressive prostate cancer in older men.

Bladder and Urothelial Tract Tumors Cancers involving the bladder and urothelial tract usually derived from the urothelial lining and these are the most common types of tumors in these regions making up about 90% of the cancers in these sites. Increased risk of urothelial cancers may be caused by many agents including cigarette smoking, chemicals such as arylamines, parasites such as Schistosomiasis haematobium (Fig. 8.4), chronic use of analgesics, cyclophosphamides, and prior radiation to the bladder or other sites with urothelial lining [9]. Papillary neoplasms are the most common. They can range from benign papillomatous lesions to highly aggressive anaplastic urothelial cancers. The noninvasive papillary tumors are the most common precursors of invasive bladder cancer. Flat noninvasive urothelial carcinoma (carcinoma in situ) is the second most common precursor for bladder cancer. When the pathologist examines a biopsy for invasive bladder cancer, evaluation for smooth muscle invasion is a critical factor for prognosis of the carcinoma in a particular patient. Invasion into the lamina propria beneath the epithelium is also important, but not as prognostic as smooth muscle invasion. High-grade urothelial cancers are a lot more likely to be muscle-invasive compared to low-grade urothelial cancers. There are several variants of urothelial cancers and many of these are associated with a worse prognosis than the common papillary urothelial carcinoma. Some of these include the nested variant, lymphoepithelioma-like carcinomas, micropapillary variant, and the plasmacytoid variant of urothelial carcinoma. Molecular studies of urothelial carcinomas have shown high rates of somatic mutations which are higher only in lung carcinomas and melanomas [10]. The cell cycle regulator TP53 is one of the most

Renal Cell Carcinomas

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Fig. 8.4  Section of urinary bladder tissue showing the calcified remnants of a parasitic infection with calcified Schistosoma haematobium. This infection is associated with squamous cell carcinoma of the urinary bladder. Portions of benign bladder urothelium is present in the upper right of the figure

commonly mutated genes in urothelial carcinomas; it is mutated in around 50% of these tumors [10]. Other genes such as RB1, CDKN2A, and FGFR3 are also commonly mutated in these carcinomas. TERT promoter mutations were present in many urothelial carcinomas of varying grades and stages, but were not present in benign urothelial lesions such as papillomas. Most of the aggressive variants of urothelial carcinomas are associated with a worse prognosis [11]. However, survival with the more aggressive variants do not differ that significantly compared to the conventional urothelial carcinomas.

Renal Cell Carcinomas Although there are many variants of renal cell carcinomas, clear cell carcinomas of the kidney are the most common type and makes up about 70% of primary renal cancers (Figs. 8.5 and 8.6). The gross appearance includes a yellow color and the tumors are often focally hemorrhagic with old and recent hemorrhage. Histologically, the tumors consist of clear cells with varying degrees of granular eosinophilic cytoplasm. The clear cells reflect glycogen and lipid in the cytoplasm of the tumor cells. The cells usually have centrally located nuclei and some suggestions of glandular cytoplasm. Immunohistochemical stains are very useful in separating different variants of renal cell carcinomas. Typical clear cell carcinomas are positive for PAX8-a transcription factor, CD10, and for carbonic anhydrase IX.

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Fig. 8.5  Normal kidney with a glomerulus in the center of the field (G). The glomerulus is surrounded by renal tubules. Bowman’s pace which is the space between the visceral and parietal epithelium of the glomerulus is shown (arrow)

Fig. 8.6  Renal cell carcinoma, clear cell type. The tumor is composed of nest of clear cell. This is the most common variant of renal cell carcinoma. The tumor cells have clear cytoplasm due to the presence of glycogen and lipids. The tumor cells have a distinct cytoplasmic membrane. The brown pigment in the upper right of the image represents hemosiderin pigment from the break down of red blood cells. Patients with this cancer may present with blood in the urine

Clear Cell Papillary Renal Cell Carcinoma This is a recently described new variant of clear cell renal cell carcinoma. It has combined features of clear cell and papillary variant of renal cell carcinomas. Histological features of clear cell papillary carcinoma include cells with clear or

Donald F. Gleason (1920–2008)

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eosinophilic cytoplasm lining papillary structures with the nuclei oriented toward the basement membrane. Immunohistochemical features include positive staining for CK7, CD10, and carbonic anhydrase IX with a “boxlike” pattern of staining with CK 7 antibody. AMACR and vimentin are also positive [12–14]. The ability to distinguish different types of renal cell carcinomas by immunohistochemical staining illustrates the power of immunophenotyping in separating various diagnostically difficult subtypes of renal cell carcinomas with overlapping histological features. There have been many genitourinary pathologists that have contributed to progress in this field. A summary of the lives of two of these genitourinary pathologists is summarized below.

Donald F. Gleason (1920–2008) Donald Gleason was born in 1920 in Spenser, Iowa, a small town at the confluence of two rivers. He attended college at the University of Minnesota and graduated with high honors. He then attended the University of Minnesota Medical School and received his MD degree in just 2 years, since this was during World War II. He was a member of the US Army’s Specialized Training Program [15]. After completing his internship, he served as a lieutenant in the Army Medical Corp Reserve at the University of Maryland in Baltimore, MD. After completing his service, he did a 3-year residency in pathology at the University of Minnesota and the Minneapolis Veterans Affair Hospital. However, after finishing his residency, he could not get a job as a pathologist in the Minneapolis area, so he went to Paris to learn more about art. His time in Paris was interrupted after he was notified that he had a job in the department of pathology of the VA Hospital in Minneapolis. He was invited to be the chief of the Minneapolis VA. He was named as an instructor in the department of pathology and laboratory medicine at the University of Minnesota in 1952. Over the next few years, he also worked on his PhD degree at the University of Minnesota which he completed in 1966. His thesis was on studies of the human heart, a subject that was far from the organ that later made him famous. One year later, he was promoted to associate professor of pathology at the University of Minnesota. He had married his sweetheart while he was in the army and they raised three children. In 1967, Dr. Gleason was appointed as the referee pathologist for the VA Cooperative Research Group, a position that he held until 1975. Dr. Gleason remained as a pathologist at the VA Hospital until 1975 and then he moved to Fairview Hospital in Minneapolis until he retired in 1986. Earlier in his career at the VA Hospital, Dr. Gleason had been asked to develop a standardized histopathologic grading system for prostate cancer by the chief of urology at the VA, Dr. George Melling. He accepted the job and worked on the project with a great deal of enthusiasm. At that time there were multiple systems for grading prostate cancer and they were not very reliable, which led to a great deal of confusion in the diagnosis and treatment of patients with prostate cancer. Gleason worked diligently on prostate cancer grading

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and developed a system using a primary and secondary patterns which were designated by the most abundant primary and secondary patterns of cancer present in the specimen. The numbers were added together to yield a combined Gleason score [16, 17]. He published the first paper using this system in 1966 [18]. By 1974, his studies on grading prostate cancer included 1032 patients with an impressive follow-up period of 4193 follow-up years. The study ended in 1975 with close to 5000 patients. One of the reasons that some experts think that the Gleason grading system was so successful was the unique approach that he used in summarizing the five patterns of tumors that he had recognized under the microscope. He used the “Gleason schedule” to summarize the five patterns with a drawing on the same page. His interest in art and his artistic talents probably contributed to the excellent quality of his sketches. His approach helped pathologists, urologists, and other physicians to quickly visualize the variation in the grading patterns in prostate cancer. Over the next few years, the Gleason grading system became well known and was widely adapted. After 1978, the American Cancer Society recommended that the system be used as the standard system and the Journal of Urology published a letter from seven leading urologists recommending that this system be used in all publications which included the histopathology of prostate cancer [15]. The first major revision of the Gleason grading system was in 2005 after the International Society of Urologic Pathology had a meeting about the Gleason grading system. The system has been modified slightly over the years and the grade group has been added to the grading of prostate cancer to address some of the minor problems in grading prostate cancer that remained. However, the Gleason grading system has remained as the most widely accepted grading system in prostate pathology until the current time. Dr. Gleason retired in 1986. He had published only 29 research papers and 9 book chapters during his career. He taught his grading system to medical students, residents, and many pathologists throughout his life at national and international meetings. He received many honors during his career, especially after his retirement, because of his outstanding contributions to urologic pathology. Some of these include the Presidential Citation Award from the American Urologic Association in 1991, A Lifetime Achievement Award from the International Society of Urologic Pathology in 2002, and the Outstanding Achievement Award from the University of Minnesota in 2001. A professorship in surgical pathology was created at the University of Minnesota Medical School Department of Laboratory Medicine and Pathology in his honor in 2009.

Fathollah Keshvar Mostofi (1911–2003) Dr. Mostofi was born in Teheran, Iran, in 1911. His father was a urologist in Iran. He moved to the USA when he was 20  years old and enrolled in college at the University of Nebraska. After graduation, he was accepted to Harvard medical school and graduated in 1939. He then completed an internship at St. Luke’s

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Hospital in Pennsylvania and then went on to train in pathology in Boston at three hospitals including Peter Brent Brigham, Boston Lying-in, and Free Hospital for Women. He then worked as an assistant pathologist at Massachusetts General Hospital for 1 year in 1943 [18–20]. Dr. Mostofi joined the US Army for 3 years. Dr. Fred Stewart assisted him in becoming a research fellow at the National Cancer Institute in Bethesda, MD.  After completing his tour of duty with the army, Dr. Mostofi joined the Armed Forces Institute of Pathology in 1948. He was asked to establish a genitourinary pathology section. He realized that he had a great deal to learn in this new specialty but responded to the challenges and soon became the leading genitourinary pathologist in the USA and the world because of his hard work in the field. He served as chair of genitourinary pathology at the AFIP from 1948 until 2003. His careful studies and hard work made the genitourinary section of the AFIP into an internationally recognized subspecialty that attracted visitors from many countries [21]. Dr. Mostofi became the head of the International Association of the Medical Museum (IAMM) in 1952. This organization had been founded by Dr. Maude Abbott. The influence of the IAMM had declined after World War II. As the leader of the IAAM, Dr. Mostofi helped to develop this organization into the International Academy of Pathology (IAP) with the US and Canadian (USCAP) branch becoming the largest and most influential branch of this prestigious organization. Dr. Mostofi was the secretary-treasurer of the IAP from 1954 to 1970. He also developed an interest in a new branch of pathology, aviation pathology, which studied the role of human error in aircraft accidents. During the 1950s, he became one of the organizers of the Joint Committee on Aviation Pathology and went on to serve as the secretary of the committee for 6 years [22]. In spite of his busy schedule with many organizations, Dr. Mostofi’s academic productivity was outstanding. He authored more than 200 original scientific papers and 15 books including the Armed Forces Institute of Pathology Fascicle on Tumors of the Male Genital system in 1973 and the Atlas of Kidney Biopsies in 1980. He was very innovative with many of the organizations that he was involved in. He introduced the first long course which was a detailed presentation on the pathology of one organ system presented at meetings of the USCAP and IAP. He also introduced “kidney night” at the USCAP which was the prototype for subsequent evening sessions as specialty conferences at the USCAP, a very popular part of the annual USCAP meetings. Dr. Mostofi received many honors throughout his career including the Distinguished Executive Rank Award in 1989. He was presented with the Presidential Honor Award from the American Urologic Association and the Presidential Award from the American Foundation for Urologic Diseases. He also received the gold medallion from the USCAP for his work with the IAMM, IAP, and USCAP. The USCAP also initiated the F.K. Mostofi Award for distinguished service to the academy which is presented annually to an outstanding member of the USCAP. Dr. Mostofi died in 2003 at the age of 91 from complications of prostate cancer [20].

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References 1. Torre LA, Bray F, Siegel RI, et  al. Global cancer statistics, 2012. CA Cancer J Clin. 2015;65:87–108. 2. Gandaglha G, Leni R, Bray F.  Epidemiology and prevention of prostate cancer. Eur Urol Oncol. 2021;4(6):877–92. 3. Humphrey PA.  Histopathology of prostate cancer. Cold Spring Harb Perspect Med. 2017;7:1–21. 4. Epstein JL, Allsbrook WCJ, Amin MB, et al. ISUP grading committee. The 2005 International Society of Urologic Pathology (ISUP) consensus conference on Gleason grading of prostate adenocarcinoma. Am J Surg Pathol. 2005;29:1228–42. 5. Haffner MC, Zwart W, Roudier MP, et al. Genomic and phenotypic heterogeneity in prostate cancer. Nat Rev Urol. 2021;18(2):79–92. 6. Merrill RM, Wiggins CL. Incidental detection of population-based prostate cancer incidence rates through transurethral resection of the prostate. Urol Oncol. 2002;7(5):213–9. 7. Jenck S, Helgstrand JT, Rader MA, et  al. The prognostic impact of incidental prostate cancer following radical cystoprostatectomy: a nation-wide analysis. Scand J Urol. 2018;52(5–6):358–63. 8. Bell KJL, DelMar C, Wright G, et al. Prevalence of incidental prostatic carcinoma: a systemic review of autopsy studies. Int J Cancer. 2015;137:1749–57. 9. Lenis AT, Lec PM, Chamie K, et al. Bladder cancer: a review. JAMA. 2020;324(10):1980–91. 10. Al-hamadi H, Netto GJ.  Molecular pathology of urothelial carcinoma. Surg Pathol Clin. 2021;14(3):403–14. 11. Lobo N, Shariat SF, Guo CC, et al. What is the significance of variant histology of urothelial carcinoma? Eur Urol Focus. 2020;6:653–63. 12. Massan F, Ciccorese C, Hes O, et al. The tumor entity denominated “clear cell papillary renal cell carcinoma” according to the WHO 2016 new classification have the clinical characters of a renal cell adenoma as does harbor benign outcome. Pathol Oncol Res. 2018;23(3):447–56. 13. Alshenawy HA.  Immunohistochemical panel for differentiating renal cell carcinoma with clear cell papillary features. Pathol Clin Oncol Res. 2015;21(4):893–9. 14. Williamson SR, Zhang S, Eble JN, et al. Clear cell papillary renal cell carcinoma-like tumor in patients with Von Hippel-Lindau disease are unrelated to sporadic clear cell papillary renal cell carcinomas. Am J Surg Path. 2013;37(8):1131–9. 15. Farre X, Sinha AA. Gleason, Donald A. (1920–2008). In: van den Tweel JG, editor. Pioneers in pathology. In: van Krieken JHJM, series editor. Encyclopedia of pathology. Cham:Springer; 2017. p. 185–188. 16. Snyder A. Obituary. Donald Gleason. Lancet. 2009;373:540. 17. Phillips JL, Sinha AA. Patterns, art and context. Donald Floyd Gleason and the development of the Gleason grading system. Urology. 2009;74:497–503. 18. Gleason DF. Classification of prostate carcinomas. Cancer Chemother Rep. 1966;30:125–8. 19. Harley RA. Mostofi, Fathollah Keshvar (1911–2003). In: van den Tweel JG, editor. Pioneers in pathology. In: van Krieken JHJM, series editor. Encyclopedia of pathology. Cham:Springer; 2017. p. 395–399. 20. Sesterhenn I.  Legends in urology. Fatollah Keshvar “Kash” Mostofi MD.  Can J Urol. 2014;21(3):7256–8. 21. Mostofi FK. A study of 2678 patients with initial carcinoma of the bladder survival rates. J Urol. 1956;75(3):480–91. 22. Mostofi FK, Townend FM, Sternbridge V. Causes of sudden and unexpected death in aircrew. Aerosp Med. 1960;31:745–8.

Chapter 9

Medical Kidney Diseases

Medical kidney biopsy and interpretation are highly subspecialized areas of pathology in which the pathologist interacts with nephrologists and radiologists in taking small needle core biopsies and using sophisticated interpretations of the needle cores along with clinical signs and symptoms to make diagnoses of medical glomerular and tubular-interstitial renal diseases. The examination and final diagnoses require complex interpretations with the use of light microscopy, immunohistochemistry, and electron microscopic studies. The processing of minute tissue fragments with precise localization of glomeruli and other basic anatomic structures requires a great deal of technical skills. The correct interpretation of these minute biopsies requires highly trained renal pathologists with knowledge of renal medicine as well as renal pathology. The renal pathologist correlates the clinical data with information from histopathology, immunohistochemistry, and electron microscopy in order to obtain the most accurate diagnosis for treating the patient. The basic stains used for light microscopy include hematoxylin and eosin, periodic acid Schiff (PAS) reaction, methenamine silver, and trichrome [1]. Other stains such as von Kossa, toluidine blue, and Congo Red whenever amyloid is suspected as well as other histochemical stains are used by the renal pathologist to arrive at the correct diagnosis [2, 3]. A new and exciting direction of renal biopsy interpretation in the last few years is the use of deep learning or artificial intelligence (AI) for the histopathological assessment of renal biopsies [4]. In a recent study, the authors used conventional neural network for multiclass segmentation of the kidney tissues stained with periodic acid Schiff. They showed that this approach can be used in renal biopsies as well as nephrectomy specimens [4]. Although the results are preliminary, the analyses suggest that deep learning to make diagnoses of specific diseases in renal tissues may be feasible in the near future [4].

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Examples of Well-Characterized Renal Glomerular Diseases Minimal Change Disease This disease is the most common cause of the nephrotic syndrome in children. In the nephrotic syndrome patients usually present with a great deal of extra protein in the urine. They also have markedly decreased serum albumin levels due to losses in the urine. As a result, they develop generalized edema or fluid accumulation and increased serum lipids. Many nephrologists maintain that minimal change disease can be diagnosed without a renal biopsy [5, 6]. However, the changes seen in the biopsy are highly diagnostic of minimal change disease. At the light microscopic level, there are very few pathologic changes noted (Fig.  9.1). Although immune complexes are not present in this glomerular disease, an immunologic basis of the disease has been suggested. There is sometimes a clinical association with respiratory infection with this disease. Electron microscopic examination reveals the diagnostic morphological findings with effacement or loss of the foot processes of the visceral epithelial cells in the glomeruli even though the glomeruli appear normal at the light microscope level. Although effacement of the foot processes can be seen in other renal disease such as membranous glomerulopathy and diabetic renal disease, other changes are present at the light microscopic level in these diseases to help

Fig. 9.1  Glomerulus from a patient with a diagnosis of minimal change glomerulonephritis. The glomerulus in the center of the field appears normal at the light microscopic level, although the patient has increased protein in the urine. The disease can be diagnosed with an electron microscope that usually shows fusion of foot processes of the epithelial cells in glomeruli. The ultrastructural findings usually support the diagnosis of minimal change glomerulonephritis. This disease is most common in pediatric patients. It is the most common cause of idiopathic nephrotic syndrome in children

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make the correct diagnoses. It is only in minimal change disease that there are no obvious changes at the light microscopic level. Most children with minimal change disease respond to corticosteroid therapy and their prognosis is usually excellent. Minimal change disease in adults may be associated with different types of lymphomas and the prognosis is usually not as good as in children [7].

Acute Proliferative (Post-Streptococcal) Glomerulonephritis This is another glomerular disease that primarily affects children. Patients develop glomerular disease usually 1–4 weeks after a streptococcal infection involving the throat or skin. Histologic and immunohistochemical findings include granular immune deposits in glomeruli consistent with immune complex deposition [7, 8]. The renal biopsy shows hypercellular glomeruli with infiltration of neutrophils and macrophages (Figs. 9.2 and 9.3). There is proliferation of endothelial cells from the blood vessels and mesangial cells from the glomeruli. Immunofluorescence analysis shows deposition of immunoglobulins including IgG, IgM and complement (C3) in

Fig. 9.2  Acute post-­streptococcal glomerulonephritis. The glomerulus in the center of the field is hypercellular due to proliferation of cells in the glomerulus including mesangial and endothelial cells as well as the presence of inflammatory cells. The disease can develop after an infection with streptococcal bacteria

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Fig. 9.3  Histochemical staining of poststreptococcal glomerulonephritis with a silver stain shows basement membrane thickening indicated by the black lines. The electron microscopic examination showed dense deposits indicative of antigen-­antibody complexes

the mesangial cells and in the basement membrane of glomeruli. Electron microscopic studies show electron dense immune complex deposition on the epithelial side of the basement membrane along with subendothelial and intramembranous deposits These dense deposits are thought to represent antigen-antibody complexes. Most affected children usually recover from their renal problems with less than 1% going on to develop progressive renal failure and chronic glomerulonephritis. In adults, the disease is more aggressive with a higher proportion of adults going on to develop chronic renal failure [7, 8].

Membranoproliferative Disease Membranoproliferative glomerulonephritis is an uncommon variant of renal glomerular disease that develops more commonly in adolescents and young adults. Patients present with the nephrotic syndrome and some component of the nephritic syndrome such as blood in the urine (hematuria). Histological examination shows alterations in the glomerular basement membrane, as well as glomerular proliferation mainly in the mesangium, but this may also involve the capillary loops. Some cases may have proteins associated with hepatitis C implicating this viruses in the

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pathogenesis. Membranoproliferative disease has been divided into three types with subtypes 1 and 3 overlapping, while subtype II is known as dense deposit disease. Electron microscopic studies show electron-dense material in the glomerular basement membrane. Special stains such as silver or PAS stain show a double contour which is often described as “tram-track” appearance of the glomerular capillary wall [7, 9]. Complement and immune complex deposition in the subendothelium of glomeruli are often present. Electron microscopy show subendothelial electron dense deposits in Types I and III, while Type II shows a homogeneous deposition of electron dense material within the basement membrane [7, 9]. Cases of complement 3 (C3) deposition, without immunoglobulin deposition has been referred to as C3 glomerulonephritis [9]. Patients with membranoproliferative glomerulonephritis have a slowly progressive disease and about half of the cases develop renal failure within 10 years of diagnosis. Patients receiving renal transplantation for their renal failure may go on to develop recurrent disease in the transplanted kidney.

Diabetic Nephropathy Diabetes mellitus is the leading cause of end-stage renal disease. Diabetic nephropathy is a major cause of end-stage renal disease in both developing and developed countries [10]. The morphological changes in the kidney in insulin-dependent (type 1) and insulin-independent (type 2) diabetic nephropathy are similar. The histological features of diabetic nephropathy include nodular expansion of the mesangium leading to nodules that were historically termed Kimmestiel–Wilson nodules (Figs. 9.4 and 9.5). There is also diffuse thickening of the glomerular and tubular

Fig. 9.4  Diabetic glomerulopathy showing glomerulus with increased mesangial matrix, thickened capillary walls, and nodular fibrosis (Kimmelstiel–Wilson lesions) in a patient with long-standing diabetes mellitus

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Fig. 9.5  Histochemical stain of a glomerulus from a patient with diabetic glomerulopathy using periodic acid Schiff (PAS). The stain highlights the Kimmelstiel–Wilson nodules (arrow) and the increased mesangial matrix. The nodules were formed in regions of glomerular capillary loops and these lesions are associated with decreased renal function (nephropathy)

basement membranes [11]. Diabetic kidney disease in most patients usually progress at variable rates to end-stage renal disease. Recent studies have shown that progression can be slowed or inhibited by very rigorous control of serum glucose levels [11]. The nodular glomerulosclerosis seen in this disease include nodules of matrix located in the periphery of glomeruli. These nodules can be more readily visualized with PAS histochemical stains (Fig. 9.5). As the disease progresses, the nodules enlarge and the glomerular tufts become obliterated as the adjacent capillaries become entrapped by the enlarging nodules. Many outstanding medical renal pathologists have contributed to progress in this field. The life of one of these outstanding individuals who has contributed greatly to progress in medical renal pathology is summarized below.

Dr. Robert Hodgson Heptinstall (1920–2021) Dr. Robert Heptinstall was born in Keswick, Cumberland, England in 1920 [12, 13]. He attended London University and in 1943 earned his Bachelor of Medicine and Bachelor of Science degrees. He started postgraduate training at Charing Cross Hospital Medical School at London University with plans to become a surgeon, but his training was interrupted by World War II when England became involved in the war. He joined the military and served as a regimental medical officer in Asia for 3 years until 1947. His service responsibilities took him to India, Burma, Siam, and the Dutch East Indies [13]. His principal duty was to take care of prisoners of war.

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After the war he returned to Charing Cross Hospital. However, he decided to change his specialty area from surgery to pathology with training at St. Mary’s Hospital. This was a major victory for pathology because of his many contributions to this field during his career. After completion of his training, he joined the faculty in 1947 and worked at St. Mary’s Hospital until 1960. It was during this time that he met his future wife, Ann Porter, and they were married in 1950. Under the influence of George Pickering who was studying hypertension at that time, he became involved in research work and subsequently published several outstanding papers in his chosen research field. Some of his other mentors included Marc Joekes, a nephrologist, and Kendric Porter, a pathologist. Both of these individuals were involved in percutaneous needle biopsies of the kidney which was becoming an increasingly popular approach to study and diagnose renal diseases. He subsequently received an Eli Lilly traveling fellowship that enabled him to spend a year at Johns Hopkins Hospital in the pathology department in 1954 where he established several connections that would help to determine his future career directions. He was impressed with the practice of medicine and research in the USA and elected to spend a year at Washington University School of Medicine in St. Louis, MO in 1960 [14]. In 1962, he was appointed as an associate professor of pathology at Johns Hopkins University School of Medicine. He was subsequently promoted to professor of pathology and acting director of pathology in 1966. He continued to advance in his field and was promoted to Baxley Professor of Pathology and as director of the department of pathology and pathologist-in-chief in 1969. Dr. Heptinstall remained at Johns Hopkins for the rest of his academic career, serving as distinguished service professor of pathology until 2008. He became an emeritus professor of pathology in 2008 [13]. Dr. Heptinstall’s research in pathology covered a broad range of areas including renal injuries secondary to hypertension and covered a broad spectrum of renal diseases including studies of renal infections, glomerular diseases, tubular epithelial injury, and atherosclerosis. Many of his studies were quite basic with the use of animal models of diseases such as experimental pyelonephritis [14] and studies of hypertension using animal models [15]. His greatest contribution to academic pathology was his book Pathology of the Kidney, which was first published in 1966. The fifth edition was published in 1998 with the title changed to Heptinstall’s Pathology of the Kidney. This edition was edited by Dr. J. Charles Jennette and three other outstanding renal pathologists. He was an active member of some of the most influential pathology societies. He was editor of Laboratory Investigation, one of the premier journals in pathology, as well as associate editor of Nephron, a premier journal in the renal field. He served as president of the American Society of Nephrology as well as president of the International Society of Nephrology. He received many prestigious awards including the David Hume Award from the National Kidney Foundation and the John P.  Peters Award from the American Society of Nephology as well as the Lifetime Achievement Award of the Renal Pathology Society. Dr. Heptinstall died in 2021 at the age of 100.

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References 1. Walker PD, Cavallo T, Bonsib SM. The Ad Hoc Committee on renal biopsy guidelines of the renal pathology society. Mod Pathol. 2004;17:1555–63. 2. Cathro HP, Shen SS, Truong LD. Diagnostic histochemistry in medical diseases of the kidney. Semin Diagn Pathol. 2018;35(6):360–9. 3. Truong LD, Herrera GA. The evolving revolution of pathology role in renal medical diseases. Arch Pathol Lab Med. 2009;133(2):178–80. 4. Herson M, de Bel T, den Boer M, et al. Deep learning-based histopathologic assessment of kidney tissues. J Am Soc Nephrol. 2019;30(10):1968–79. 5. Wenderfer EE, Gault JP.  Glomerular disease in children. Adv Chronic Kidney Dis. 2017;24(6):364–71. 6. Grimbert P, Audard V, Remy P, Lang P, Sahali D. Recent approaches to the pathogenesis of minimal change nephrotic syndrome. Nephrol Dial Transplant. 2003;18:245. 7. Alpers CE. The kidney. In: Kumar V, Abbas AF, Fausto N, Aster TC, Robbins and Cotran, editors. Pathologic basis of disease. 8th ed. Philadelphia: Elsevier; 2010. p. 905–67. 8. Eison TM, Ault BH, Jones DP, et al. Post-streptococcal acute glomerulonephritis in children. Pediatr Nephrol. 2011;26(2):165–80. 9. Sethi S, Nester CM, Smith RJH. Membranoproliferative glomerulonephritis and C3 glomerulopathy. Resolving the confusion. Kidney Int. 2012;81(5):434–41. 10. Qi C, Mao X, Zhang Z, Wu H. Classification and differential diagnosis of diabetic nephropathy. J Diabetes Res. 2017:8637138. https://doi.org/10.1155/2017/8637138. 11. Akhtar M, Taha NM, Nauman A, et al. Diabetic kidney disease: past and present. Adv Anat Pathol. 2020;27(2):87–97. 12. Jennette C, Weening JJ. Heptinstall, Robert H. (alive). In: van den Tweel JG, editor. Pioneers in pathology. In: van Krieken JHJM, series editor. Encyclopedia of pathology. Cham:Springer; 2017. p. 212–14. 13. Racusen LC. In memoriam-Dr. Robert Hodson Heptinstall. BMC Nephrol. 2021;22:163. 14. Heptinstall RH, Gorrill RH. Experimental pyelonephritis and its effects on the blood pressure. J Pathol Bacteriol. 1955;69:191–8. 15. Hill GS, Heptinstall RH.  Steroid-induced hypertension in the rat. A microangiopathic and histologic study of the pathogenesis of hypertensive vascular and glomerular lesions. Am J Pathol. 1968;52:1–40.

Chapter 10

Gastrointestinal Tract, Liver, and Pancreas

Gastrointestinal tract, liver, and pancreatic pathology are popular subspecialty areas of surgical pathology, although there is no official certification in this subspecialty. Pathologists in this subspecialty examine and diagnose lesions affecting these three organs. They work closely with gastroenterologists, hepatologists, radiologists, and surgeons who specialize in surgery of these organs and in treating patients with diseases affecting these organs. The types of diseases the pathologist examines under the microscope range from infections, cysts, inflammatory conditions autoimmune diseases, and neoplasms. A few examples of diseases affecting the gastrointestinal tract, liver, and pancreas in the practice of pathology today are summarized below.

Helicobacter Pylori Infection The stomach was historically considered as a sterile environment free of infectious organisms, because of its high acidity. However, the past few decades have shown that Helicobacter pylori (H. pylori) is a common resident of the stomach and can lead to development of infectious diseases and neoplasms. H. pylori is present in gastric biopsies of most patients with duodenal ulcers, gastric ulcers, and chronic gastritis. H. pylori is a gram-negative bacterium that colonizes the gastric epithelium. It is acquired in childhood and can persists for the lifetime of the host, in spite of the harsh gastric environment. About half of the world’s population is infected with H. pylori [1]. The connection between H. pylori and chronic gastritis led to the awarding of the Nobel Prize to Marshall and Warren [2, 3]. H. pylori is typically found in the antrum of the stomach. On biopsy, the bacteria are usually present in the superficial mucus associated with the epithelial cells on the surface of the neck regions of the glands (Fig. 10.1). Special stains such as the Warthin–Starry silver stain or immunohistochemical staining with specific antibodies directed against © The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 R. V. Lloyd, Pathology: Historical and Contemporary Aspects, https://doi.org/10.1007/978-3-031-39554-3_10

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Fig. 10.1  Chronic gastritis secondary to Helicobacter pylori infection. Inflammatory cells including lymphocytes and plasma cells are present in the lamina propria and in the mucosal glands

Fig. 10.2  Chronic gastritis secondary to Helicobacter pylori infection. Immunohistochemical staining with an antibody specific for Helicobacter demonstrate the bacteria in the lumen of the glands (arrow)

H. pylori can be used to visualize the bacteria (Fig.  10.2). The areas next to the organisms in the tissues usually contain intraepithelial neutrophils and subepithelial plasma cells along with lymphoid cells and germinal centers. Some patients with H. pylori go on to develop gastric carcinomas or mucosal-associated gastric lymphomas.

Large Intestinal Polyps The large intestine is the most frequent location for polyps, although polyps are also present in the small intestine and stomach and in other areas of the gastrointestinal tract. Polyps in the colon have been classified into inflammatory, hamartomatous, hyperplastic, and neoplastic types. Hyperplastic polyps are usually present in the right colon and are usually less than 5 mm in diameter. Microscopically, they are

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Fig. 10.3  Sessile serrated polyp with superficial serrated crypts showing asymmetric proliferation and deep serrations with basilar crypt dilatation

Fig. 10.4 Adenomatous polyp (tubular adenoma). Crowded glands with enlarged cells showing elongated nuclei are present. Focal areas with villous configuration are also present

made up of mature goblet cells and absorptive cells. These lesions should be distinguished from serrated adenomas which are true neoplasms. Serrated polyps include hyperplastic polys, traditional serrated adenomas (Fig. 10.3), and sessile serrated adenomas and they show molecular changes seen in colorectal carcinomas [4]. Neoplastic polyps include tubular (Figs. 10.4 and 10.5), tubulovillous, and villous adenomas (Fig.  10.6). These neoplastic polyps can progress from low-grade to high-grade dysplasia and on to intramucosal carcinoma and invasive carcinomas. Dysplastic changes in neoplastic polyps are often diagnosed by the gastrointestinal pathologist and include cells with enlarged, elongated, and stratified nuclei with hyperchromasia and prominent nucleoli. Tubular adenomas are usually small pedunculated polyps with rounded tubular glands, while villous adenomas are usually larger sessile polyps that are covered with slender villi. Tubulovillous adenomas have a mixture of tubular and villous features. The sessile serrated adenomas have some features like hyperplastic polyps, but do not appear as dysplastic as other

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Fig. 10.5 Tubular adenoma with high-grade dysplasia showing prominent dysplastic glands

Fig. 10.6  Villous adenoma with papillary-like projections. There are long papillary growth and cells with increased nuclear sizes. Invasive growth is not present

neoplastic polyps. They appear serrated or sawtooth throughout the full length of the glands from the base to the surface. Crypt dilatation is usually a prominent feature of serrated adenomas. Intramucosal invasion can develop when the adenoma shows high-grade dysplasia, extends beyond the basement membrane and into the lamina propria [4]. However, because of the absence of lymphatic channels in the colonic mucosa there is a very low metastatic potential at this stage of growth. However, these neoplastic polyps are capable of metastasizing when they extend into the submucosal stalk of the polyp. There are several molecular pathways leading to the development of colorectal carcinomas. The serrated pathway includes mutations in RAS, disruption of Wnt signaling pathway, and extensive methylation of CpG islands [5]. Colorectal serrated polyps are classified as hyperplastic polyps, sessile serrated lesions, and traditional serrated adenomas. In the serrated neoplastic pathway, the latter two are considered as premalignant lesions.

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Colonic Adenocarcinomas Colonic adenocarcinoma is the most common malignant lesion of the gastrointestinal tract and is a major cause of mortality worldwide (Figs. 10.7 and 10.8). In the USA, there are over 55,000 deaths from colonic adenocarcinoma with around 130,000 patients developing this disease annually. Compared to the USA and other developed countries, the incidence of colonic adenocarcinoma is much lower in Africa, South America, and India. Earlier studies highlighted the molecular alterations during progression from colonic adenomas to carcinomas with involvement of specific genes including APC, BETA CATENIN, K-RAS, TP53, SMAD 2,4, and TELOMERASE [6, 7]. Another pathway for the development of colorectal carcinomas involves DNA mismatch repair deficiency with microsatellite instability. Tumors belonging to this group can be detected by loss of staining for mismatch repair proteins or by Fig. 10.7  Invasive colonic adenocarcinoma. The nests of cancer cells invade into the lamina propria (arrow). Normal colonic epithelium (NC) is also present

Fig. 10.8  Invasive colonic adenocarcinoma showing foci of angiolymphatic invasion (arrow). Normal colonic crypts are present in the lower right-hand corner of the image

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molecular analysis of microsatellite sequences [7]. The morphological features of colonic adenocarcinomas with microsatellite instability include location in the right proximal colon, abundant lymphocytic infiltration, and a poorly differentiated mucinous or signet ring appearance [7]. Tumors with microsatellite instability constitute about 15% of colorectal adenocarcinomas [8, 9].

Liver Diseases The liver is one of the rare organs in humans that is capable of regenerating. After partial hepatectomy, the liver may regenerate completely in a few weeks. The range of liver diseases range from infectious, inflammatory, cystic, and neoplastic lesions.

Alcoholic Liver Disease Alcoholic liver disease is a complex process that involves changes in the liver induced by excessive consumption of alcohol. The spectrum of morphological changes range from inflammation to steatosis (Fig. 10.9) then cirrhosis (Figs. 10.10 and 10.11) which may go on to hepatocellular carcinoma (Fig. 10.12) [10]. Alcoholic hepatitis is the most severe form of alcohol-induced liver injury. The histologic spectrum includes fatty liver, alcoholic hepatitis, and cirrhosis. Patients with alcoholic liver disease are usually diagnosed at an advanced stage. The diagnosis requires documentation of heavy drinking and exclusion of other causes of liver disease [11]. Treatment with corticosteroids may increase the chances of short-term survival. Alcoholic liver disease is one of the major causes of chronic liver disease worldwide. It is associated with up to half of the cirrhosis-associated deaths in the USA [11]. The presence of other diseases such as hepatitis C virus infection usually Fig. 10.9 Hepatic steatosis in a patient with a history of alcohol abuse. There is predominantly macro-vesicular steatosis with a few foci of micro-vesicular steatosis also present

Alcoholic Liver Disease Fig. 10.10  Alcoholic liver disease with extensive fibrosis (F) in the background. Foci of macro-vesicular steatosis are present in the right upper portion of the image

Fig. 10.11 Alcoholic cirrhosis highlighted with trichrome histochemical stain (blue). Two foci of hepatic nodules (red color) are present in the field. Steatosis is also present in the nodules

Fig. 10.12 Hepatocellular carcinoma showing nests of tumor cells infiltrating the stroma (arrow). This carcinoma is from a patient without cirrhosis and is consistent to be a fibrolamellar variant of hepatocellular carcinoma which is associated with a better prognosis than hepatocellular carcinomas arising from cirrhosis caused by alcohol abuse

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makes liver fibrosis worse. Alcoholic fatty liver is usually seen within 2 weeks of heavy and regular drinking of alcohol [12, 13]. Alcoholic cirrhosis is characterized by the accumulation of extracellular matrix with a diffuse accumulation of excess fibrous tissue with parenchymal nodules consisting of regenerating hepatocytes and changes in the vascular architecture [14, 15]. Alcoholic cirrhosis is associated with an increased risk of developing hepatocellular carcinoma. It usually increases with age and with the quantity and duration of alcohol abuse. The histologic features of alcoholic cirrhosis include bridging fibrosis with scars lining portal tracts with each other and with portal tracts with hepatic veins. Fibrosis usually involves collagen deposition and remodeling. The presence of parenchymal nodules results from hepatocyte regeneration and scarring which usually leads to disruption of the architecture of the liver (Fig. 10.11). Recent studies have shown that fibrosis in alcoholic cirrhosis may be reversible [15]. The causes of death in patients with cirrhosis include liver failure and development of portal hypertension.

Viral Hepatitis Viral hepatitis can be caused by various types of viruses including Epstein–Barr virus, cytomegalovirus, Hepatits A, Hepatitis B, and Hepatitis C viruses as well as less commonly Hepatits E, D, and G viruses. Hepatitis C virus causes liver disease in more than 170 million people worldwide [16, 17]. Hepatitis C virus is the most common chronic bloodborne infection and is the etiology of chronic liver diseases in around half of the patients in the USA. About one of five patients with Hepatitis C virus infection go on to develop cirrhosis. This virus is unstable with multiple genotypes and subtypes, because of the poor fidelity of Hepatitis C virus RNApolymerase. Transmission of Hepatitis C virus is often done by blood transfusion worldwide, but this is uncommon in the USA because of careful screening. Patients with Hepatitis C virus infection usually have persistent infection with chronic hepatitis. Patients with acute illnesses may be asymptomatic in up to 80% of cases. Cirrhosis usually develops between 5 and 20 years after acute infection. Hepatocellular carcinoma which can be caused by Hepatitis C virus is the second leading cause of death worldwide [17, 18].

Hepatocellular Carcinoma Hepatocellular carcinoma is the most common primary liver cancer with more than 600,000 new cases per year worldwide [19]. Most patients with this cancer die of their disease in a relatively short time. The range of etiologies include chronic viral hepatitis with Hepatitis B and C viruses, chronic alcoholism, nonalcoholic steatohepatitis, and food contamination such as aflatoxin. Hepatocellular carcinoma may be unifocal or multifocal in the liver or it may be infiltrative. The tumor cells are

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usually paler than the surrounding liver and the tumor may have a green appearance due in part to the accumulation of bile pigment. Vascular invasion is common in hepatocellular carcinoma. The tumor cells may invade the portal vein. The tumor cells may range from well-differentiated to poorly differentiated anaplastic carcinomas. Molecular studies of hepatocellular carcinomas have shown the majority of cancers have TERT promoter mutations. In addition, TP53 and CTNNB1 genes are frequently mutated in many hepatocellular carcinomas [20]. The fibrolamellar variant of hepatocellular carcinoma is a unique form of liver carcinoma (Fig. 10.12). This variant is present in only a small percentage of cases. These uncommon tumors usually develop in younger patients between 20 and 40 years of age and an underlying liver disease such as cirrhosis is usually not present as in most other cases of hepatocellular carcinomas. The prognosis is also more favorable compared to other cases of hepatocellular carcinomas [19]. Histological examination shows large polygonal cells with abundant eosinophilic cytoplasm and prominent nucleoli with pale inclusion bodies. Intratumoral fibrosis is often prominent, but the fibrosis is not present in the nonneoplastic liver (Fig. 10.12). Molecular studies have shown a DNAJB1 and PRKACA fusion on chromosome 19 with a deletion between these two genes [21]. The fusion is highly specific for fibrolamellar hepatocellular carcinomas [22].

Pancreatic Neoplasms Pancreatic neoplasms include two major types of tumors—those arising from the exocrine pancreas which are predominantly adenocarcinomas and tumors arising from the neuroendocrine cells or their precursors cells in the pancreatic islets giving rise to neuroendocrine tumors. Pancreatic adenocarcinomas have a very high mortality rate and it is the fourth leading cause of death in the USA [23]. Only about 5% of patients with pancreatic adenocarcinomas survive for 5 years. One of the major reasons for the high mortality is that the disease remains asymptomatic until it invades structures adjacent to the pancreas [23, 24], so many patients have inoperable disease at the time of diagnosis. The progression from a precursor lesion designated as pancreatic intraepithelial neoplasia (PanIN) to carcinoma has been well documented [24]. Pancreatic adenocarcinoma is most common in the head of the pancreas which accounts for 60% of cases. Carcinomas are uncommon in the tail of the pancreas (5% of cases). The histological features include moderately to poorly differentiated glands with a deeply infiltrative pattern of growth. Perineural invasion with lymphatic and vascular invasion are commonly present (Figs. 10.13 and 10.14). Molecular changes in pancreatic adenocarcinoma include mutations of KRAS, p16CDKN2A, SMAD4, and TP53 as common molecular alterations [25]. However, many other genetic alterations can be detected in a smaller percentage (5–10%) of pancreatic adenocarcinomas.

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Fig. 10.13 Pancreatic adenocarcinoma composed of ductal cells with enlarged nuclei showing infiltration into a desmoplastic stroma

Fig. 10.14 Pancreatic adenocarcinoma showing perineural invasion. The cancer cells (arrow) have invaded a nerve (N)

Neuroendocrine Tumors of the Pancreas Pancreatic neuroendocrine tumors include well-differentiated and poorly differentiated pancreatic endocrine tumors which are distinguishable by grade using mitotic activity and Ki-67 proliferation index [26]. Pancreatic endocrine tumors constitute about 1–3% of pancreatic neoplasms each year. The grade 1 tumors include well-­ differentiated (Figs. 10.15 and 10.16), while the grade 2 tumors include other well-­ differentiated neoplasms with a higher mitotic index and Ki67 proliferative index. A third category of well-differentiated high-grade neuroendocrine tumors (G3) has been recently added to the grading scheme. Morphologically neuroendocrine tumors have dense core secretory granules when they are examined by electron microscopy. They are positive for biomarkers such as chromogranin, synaptophysin, and INSM1. Some of the tumors produce specific hormones such as insulin, glucagon, somatostatin, gastrin, vasoactive intestinal polypeptide, serotonin, and rarely ACTH. Most

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Fig. 10.15  Normal islet cells (I) composed of neuroendocrine cells secreting insulin, glucagon, somatostatin, and pancreatic polypeptide. Most of the cells within the islet are insulin-producing

Fig. 10.16 Neuroendocrine tumor of the pancreas composed of nests of tumor cells with moderate amounts of cytoplasm and slightly enlarged nuclei. This was a nonfunctioning tumor which was positive for the broad spectrum markers chromogranin A and synaptophysin. The tumor cells were negative for pancreatic hormones

tumors arise sporadically, but a small percentage may be familial such as in multiple endocrine neoplasia type I, von Hippel Lindau disease, tuberous sclerosis, neurofibromatosis type I, and glucagon cell hyperplasia and neoplasia syndromes [27]. Genes involved in the pathogenesis of these tumors include MENI, DAXX/ATRX, and the mammalian target of rapamycin (mTOR) pathway [28]. Neuroendocrine tumors are characterized by small to medium size cells with a “salt-and-pepper” nuclear pattern and small nucleoli. Mitotic activity and Ki-67 labeling index are used to grade these tumors. Many pathologists have contributed to progress in gastrointestinal, liver, and pancreatic pathology. The biographies of two of these individuals are summarized below.

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Morison, Basil Clifford (1921–2016) Dr. Morison was born in 1921 in the UK. His father was a consultant surgeon. He graduated from Middlesex Hospital Medical School in London in 1949. He started training in pathology at the Middlesex Hospital a year later. He also continued his academic studies and received a doctor of medicine degree from Oxford University in 1955 [29, 30]. His first research project in pathology involved studies of gastric and esophageal pathology. Early on in his career, he described a gastric-type metaplasia which was present in Barrett’s esophagus. During the early part of his career endoscopic biopsies were becoming more widespread. Dr. Morison soon became an expert in interpreting endoscopic biopsies. He worked with Dr. Cuthbert Dukes initially and when Dr. Dukes retired from St. Mark’s Hospital in London, Dr. Morison succeeded him. He also continued Dr. Duke’s work on colorectal cancer. Although Dr. Morison was the only consultant pathologist at St. Mark’s Hospital in gastrointestinal lesions, he was extremely productive in his studies of gastrointestinal pathology. Some of his studies ranged from Crohn’s disease and ulcerative colitis to studies of colorectal carcinoma development [31, 32]. He also did studies in other areas of gastrointestinal pathology including diverticular diseases and familial polyposis syndrome [29, 30]. Dr. Morison was called a “surgeon’s pathologist“because of his willingness to discuss the clinical significance of unusual biopsy findings. He was always willing to discuss pathological findings in unusual cases with his colleagues. His textbook on gastrointestinal pathology written with Dr. Dawson was a major contribution to the field, since it was the first major textbook in the field [33]. The first edition was published in 1972. He wrote over 200 original papers and 11 books in his field. Although he retired in 1985, he was sought after because of his knowledge and vast experience in gastrointestinal pathology. He received many honors including president of the Royal Society of Medicine-Pathology section, treasurer and vice president of the Royal College of Pathology. He was the first pathologist to become president of the British Society of Gastroenterology and a lecture was named in his honor. He was also awarded the commander of the order of the British Empire for Services in Medicine in 1987. Dr. Morison had many outstanding visiting fellows in pathology from all around the world. Many of these fellows became outstanding leading gastrointestinal pathologists in their own countries. After retiring in 1985, he continued to pursue many of his other interest which included ornithology and gardening. He was married twice and had three children. He died in 2016 at the age of 93.

Popper, Hans (1903–1988) Dr. Hans Popper was born in Vienna, Austria, in 1903. His father was a physician and he followed in his father’s footsteps and attended medical school at the University of Vienna and graduated in 1928. While in medical school he worked in a biochemistry laboratory. After graduation, he studied anatomic pathology for

References

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5 years and he established a biochemistry section in the pathology services [34]. One of his mentors was Dr. Hans Eppinger who would later become involved with the medical experiments under Hitler during World War II. Dr. Eppinger was also involved in the Nuremberg War Criminal Trials at the end of World War II.  Dr. Eppinger would later commit suicide [34]. It was during the time that he worked with Dr. Eppinger that Dr. Popper developed the creatinine clearance test to measure renal function by combining his skills in pathology and biochemistry. This was a major contribution to clinical laboratory testing. With the rise of Hitler and the Third Reich in 1934, and because he was of Jewish ethnicity, Dr. Popper migrated to the USA. He obtained a research fellowship at the Cook County Hospital in Chicago and worked on his PhD degree at the University of Illinois. He enlisted in the Armed Forces in 1943 during World War II and served until 1947. He later became a professor of pathology at Northwestern University and served as the director of the Hektoen Institute for Medical Research. In 1942 while he lived in Chicago, Dr. Popper married Lina Billig who was also originally from Austria and they had two sons. In 1957, he and his family moved to New York for a position at Mount Sinai Hospital. He served as the pathologist-in-chief and continued with his rigorous schedule which soon made him the most outstanding hepatopathologist in the world. His schedule was amazing. He worked 7 days a week performing diagnostic service, research, and teaching. He also worked more than 11 hours each day during the week, all day on Saturday and half the day on Sundays. He became the first chairman of pathology at Mount Sinai School of Medicine and the first president of the medical center and dean of the medical school [35, 36]. Dr. Popper received many honorary degrees from over 15 universities from around the world. He also published more than 800 original papers and 28 books. Dr. Popper received many awards and honors including election to the prestigious National Academy of Science. He received the Gold Medal Cane Award from the American Association of Pathologist and Bacteriologist, the Distinguished Service Award from the International Association for the study of the liver, and the USCAP Distinguished Pathologist Award as the inaugural recipient. He trained many medical students, residents, and pathologists as well as other physicians. A few of his former students formed the Hans Popper Hepatology Society which has been in existence for several decades as one of the leading liver pathology organizations. Dr. Popper died in 1988 of pancreatic cancer.

References 1. Wroblewski LE, Peek RM.  Helicobacter pylori, cancer and the gastric microbiota. Ad Exp Med Biol. 2016;908:393–408. 2. Marshall BJ, Warren JR. Unidentified curved bacilli in the stomach of patients with gastritis and peptic ulcer. Lancet. 1984;323(8390):1311–5.

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3. Uemura N, Okamoto S, Yamamoto S, et al. Helicobacter pylori infection and the development of gastric cancer. N Engl J Med. 2001;345(11):784–9. 4. Nossfinger AE. Serrated polyps and colorectal cancer. New pathway to malignancy. Ann Rev Pathol Mech Dis. 2009;4:343–68. 5. Crockett SD, Nagtegaal ID. Terminology, molecular features, epidemiology, and management of serrated colorectal neoplasia. Gastroenterology. 2019;157:949–66. 6. Vogelstein B, Fearon ER, Hamilton SR, et  al. Genetic alterations during colorectal-tumor development. N Engl J Med. 1988;319(9):525–32. 7. Fearon ER, Vogelstein B. A genetic model for colorectal tumorigenesis. Cell. 1990;61:759–67. 8. Boland CR, Goel A.  Microsatellite instability in colorectal cancer. Gastroenterol. 2010;138(6):2073–87. 9. Thibodeau SN, Bren G, Schaid D. Microsatellite instability in cancer of the proximal colon. Science. 1993;260:816–9. 10. Louvet A, Mathurin P. Alcoholic liver disease mechanisms of injury and targeted treatment. Nat Rev Gastroenterol Hepatol. 2015;12(4):231–42. 11. Singal AK, Bataller R, Ahn J, et  al. ACG clinical guide. Alcoholic liver disease. Am J Gastroenterol. 2018;113(2):175–94. 12. Lane BP, Lieber CS. Ultrastructural alterations in human hepatocytes following ingestion of ethanol with adequate diets. Am J Pathol. 1966;49:593–603. 13. Rubin E, Lieber CS. Alcohol-induced hepatic injury in nonalcoholic volunteers. N Engl J Med. 1989;278:869–76. 14. Osna NA, Donohue TM Jr, Kharbanda KK. Alcoholic liver disease: pathogenesis and current management. Alcohol Res. 2017;38(2):147–61. 15. Lackner C, Tiniakos D. Fibrosis and alcohol-related liver disease. J Hepatol. 2019;70:294–304. 16. Pol S, Lagaye S. The remarkable history of hepatitis C virus. Genes Immun. 2015;20(5):436–46. 17. Goossens N, Hoshida Y.  Hepatitis C virus-induced hepatocellular carcinoma. Clin Mol Hepatol. 2015;21(2):105–14. 18. Bailey JR, Barnes E, Cox AL. Approaches, prognosis and challenges to hepatitis C virus vaccine development. Gastroenterol. 2019;156(2):418–30. 19. El Jabbour T, Lagana SM, Lee H. Update on hepatocellular carcinoma Pathologist’s review. World J Gastroenterol. 2019;25(14):1653–65. 20. Vyas M, Zhang X. Hepatocellular carcinoma: role of pathology in the era of precision medicine. Clin Liver Dis. 2020;24(4):591–610. 21. Rebouissou S, Nault J-C.  Advances in molecular classification and precision oncology in hepatocellular carcinoma. J Hepatol. 2020;72(2):215–29. 22. Graham RP, Jin L, Knutson DL, et al. DNAJB-1-PRKACA is specific for fibrolamellar carcinoma. Mod Pathol. 2015;28:822–9. 23. McGuigon A, Kelly P, Turkington RC, et al. Pancreatic cancer: a review of clinical diagnosis, epidemiology, treatment and outcomes. World J Gastroenterol. 2018;24(43):4846–61. 24. Luchini C, Capella P, Scarpa A. Pancreatic ductal adenocarcinoma and its variants. Surg Pathol Clin. 2016;9(4):547–60. 25. Hezel AF, Kimmelman AC, Stanger BZ, et al. Genetics and biology of pancreatic ductal adenocarcinoma. Genes Dev. 2006;20:1218–49. 26. Guilmette J, Nose V. Neoplasms of the neuroendocrine pancreas: an update in the classification, definition and molecular genetic advances. Adv Anat Pathol. 2019;26:13–30. 27. Kloppel G, Couveland A, Hruban RH, et al. Neoplasms of the neuroendocrine pancreas. In: Lloyd RV, Osamura RY, Kloppel G, Rosai J, editors. WHO classification of tumours of endocrine organs. Lyon: International Agency for Cancer; 2017. p. 209–39. 28. Zhang N, Francois SR, Lyer R, et al. Current understanding of the molecular biology of pancreatic neuroendocrine tumors. J Natl Cancer Inst. 2013;105(14):1005–17. 29. Gillain S. Morison. Basil Clifford 1921–2016. Colorectal Dis. 2017;19(1):5.

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30. Shepherd NA. Morison Basil Clifford (1921-2016). In: van den Tweel JG, editor. Pioneers in pathology. In: van Krieken JHJM, series editor. Encyclopedia of pathology. Cham:Springer; 2017. p. 392–95. 31. Morison BC, Pang LSC. Rectal biopsy as an aid to cancer control in ulcerative colitis. Gut. 1967;8:423–34. 32. Muto T, Bussey H, JR, Morison BC. The evolution of cancer of the colon and rectum. Cancer. 1975;16:2251–70. 33. Morison BC, Dawson JMP. Gastrointestinal pathology. Oxford: Blackwell Scientific; 1972. 34. Geller Sa Popper. Hans (1903–1988). In: van den Tweel JG, editor. Pioneers in pathology. In: van Krieken JHJM, series editor. Encyclopedia of pathology. Cham:Springer; 2017. p. 442–49. 35. Schmid R.  Hans Popper November 1903–1988: a biographical memoir. Washington D.C.: National Academic Press; 1994. 36. Kaiser S, Sziranyi J, Grob D. The hepatopathologist Hans Popper (1903-1988). An early victim of National Socialism in Austria. Pathologe. 2020;41(Suppl 1):530–8.

Chapter 11

Lung Pathology

Surgical pathology of lung diseases involves one of the most critical organs in the human body. The major conditions studied in lung pathology include inflammatory, autoimmune, infectious, and neoplastic diseases. The pulmonary pathologist works closely with radiologist, pulmonologists, and thoracic surgeon in the diagnosis and management of difficult and challenging cases of lung diseases. In the area of inflammatory lung diseases, the radiologist has a major role in establishing the diagnosis, especially if the pathologist has only a needle biopsy of lung tissue. Classification of neoplastic lung diseases is done mainly by the pathologist, since microscopic examination is needed to classify these neoplasms. A few examples of pulmonary diseases are summarized below.

Acute Pneumonia The etiology of pneumonias includes bacterial, viral, or of atypical origin such as mycoplasma or chlamydia. In debilitated patients, bacterial pneumonia may coexist with viral pneumonia. Community-acquired acute pneumonia may include various bacteria such as Streptococcus, Haemophilus influenzae, Staphyloccocus aureus, Klebsiella, and Legionella pneumonia [1]. Anatomically, there may be bronchopneumonia with a patchy distribution in parts of the lungs or lobar pneumonia with involvement of the entire lobe. Bacterial pneumonia in older adults may be associated with all of the above organisms usually associated with a higher mortality [2]. The histopathological features of bacterial pneumonia include the acute phase with edema and neutrophils in alveolar spaces, as well as congestion of small vessels in the alveolar walls. This is referred to as red hepatization. The next phase is early organization of the inflammatory cells and early fibrin formation. The advance stage includes accumulation of fibrin and myxoid material with macrophages and

© The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 R. V. Lloyd, Pathology: Historical and Contemporary Aspects, https://doi.org/10.1007/978-3-031-39554-3_11

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fibroblasts. This is followed by resolution or organization. Organization secondary to marked deposition of fibroblast and scarring can occur. When patients are treated with antibiotics, this cycle is disrupted and the clinical signs are decreased 2–3 days after initiation of antibiotics.

Viral Pneumonia Viral pneumonia is more common in pediatric patients. There are many viruses involved in pneumonia and some of these include respiratory syncytial virus, influenza virus, parainfluenza virus, and coronavirus [3]. Some other organisms involved in community-acquired pneumonia (also referred to as atypical pneumonia) include Mycoplasma pneumoniae, especially in children and young adults and Chlamydia pneumonia. Histopathological changes in these atypical pneumonias include patchy or lobar involvement with the inflammatory process; some cases may even be bilateral. Pleural involvement is uncommon. Histopathological findings include inflammation in the walls of the alveoli in an interstitial location with infiltration by lymphocytes, macrophages, and plasma cells. Secondary bacterial infection may also occur with viral and atypical pneumonias, so a mixed histopathological picture may be present.

Influenza Types A, B, and C Pneumonia Influenza type A infects humans and has been a major source of pandemics and epidemic infections. Variations in specific proteins in the virus, including hemagglutinins and neuraminidases, are related to the ability of the virus to be resistant to antibodies of the host when the enzymes are replaced by recombination of RNA segments in other animal viruses and this had led to pandemics in the past [3]. Influenza B and C infect mostly children [3]. The H5N1 antigenic type of avian influenza mainly infects birds and is often associated with a lethal infection. Histopathological examination of the lung in patients with H5N1 influenza infection show an infiltrate of lymphocytes and mononuclear cells with some plasma cells. The mucosa of the trachea and adjacent areas is edematous, especially in pediatric patients. The infection usually extends to the bronchi and bronchioles and is associated with a mucous exudate [4]. The influenza virus was responsible for the pandemic of 1918 which may have killed up to an estimated 50 million people worldwide. Influenza A infection has recurred every 8–41 years for at least several centuries with infections of up to 50% of the population [4].

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Sars-CoV-2 Pneumonia Pandemics with the coronaviruses have included severe respiratory syndromes (SARS), Middle East respiratory syndrome (MERS), and currently COVID-19 [5]. The viruses, which are most likely acquired from zoonotic sources, spread by direct means after contact transmission. Symptoms usually include fever, cough, myalgia, and variable degrees of respiratory failure. SARS was first recognized in China in 2002 and spread to 30 countries infecting 79,000 people by 2003 with a 9.5% fatality rate. The sources of SARS involved dogs, racoons, badgers, and also humans working at the same commercial market [5]. MERS was associated with a higher fatality rate than SARS (34%). Camels were thought to be the potential source of infection for humans [5]. SARS-CoV-2 (COVID-19) first appeared in Wuhan, China in December 2019. A pandemic developed within a few months. Infected patients developed pneumonia, acute respiratory distress syndrome, and respiratory failure [6, 7]. In contrast to acute COVID-19 infection, long COVID is associated with a more chronic disease lasting weeks to months or even longer. COVID-19 pulmonary disease is associated with excess vascular permeability leading to microthrombi and pulmonary edema with secondary bacterial infection in some cases. Extrapulmonary manifestations include olfactory dysfunction, gastrointestinal syndromes, and cardiac, liver, and renal dysfunction [7]. The main pathologic lung findings include reactive epithelial cell changes and diffuse alveolar damage. Microvascular changes, organizing pneumonia, and interstitial fibrosis may also be present [7].

Chronic Pneumonia Chronic pneumonia may be associated with bacteria such as tuberculosis and with fungal infections such as histoplasmosis, blastomycosis, and coccidiomycosis. These pneumonias are usually associated with a granulomatous inflammation with multinucleated histiocyte-derived cells. This histologic feature is usually not present with bacterial or viral pneumonias.

Interstitial Pulmonary Fibrosis (IPF) Interstitial pulmonary fibrosis (IPF) is a syndrome with characteristic pathologic, radiologic, and clinical features [8]. The histopathological pattern of fibrosis is often referred to as usual interstitial pneumonia (UIP), although UIP is usually not unique for IPF. The correlation of pathologic, radiologic, and clinical findings is essential to establish a correct diagnosis [8]. Histopathological findings include a cobblestone pattern on the pleural surface due to retraction of fibrotic tissues. Subpleural fibrosis is

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usually seen with a characteristic patchy distribution. Microscopic features include patchy interstitial fibrosis which is a distinctive finding. Early in the disease there is focal fibroblastic proliferation; there is subsequent increased fibroblastic proliferation with disease progression. There is often an admixture of early and late lesions in biopsy specimens or at autopsy. Alveolar destruction develops with progressive fibrosis and there is formation of cystic spaces leading to a honeycomb pattern of fibrosis radiologically and under the microscope (Figs.  11.1 and 11.2). Pulmonary artery hypertensive changes are often seen as well as smooth muscle hyperplasia.

Fig. 11.1 Interstitial pulmonary fibrosis (usual interstitial pneumonia) showing subpleural accentuation of dense fibrous tissue and irregular spaces. A prominent myxoid matrix is present focally (arrow)

Fig. 11.2  Interstitial pulmonary fibrosis (usual interstitial pneumonia) with a prominent myxoid stroma and dilated cystic spaces. The epithelial cells lining the dilated cystic spaces (arrow) show prominent reactive changes. This nonneoplastic disease is of uncertain etiology and is associated with death of the patient in 3–5 years after diagnosis

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Molecular studies have shown shortened chromosomal telomeres with mutation of TERT, TEAC, PARW, and RTEL genes which are important for maintenance of telomere length. Mutations of these genes also indicate an increased risk of IPF before the diagnosis is made [8]. Risk factors for development of IPF include male sex, older age, and cigarette smoking [8, 9]. Patients with IPF usually survive less than 4 years after diagnosis, especially when the disease is diagnosed after age 65. Lung transplantation is one of the ways that IPF has been treated in some patients. However, long-term survival for more than 5 years is poor even with lung transplantation [9, 10].

Lung Neoplasms Lung cancer is the most commonly diagnosed malignant neoplasm worldwide and the most common cause of death from cancer [11]. Cigarette smoking is a major contributor to this morbidity and mortality, but other etiologies include industrial chemicals, radiation, asbestos, and other pollutants including radon may also be important in the etiology of this highly lethal disease. Adenocarcinoma has become the most common type of malignant lung cancer in the USA. This is followed by squamous cell carcinoma which decades ago was the most common cancer type. Small-cell carcinoma and large-cell carcinoma are both less common.

Squamous Cell Carcinoma Development of squamous cell carcinoma is usually preceded by squamous cell metaplasia in which change from one type of mature epithelium to another type of mature epithelium occurs. In the lungs, this change is from respiratory epithelium to squamous epithelium. Metaplasia is followed by squamous dysplasia, if the toxic stimuli continue to affect the cells. The next stage of progression is carcinoma in situ followed by invasive carcinoma. The last stage in the progression is metastatic carcinoma which often becomes lethal at this stage. Squamous cell carcinoma has several diagnostic features. The tumor is composed of large eosinophilic cells with large nuclei. The cells have variable degrees of keratinization manifested by bright eosinophilic cytoplasm and they may show formation of keratin pearls (Fig. 11.3). Mitotic activity is usually easily found with more mitoses in higher grade cancers. Although most squamous cell carcinomas are centrally located in the lungs, there is an increasing number of peripherally located squamous cell carcinomas. Since many cancers metastasize to the lung, it is important for the pathologist to distinguish between a primary lung cancer and a metastasis. The presence of carcinoma in situ in adjacent bronchial epithelium is strong supportive evidence of a primary site in the lungs.

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Fig. 11.3  Squamous cell carcinoma of lung with nests of infiltrating squamous cells. This welldifferentiated carcinoma shows nests of keratin material in the center of a focus of cancer cells (arrow)

Molecular findings in squamous cell carcinomas include mutations in TP53 (in up to 50% of cases), RB1 (in 15% of cases), and inactivation of p16/INK4a [11]. Other molecular changes include amplification of FGFR1, mutations of PIK3CA, DDR2, and PTEN [12]. DNA methylation in squamous cell carcinoma has been associated with tumor development and progression [13].

Adenocarcinomas Adenocarcinomas are now the most common malignant lung neoplasms in both men and women in the USA. Many adenocarcinomas arise in the periphery of the lungs in contrast to squamous cell carcinomas that are often centrally located. However, with the increasing number of squamous cell carcinomas, histological examination of the carcinoma is essential for diagnosis. Adenocarcinomas show glandular differentiation (Fig.  11.4) and/or mucin production in the tumor cells. Histochemical stains for mucin is often positive. Immunohistochemical stains for TTF-1 is usually positive in lung adenocarcinomas in contrast to squamous cell carcinomas that are negative for this biomarker. However, this biomarker is not totally specific for lung adenocarcinoma, because thyroid carcinomas and a few other carcinomas are also be positive for TTF-1. Precursor lesions for lung adenocarcinomas include atypical adenomatous hyperplasia with transition to bronchoalveolar carcinoma. Adenocarcinoma in situ usually progresses to minimally invasive adenocarcinoma and then invasive adenocarcinoma [14]. Molecular alterations in pulmonary adenocarcinoma include mutations of KRAS (29%), EGFR (14%), and BRAF (7%) [14]. EGFR and KRAS mutations in these carcinomas are usually mutually exclusive [15]. EGFR mutations are most common in women, especially of Asian descent, as well as nonsmokers. Other common gene abnormalities in lung

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Fig. 11.4  Lung tissue with adenocarcinoma. Adjacent normal lung (NL) is present in the upper right-hand corner of the image. The tumor cells are present in nests and sheets with focal areas of gland formation. Adenocarcinoma is currently the most common type of lung cancer in the USA

adenocarcinomas include TP53, RB1, and p16 mutations, and they are more common in smokers compared to nonsmokers [15]. Other gene abnormalities with therapeutic implications in lung adenocarcinomas include ALK, ROS1, ERBB2, and MET [12].

Lung Neuroendocrine Tumors Lung neuroendocrine tumors comprise about 1–2% of lung cancers [16]. They make up about 39% of neuroendocrine tumors, the second most common site after the gastrointestinal tract [16]. The International Agency for Research of Cancer and the World Health Organization recently recommended that the names of lung neuroendocrine tumors be modified to follow many other neuroendocrine tumors including low-grade neuroendocrine tumors (carcinoids and atypical carcinoids) and neuroendocrine carcinomas to include small- and large-cell neuroendocrine carcinomas [17]. The low-grade neuroendocrine lesions are referred to as tumors while the high-grade lesions are designated as carcinomas. Neuroendocrine tumors of the lungs, like other neuroendocrine tumors, have dense core secretory granules that can be identified by electron microscopy. They usually express chromogranin, synaptophysin, and INSM1 which are universal neuroendocrine biomarkers. Tumorlets are neuroendocrine tumors that are 5  mm or smaller in size. Neuroendocrine tumors may arise centrally or peripherally, but location next to a bronchus or bronchiole is common. Tumors are graded by mitotic activity and Ki-67 proliferative index. Typical carcinoids have less than 2 mitoses per high power fields and a Ki67 index of less than 10%. Atypical carcinoids have 2–10 mitoses per 2 mm square and a Ki67 index of 10–25%. Most low-grade neuroendocrine tumors are nonfunctional, although a few tumors may secrete ACTH ectopically as well as other hormones (Fig. 11.5).

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Fig. 11.5 Neuroendocrine tumor (carcinoid tumor) of lung. Sheets of tumor cells with neuroendocrine-­appearing uniform nuclei and moderate amounts of cytoplasm. There is a low mitotic rate in this low-grade tumor

Fig. 11.6  Small-cell carcinoma of lung. This neuroendocrine carcinoma consists of nests of small tumor cells with large nuclei and scant amounts of cytoplasm. This high-grade tumor cells has a high mitotic rate (arrow). Cigarette smoking is one of the etiologic agents for the development of this cancer

High-grade neuroendocrine carcinomas or small- and large-cell neuroendocrine carcinomas are tumors with more than 10 mitoses per 2 mm square or a Ki67 index of 25–100%. They are highly malignant cancers [18]. The small-cell neuroendocrine carcinoma or high-grade neuroendocrine carcinoma are composed of round, oval, or spindle-shaped cells with prominent nuclear molding (Figs.  11.6, 11.7, 11.8, and 11.9). Necrosis is a common finding. Electron microscopy shows variable numbers of neurosecretory granules in around 70% of these carcinomas. A small percentage of the tumors are negative for Chromogranin A and probably correlates

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Fig. 11.7  Small-cell carcinoma of lung showing diffuse positive immunohistochemical staining for synaptophysin. This is a broad spectrum neuroendocrine marker present in the cytoplasm of neuroendocrine cells and tumors

Fig. 11.8  Small-cell carcinoma of lung showing positive staining for thyroid transcription factor I (TTF-1). About 60% of the malignant tumor cell nuclei are positive for this transcription factor. This immunohistochemical stain is not specific for small-cell carcinoma, since other cancers such as those in the thyroid are also positive for TTF-1

with tumors without many secretory granules by electron microscopy. They sometimes produce hormones ectopically including ACTH, parathyroid hormone-­related peptide, calcitonin, gonadotropins, serotonin, and others [19]. Large-cell neuroendocrine cancers have large nuclei, prominent nucleoli, and moderate amounts of cytoplasm. Molecular alterations in these carcinomas in addition to those in small-cell neuroendocrine carcinomas include mutations of NOTCH, MYC, TP53, RB1, and KMT2. They also include amplification of MYC, MYCN, and loss of PTEN [18, 19].

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Fig. 11.9  Small-cell carcinoma of lung showing strong immunohistochemical staining for the proliferating marker Ki-67(MIB-1). More than 90% of the cancer cells show positive nuclear staining for this proliferation marker

Fig. 11.10  Pleural tissue of lung showing a malignant mesothelioma. The cancer cells of this epithelial variant of mesothelioma include prominent tubular structures in a desmoplastic stromal background

Mesotheliomas Malignant mesotheliomas of the thorax can arise from the visceral or parietal pleura [20, 21]. Exposure to asbestos is one of the major risk factors for the development of mesotheliomas. There is a long latency period between exposure to asbestos and the development of malignant mesotheliomas usually around 30 years [22]. There are several subtypes of malignant mesotheliomas; the epithelioid subtype is the most common and make up about 80–90% of mesotheliomas (Figs.  11.10 and 11.11). Because mesotheliomas have

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Fig. 11.11  Higher magnification of a malignant mesothelioma of pleura with atypical mesothelial cells and a vascularized fibrous stroma cells with red blood cells in the endothelial-lines vessels. These cancers are associated with a very poor prognosis

overlapping histological features with lung adenocarcinomas, immunohistochemical staining becomes very important, especially with small biopsy specimens. Positive staining for calretinin, cytokeratin 5,6, and D2-40, as well as electron microscopic findings of cells with prominent microvilli are helpful diagnostic features for mesotheliomas. TTF-1 and CEA are helpful markers for adenocarcinoma. Molecular findings for mesothelioma include deletions on chromosomes 1p, 3p, 6q, 9p, and 22q which may be present in about a third of mesotheliomas. Mutations of p16 and TP53 are uncommon; however, overexpression of p53 protein may be present in the majority of cases (70%). Mesotheliomas usually have a poor prognosis with a 5-year survival of about 5% [20–22].

Outstanding Pulmonary Pathologist Herbert Spencer (1915–1993) Herbert Spencer was born in Barnet Hertfordshire, England in 1915. His father was a coat manufacturer. Herbert attended Highgate School and trained in medicine at St. Mary’s Hospital in London and he graduated with honors from medical school [23]. He met his future wife at St. Mary’s Hospital. They were married in 1940 and had four children. He started training to be a surgeon, but contracted a severe case of typhoid fever. When he recovered, he decided to go into pathology and received his training at Park Prewett Hospital in Basingstoke, England. He was called to serve in the army after 18 months of pathology training. He served in the Middle East in Iran, Iraq, and Egypt until 1946 [23]. He was the assistant director of pathology and officer in charge of the central laboratory in Cairo, Egypt. After leaving the service he became the pathologist to the Arching group of hospitals. He subsequently became the senior lecturer, reader, and professor of morbid anatomy at St. Thomas’s Hospital [23]. One of his main areas of interest was in the pathology of lung diseases. He was

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an early pioneer in the use of the electron microscope for pathological studies. His major publication was the book Pathology of the Lung, which was published in 1962. It became an authoritative text and underwent many editions over the years as a major pathology textbook. He also had expertise in tropical medicine and published a textbook on Tropical Pathology in 1973 [23]. Dr. Spencer served as an examiner in pathology to the conjoint board as well as a primary FRCS examiner. He was also an examiner for the MRCP. One of his other academic involvements included serving on the editorial board of the journal Thorax, which included studies in lung pathology. Dr. Spencer retired in 1980 from St. Thomas’s. He continued working in the pathology department at St. Mary’s Hospital Medical School and was also a consultant to the histopathology unit at the Royal College of Surgeons/Imperial Cancer Research Fund where he provided expert pathology consultation. Dr. Spencer was also a founder fellow of the Royal College of Pathologists. Dr. Spencer was a visiting associate professor of pathology at Yale University Medical School in New Haven, the USA in 1961. He and Dr. Liebow, who worked at Yale University Medical School at that time, became friends and collaborators. Dr. Liebow wrote the forward to Dr. Spencer’s book, Pathology of the Lung, in the 1968 edition. Because of his expertise in pulmonary pathology, Dr. Spencer received many pathology specimens from many countries for his expert opinion. He used many of the cases for teaching medical students and postgraduate doctors. Dr. Spencer died on June 1, 1993.

Avrill / Abraham Liebow (1911–1978) Dr. Liebow, who is considered by some authorities to be the father of pulmonary pathology, was born in the Austria-Hungarian Empire in 1911. His family was very poor and they emigrated to the USA in 1920. He graduated from medical school in 1935. He graduated from the City College of New  York with honors and then enrolled in Yale University Medical School in New Haven, Connecticut, in 1935. One of his mentors was Milton Winternitz, Dean at Yale Medical School (1920–1935) and this influenced his decision to go into pathology [24]. Liebow also won an award as the outstanding student in pathology as sophomore in Medical School. After graduation, he became an assistant in pathology at Yale. He moved through the academic ranks to become a professor and remained at Yale for most of his career. However, he did move to The University of California, San Diego to become chairman of pathology in 1968. When the USA entered World War II, Dr. Liebow joined the army and served in the South Pacific. He developed a medical interest in infectious diseases including coccidiomycosis [25, 26]. He performed studies on cutaneous diphtheria and helped to make possible a treatment for “jungle rot,” a chronic ulcerative skin lesion with many etiologies including Mycobacteria, which was a major health issue in the South Pacific at this time.

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During the war he met and married Carolyn Gott, who was an army nurse, and they had three sons. Soon after the war was over, he was recruited to be a member of the Joint Commission for the effects of the atomic bomb in Japan. The commission did an in-depth study about the consequences of the explosion of the atomic bombs in Nagasaki and Hiroshima. After he returned to Yale, he was appointed as a member of the Atomic Bomb Casualty Commission. He later wrote a book about the casualties of the atomic bomb explosion. He developed interest and expertise in lung diseases at Yale, having published his first paper on lung diseases in 1948 in the American Journal of Pathology, a leading journal in the field. Dr. Liebow was an outstanding teacher and investigator. He taught graduate students, medical students, practicing pathologists as well as surgeons, internists and radiologists. His interest in pulmonary diseases ranged wide and broad. He frequently used plastic casts of the human (and animal) tracheobronchial tree and its vascular supply. These casts were used for many of his studies in pulmonary pathology, since they were very durable models. Dr. Liebow was a prolific writer. One of his most influential publications was “Tumors of the Lower Respiratory Tract” published in 1952 [26]. This elegantly written fascicle had a major influence in the field for many years. He was a keen observer and described many new diseases in pulmonary pathology, especially about medical aspects of lung diseases. His interests were very broad and included experimental aspects of lung diseases. A few new entities described by Dr. Liebow included desquamative interstitial pneumonia and nonspecific interstitial pneumonia [27, 28]. He did an extensive study of lymphomatoid granulomatosis which was published posthumously [29]. Many students came to study with him in New Haven and San Diego. Some of these students went on to be outstanding academic pulmonary pathologist such as Dr. Anna Katzenstein [29] and Dr. CB Carrington as well as doctors from many different countries. Dr. Liebow received many honors including the Francis Blake award for teaching excellence from the graduating class at Yale, the California Physician of the Year Award and the Middleton Goldsmith Medal from the New  York Pathological Society. Dr. Liebow had a massive stroke while teaching a course. Teaching was one of his favorite activities. He died in 1978.

References 1. Bartlett JG, Breiman RF, Mandell LA, et  al. Community acquired pneumonia in adults. Guideline for management. The infectious disease Society of America. Clin Inf Dis. 1998;26:811–38. 2. Henig O, Kaye KS.  Bacterial pneumonia in older adults. Inf Dis Clin North Am. 2017;31(4):684–713. 3. Ruuskanen O, Lahti E, Jennings LC, et al. Viral pneumonia. Lancet. 2011;377(9773):1264–75.

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4. Taubenberger JK, Morens DM. The pathology of influenza virus infections. Ann Rev Pathol. 2008;3:499–522. 5. Umakantham S, Sahu P, Raviade AV, et al. Origin, transmission, diagnosis and management of coronavirus disease 2019 (COVID-19). Postgrad Med J. 2020;96(142):753–8. 6. Merad M, Blish CA, Sallusto F, et al. The immunology and immunopathology of COVID-19. Science. 2022;375(6585):1122–7. 7. Borczuk AC, Salvatore SP, Seshan SV, et  al. COVID-19 pulmonary pathology: a multi-­ institutional autopsy cohort from Italy and New York City. Mod Pathol. 2020;33(1):2156–68. 8. Lederer DJ, Martinez FJ. Idiopathic pulmonary fibrosis. N Engl J Med. 2018;378:1811–23. 9. Sack C, Raghu G. Idiopathic pulmonary fibrosis: unmasking cryptogenic environmental factors. Eur Respir J. 2019;53(2):1801699. https://doi.org/10.1183/13993003.01699-­2018. 10. Wolters PJ, Collard HR, Jones KD. Pathogenesis of idiopathic pulmonary fibrosis. Annu Rev Pathol. 2014;9:157–79. 11. Jemal A, Bray F, Center MM, et  al. Global cancer statistics. CA Cancer J Clin. 2011;61(2):69–90. 12. Sholl LM.  The molecular pathology of lung cancer. Surgical Pathology Clinics. 2016;9(3):353–78. 13. Huang G, Zhang J, Gong L, et al. Specific lung squamous cell carcinoma. Prognosis-subtype distinction based on DNA methylation patterns. Med Sci Monit. 2021;27:e929524. https://doi. org/10.12659/MSM.929524. 14. Succony DM, Rassl DM, Barker AP, et  al. Adenocarcinoma spectrum lesions of the lung: detection, pathology and treatment strategies. Cancer Treat Rev. 2021;99:102237. 15. Sakamoto H, Shimazu J, Horio Y, et al. Disproportionate representation of KRAS gene mutations in atypical adenomatous hyperplasia, but even distribution of EGFR gene mutations from preinvasive and invasive adenocarcinoma. J Pathol. 2007;212(3):287–94. 16. Randhawa S, Trikalinos N, Patterson GA. Neuroendocrine tumors of the lung. Thorac Surg Clin. 2021;31(4):469–76. 17. Rindi G, Klimstra DS, Abedi-Andekavu B, et al. A common classification framework for neuroendocrine neoplasms. An international Agency for Research on Cancer (IAAC) and World Health Organization (WHO) expert consensus proposal. Modern Pathol. 2018;31(12):1770–86. 18. Metovic J, Barella M, Bianchi F, et  al. Morphological and molecular classification of lung neuroendocrine neoplasms. Virchows Arch. 2021;478(1):5–19. 19. Raso MG, Bota-Rabbasedas N, Wistuba II.  Pathology and classification of SCLC Cancers. 2021;13(4):820. 20. Beasley MB, Galateau Salle F, Dacic S. Pleural mesothelioma classification update. Virchows Arch. 2021;478:59–72. 21. Hung UP, Chirieac LR.  Pathology of malignant pleural mesothelioma. Thor Surg Clinic. 2020;30(4):367–82. 22. Robinson BW, Lake RA.  Advances in malignant mesothelioma. N Engl J Med. 2005;353:1591–603. 23. Tighe JR. Herbert Spenser Obituary(1915–1993). British Med J. 1993;307:378. 24. Harley RA. Liebow, Averill Abraham (1911–1978). In: van den Tweel JG, editor. Pioneers in pathology. In: van Krieken JHJM, series editor. Encyclopedia of pathology. Cham:Springer; 2017. p. 340–44. 25. Bloor CM. In remembrance of Avil A. Liebow. March 31, 1911-May 3,1978. Am J Pathol. 1978;92(3):577–80. 26. Smith GJ. Averill Abraham Liebow contributions to pulmonary pathology. Yale J Biol Med. 1981;54(2):139–46. 27. Liebow AA. Tumors of the lower respiratory tract. In: Atlas of tumor pathology. Washington DC: Armed Forces Institute of Pathology; 1952. 28. Liebow AA, Steer B, Billingsley AJ. Desquamative interstitial pneumonia. Am J Medicine. 1965;39:369–404. 29. Katzenstein AL, Carrington CB, Liebow AA, Lymphomatoid granulomatosis. A clinicopathologic study of 152 cases. Cancer. 1979;43(1):360–73.

Chapter 12

Neuropathology

Neuropathologists study and make diagnoses of diseases of the brain, spinal cord and skeletal muscle. They use techniques such as gross and microscopic examination, electron microscopy, histochemistry, and immunohistochemistry to assist in making diagnoses of specific diseases. The types of diseases studied by neuropathologists include neoplasms, degenerative diseases, immunologic, metabolic, and vascular diseases. Neuropathologists work closely with neurologists, neurosurgeons, and neuroradiologists to establish specific diagnoses.

Alzheimer’s Disease Alzheimer’s disease is the most common cause of dementia worldwide. The prevalence of this disease has been increasing in part due to the increasing age of worldwide populations. The disease was named after a famous pathologist, Dr. Alois Alzheimer (1864–1915), who is often considered the father of neuropathology [1]. In Germany in 1901, Dr. Alzheimer had examined a patient, who at age 51 showed loss of short-term memory and unusual behaviors. When the doctor examined her brain after she died in 1906, there were several abnormal findings. He used silver stains to show amyloid plaques and neurofibrillary tangles which were highlighted by the special silver stains. He described his findings in a scientific meeting in 1906 including the brain pathology and symptomatic presenile (because the findings were in a younger patient than expected) dementia. The disease was designated as Alzheimer’s disease in 1910  in a book chapter by Professor Kraeplin in his Handbook of Psychiatry and designated as “Presenile and Senile Dementia.” Interestingly, an American psychiatrist, Solomon Fuller, described similar morphological brain findings at a meeting about 5  months prior to Alzheimer’s meeting report [1, 2]. About 1–2% of Alzheimer’s disease is inherited as an autosomal dominant disease [3], while most patients acquire the disease with aging. In general, © The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 R. V. Lloyd, Pathology: Historical and Contemporary Aspects, https://doi.org/10.1007/978-3-031-39554-3_12

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dementia refers to a clinical syndrome which shows a progressive decline in two or more cognitive domains which may include memory, language, personality, executive and visuospatial function, and behavior. These changes can lead to loss of the ability to perform basic activities in daily living. Alzheimer’s disease account for around 80% of all dementias [2]. The definitive diagnosis of Alzheimer’s disease is dependent on postmortem examination of the brain by a neuropathologist who performs special studies to show the neurofibrillary plaques and tangles that are seen under the microscope. However, the diagnosis can be suspected in living patients by studies of the cerebrospinal fluid, positive emission tomography (PET), by specific biomarkers and by using clinical criteria [2–4]. Alzheimer’s disease is characterized pathologically by extracellular deposition of amyloid beta in the form of diffuse and neuritic plaques and intraneuronal neurofibrillary tangles, neuropil threads, and hyperphosphorylated tau proteins [4]. APOE is a strong genetic factor for the development of Alzheimer’s disease. APOE is an apolipoprotein, which functions as a lipid-binding protein that helps to transport lipid to target sites. In the brain APOE is found mainly in astrocytes, but small amounts are also present in microglia. APOE binds to amyloid beta found in the plaques in Alzheimer’s disease. There is a great deal of ongoing research about the pathogenesis and most effective treatment for Alzheimer’s disease, since it is such an important neurodegenerative disorder that affects such a very large number of patients.

Chronic Traumatic Encephalopathy A disease that shows some overlapping features with Alzheimer’s disease is chronic traumatic encephalopathy (CTE). CTE is a poorly defined disorder in which the clinical diagnostic criteria and the morphological features are not totally agreed on [5]. CTE is thought to be secondary to repetitive exposure of the head to trauma. There is no agreement as to whether a single exposure, or as is commonly seen, moderate brain trauma and multiple head trauma can lead to CTE [6]. A Hollywood movie in which a neuropathologist was the main protagonist has contributed to this confusion in the medical literature [7]. The incidence of CTE is unknown because only a limited number of cases have been studied extensively [5, 6]. A strict definition of CTE remains controversial [5]. The connection between head trauma and dementia has been known for many decades such as has been described in boxers with punch drunk syndrome [6]. The clinical picture of CTE includes personality changes, memory loss dementia, and cerebellar impairment. Recent studies have suggested that the suicide rate in individuals with autopsy-diagnosed CTE is higher than expected [6]. CTE has been reported in American football players, soccer and rugby players, and ice hockey players [6]. At autopsy, the neurofibrillary tangles in CTE may show a patchy distribution of hyperphosphorylated tau deposits in neurons and glial cells in a perivascular location. These are usually located toward the depth of the cortical sulci [6]. The tau pathology is usually considered to be useful in the diagnosis of CTE. The clinicopathologic heterogeneity of CTE is analogous to the heterogeneity seen in Alzheimer’s disease [6].

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Brain Tumors Brain tumors diagnosed by neuropathologists range from low-grade neoplasms with an excellent prognosis such as low-grade gliomas (Figs.  12.1 and 12.2) to high-­ grade cancers with a poor prognosis such as high-grade astrocytomas (Figs. 12.3 and 12.4) which includes glioblastoma multiforme (GBM). High-grade astrocytomas arise from astrocytic cells which support and interact with nerve cells or

Fig. 12.1  Pilocytic astrocytoma (low-grade glioma). The tumor cells have prominent cytoplasmic processes and mild nuclear pleomorphism. These tumors represent mainly pediatric low-grade gliomas. Molecular classification uses molecular analysis for mutations to help classify these neoplasms. The most frequent mutation is KIAA1549-BRAF, which occurs in 70–80% of these neoplasms

Fig. 12.2  Immunohistochemical staining for glial fibrillary acidic protein in a low-grade glioma showing positive staining in the tumor cell bodies and processes

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Fig. 12.3  Diffuse high-grade astrocytoma with atypical cells and marked cellular pleomorphism. These tumors usually have increased mitoses and areas of necrosis. These tumors may show isocitrate dehydrogenase (IDH) mutations. They usually also have mutations of TP53 and ATRX genes

Fig. 12.4  Immunohistochemical staining of a high-grade astrocytoma for glial fibrillary astrocytoma. There are diffuse staining of the tumor cell bodies and processes

neurons in the brain and spinal cord. High-grade astrocytomas are the most common malignant brain tumors and constitute around 12–15% of all intracranial tumors. They commonly develop in patients in their eighth decade; however, they may also occur in younger patients and even in some pediatric patients. Adult type diffuse gliomas are highly infiltrative and largely incurable malignancies. Earlier classification of brain tumors had relied mainly on microscopic features; for glioblastomas this consisted of high-grade morphology and microvascular proliferation and/or tumor cell necrosis. Since the publication of the World Health Organization

Meningiomas

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(WHO) Blue Book in 2016, the classification of brains tumors has shifted to a molecular-­oriented approach [8–13]. Classification of gliomas are dependent on isocitrate dehydrogenase 1/2 (IDH 1/2) mutations and 1p/19q codeletions states. There is a wide gap in survival with IDH mutant and IDH wild-type tumors even with the same histopathological classification. For example, infratemporal astrocytoma commonly has IDH mutations. Survival in high-grade infratemporal IDH mutant astrocytomas is much reduced compared to supratemporal high-grade astrocytomas. Supratemporal high-grade astrocytomas have less IDH mutations. For glioblastomas, common genetic changes include a loss of heterozygosity at chromosome 10q and amplification of epidermal growth factor receptor, mutations of TP53, PTEN and IDH and several other mutations [13]. The prognosis for these very aggressive cancers remains very poor, in spite of aggressive treatment with surgery, radiation therapy, chemotherapy, and targeted drug therapies [8, 9].

Meningiomas Unlike glioblastomas, meningiomas are often benign neoplasms arising from the dural lining of the central nervous system [14]. Meningiomas are the most common intracranial neoplasms [15]. Although most meningiomas are benign, many tumors are associated with focal neurological deficits, seizures, and decreased quality of life (Fig. 12.5) [14, 15]. Meningiomas comprise around 36.6% of central nervous system tumors in the USA. There is an increased incidence with age and in African-­ Americans compared to Caucasians and also in women in the USA [14]. Because

Fig. 12.5  Meningioma consisting of tumor cells with a lobular architecture composed of oval nuclei and eosinophilic cytoplasm. Whorls of tumor cells (arrow) are present in the neoplasm. The tumor cells are usually positive for epithelial membrane antigen by immunohistochemical staining

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they are much more common in women of child-bearing age and they express sex steroid hormone receptors, their growth is probably influenced by sex steroid hormones [16]. Although most meningiomas are benign, tumors with increased mitoses and proliferative indices are likely to recur [14–16]. Molecular genetic studies have shown changes in the NF2 gene with deletion of chromosome 22q. Some of the mutations in meningiomas such as NF2, KLF4, TRAF7, and AKT1 are more commonly associated with specific subtypes of meningiomas [11]. Molecular genetic studies have not changed the classification of meningiomas as they have for gliomas to date [8].

Pituitary Tumors Another common brain tumor is the pituitary adenoma located in the sella turcica beneath the hypothalamus. Pituitary adenomas constitute around 15% of brain tumors and most of them are benign neoplasms (Fig. 12.6) [17]. Although most pituitary tumors were treated by surgery historically, for the past few decades, some subtypes of adenomas such as prolactin-producing and growth hormone-producing adenomas have been treated by medical therapy for a few months to many years in patients with these tumors (Figs. 12.7, 12.8, and 12.9) [17]. About 30% of resected pituitary tumors may recur or have continued growth. Some subtypes of adenomas are more likely to recur, so precise classification by the neuropathologist can help the clinician to monitor these tumors and anticipate the subtypes that are likely to recur. Adenomas such as sparsely granulated growth hormone adenomas, Crooke’s cell adenomas, and silent corticotroph adenomas are some of

Fig. 12.6  Pituitary adenoma consisting of uniform neuroendocrine cells with moderate amounts of cytoplasm and round nuclei. A few prominent small blood vessels (arrow) are present. This neuroendocrine tumor belongs to the null cell type of pituitary adenoma because it was negative for all of the common pituitary hormones after immunohistochemical staining

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Fig. 12.7  Prolactin-­producing pituitary adenoma consisting of cells with sparse cytoplasm and enlarged round nuclei. The patient was treated with bromocriptine for more than 5 years before surgery. Bromocriptine, which inhibits prolactin secretion, is also associated with cell shrinkage

Fig. 12.8  The same tumor from Fig. 12.7 after staining for prolactin hormone. There is diffuse staining for this hormone in the tumor cells

the tumors that are more likely to recur and they are often designated as aggressive pituitary adenomas. Pituitary carcinomas which often have metastatic disease are very uncommon. The majority of pituitary carcinomas produce prolactin or ACTH [18, 19]. Molecular studies have shown mutations of the TP53 gene in some pituitary carcinomas [20]. Some of the pathologic classification of pituitary tumors remain controversial. For example, some pathologists have changed the nomenclature of pituitary adenomas to pituitary neuroendocrine tumors (PitNET), because it is sometimes difficult to predict the tumors that will recur [21]. However, many clinical pituitary endocrinologists, neurosurgeons, and pituitary researchers have objected to this change, since they indicate that most pituitary adenomas are benign

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Fig. 12.9 The same tumor from Fig.  12.7 stained for synaptophysin, a broad-­ spectrum neuroendocrine marker, shows positive cytoplasmic staining

lesions and do not affect life expectancy of patients with these tumors and that the use of this new terminology may affect patients adversely [22].

Prion Diseases Prion diseases are caused by misfolded and aggregated proteins known as prions. They cause infections that lead to neurodegenerative disorders secondary to misfolded protein aggregates. The misfolded proteins have been responsible for disease epidemics such as kuru in humans and bovine spongiform encephalopathy in cattle [23, 24]. In humans, Jakob–Creutzfeldt disease, the most common prion disease, is characterized by neuronal loss, spongiform encephalopathy, gliosis, and deposits of aggregated prion proteins. The infectious agent causing prion disease, PrP (Sc), lacks nucleic acids and is a misfolded pathogenic form of the cellular prion protein [24]. After infection with PrP (C), there is a marked increase in PrP (Sc) in the brain and spinal cord leading to neuronal death. In humans, the PrP (C) is encoded by the prion gene PRNP on chromosome 20 [25]. Prion disease has a long incubation period from infection measured in years. However, the clinical phase usually progresses rapidly from weeks to months [23]. Signs and symptoms include motor dysfunction, behavioral abnormalities, ataxia, and cognitive impairments [26, 27]. There is no definitive treatment other than palliative care. In humans, the majority of cases are sporadic such as Jakob–Creutzfeldt which has no known cause. There is usually somatic mutation in PRNP or the conversion of PrP (C) to PrP (Sc) spontaneously. The acquired disease may also be contracted by corneal transplant contaminated with prions, blood transfusions, injection with contaminated human growth hormone, or even contaminated surgical instruments [26, 27]. Genetic studies have shown that the mutations in PRNP are autosomal dominant and can involve insertions, deletions, or missense mutations [23].

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Many outstanding neuropathologists have contributed to progress in this field. The next section will summarize the lives of two of these outstanding individuals who have contributed greatly to progress in neuropathology.

Lucien J Rubinstein (1924–1990) Lucien Rubinstein was born in Antwerp, Belgium, on October 15, 1924 [28–32]. His family migrated to London when he was 15, after the German army had invaded Belgium during World War II. Lucien completed middle school and university training in England. He received a Bachelor of Medicine degree in 1948 and his MD degree in pathology in 1952 at the University of London [28–30]. He then obtained postdoctoral training at the University of London Hospital [31, 32]. He also spent time in the British Royal Army Medical Core and served with distinction. Part of his service was spent in the Military Hospital for Head Injuries at Oxford. He also trained under Dr. Dorothy Russell. These two pathologists wrote the classic Pathology of Tumors of the Nervous System. They wrote four editions of the textbook together until Dr. Russell died in 1983. Dr. Rubinstein did major revisions on the fifth edition which was published in 1989. Dr. Rubinstein and his family migrated to the USA in 1961. He worked at Montefiore Hospital for 3  years and then became a professor at Stanford University School of Medicine. He subsequently moved to the University of Virginia in 1981 where he became the director of the division of neuropathology. It was at Virginia that he undertook the herculean task of revising the fifth edition of Russell and Rubinstein’s Pathology of the Nervous System, which he completed in 1989. He also wrote the AFIP fascicle on “Tumors of the Central Nervous System.” He was on the editorial board of numerous publications in neuropathology and was advisor to the Commission on the Histopathological Classification and Nomenclature of Tumors of the World Health Organization. Rubinstein made many contributions to the diagnostic as well as scientific aspects of neuropathology. He and his second wife, Dr. Mary M. Herman, who was also a neuropathologist, set up cell culture systems for many of the central nervous system tumors that were recognized at the time including gliomas and medulloblastomas. Diagnostic immunohistochemistry was in its infancy at the time of his early practice and Rubinstein applied this emerging tool to the study of brain tumors. His work on the application of antibodies to glial fibrillary acidic protein (GFAP) in the study of brain tumors was innovative. He used this immunohistochemical approach to characterize many central nervous system and peripheral nervous system tumors [28]. He described new brain tumors such as polar spongioblastomas [33], pleomorphic xanthoastrocytomas [34], and several others [28]. Rubinstein was careful to correlate his neuropathological findings with the clinical outcomes of patients which added more impact to his original observations. For example, he suggested that anaplastic oligodendrogliomas was a diagnosis that should be reserved for patients with a rapidly growing highly cellular poorly differentiated oligodendroglioma [28]. He and his collaborators established the use of glial fibrillary acidic protein to recognize astroglial histogenesis and differentiation in brain neoplasms and used this marker to recognize tumors of astrocytic origin [28].

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Dr. Rubinstein had three children with his first wife, Dorothea, including two sons and a daughter. His second wife, Mary Herman, was his scientific collaborator and they performed many scientific studies together including cell culture studies of brain tumors [28]. Rubinstein enjoyed music, books, classical theater, and French drama. After the passing of Russell, he completed the fifth edition of Russell and Rubinstein, Pathology of the Nervous System, in 1989. He was diagnosed with a basilar aneurysm which led to his demise in 1990. His contributions to neuropathology and neuro-oncology have established him as a giant in the field.

Bernd W. Scheithauer (1946–2011) Bernd Scheithauer, a leading expert in neuropathology, had trained under Dr. Rubinstein after he decided to become a neuropathologist. Bernd was born in Geledau, Germany in 1946. His parents refused to join the Nazi party in Germany and were taxed very heavily [35–37]. After World War II, they moved from East Germany to West Germany. Scheithauer started grade school in Germany, but his family migrated to the USA when he was 7 years old and they settled in Eureka, California. Bernd loved growing up in Eureka and often spoke of returning there during some of the cold winters at the Mayo Clinic in Rochester, Minnesota. Scheithauer loved to read as a child. In high school he participated in weight lifting and became very good in the sport, winning several medals in high school. He attended Humboldt State University in California. After graduating in 1969, he attended medical school at Loma Linda University School of Medicine in California and graduated in 1973. He then trained in anatomic pathology followed by 2 years of training in neuropathology. This was followed by a fellowship year in surgical pathology at Stanford University Medical School where he came under the influence of Dr. Lucien Rubinstein. The year of working with Dr. Rubinstein provided Scheithauer with a great deal of insight into academic neuropathology and undoubtedly contributed to his outstanding success as an academic neuropathologist during his career [35–37]. Dr. Rubinstein was probably at the peak of his career and had coauthored several editions of one of the premier neuropathology textbooks with Dr. Russell. Dr. Scheithauer accepted a position in neuropathology at the Mayo Clinic after completing his training at Stanford and remained at Mayo for all of his academic career until his demise in 2011. Although he spent most of his time working in Rochester, Minnesota, his hectic lecture schedule, teaching, and research took him all over the world in the more than three decades that he was at Mayo. He was married and then divorced while at the Mayo Clinic. He had two children whom he greatly adored and always spoke lovingly about them. Dr. Scheithauer was extremely productive at Mayo having published more than 750 original manuscripts, over 80 book chapters, and several books. His studies and publications on brain tumors including the Armed Forces Institute of Pathology Fascicle on tumors of the nervous system with his friend and colleague Peter Burger and on the peripheral nervous system with his colleague James Woodruff influenced many young residents and served as authoritative reference sources for many pathologists. His studies of pituitary adenomas and carcinomas with Drs. Kovacs and Horvath led to many seminal publications about these tumors.

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Dr. Scheithauer’s diagnostic skills and original observations led him to describe several new entities in diagnostic neuropathology including clear cell meningioma [38], dysembryoplastic neuroepithelial tumors [39], chordoid gliomas [40], pituitary blastoma [41], and Crooke’s cell pituitary adenomas which had a high propensity to recur in patients with this pituitary tumor type [42] and several other tumor types. Dr. Scheithauer was one of the authors of the World Health Organization Blue Book on Neuropathology. His passion for teaching attracted many visitors to his laboratory from around the world who came and spent time with him as fellows or for shorter visits. Many of his trainees now work at major academic institutions in the USA and in many other institutions around the world and will ensure that his legacy as a major contributor to advances in neuropathology will continue far into the future. Dr. Scheithauer died in 2011 in Rochester, Minnesota.

References 1. Jones ML, Alzheimer A.  In: van den Tweel JG, editor. Pioneers in pathology. van Krieken JHJM, series editor. Encyclopedia of pathology. Switzerland: Springer International Publishing; 2017. p. 21–23. 2. Weller J, Budson A. Current understanding of Alzheimer’s disease diagnosis and treatment. F1000Research. 2018;7:1161. 3. Long JM, Holtzman DM. Alzheimer’s disease. An update on pathobiology and treatment strategies. Cell. 2019;179(2):312–39. 4. Duyckaerts C, De La Tour B, Potier MC. Classification and basic pathology of Alzheimer’s disease. Acta Neuropathol. 2009;118:5–36. 5. McKee AC, Cairns NJ, Dickson DW, Folkerth RD, et al. The first NINDS/NIBIB consensus meeting to define neuropathological criteria for the diagnosis of chronic traumatic encephalopathy. Acta Neuropathol. 2016;131:75–86. 6. Smith DH, Johen VE, Trojanowski JQ, et al. Chronic traumatic encephalopathy—confusions and controversies. Nat Rev Neurol. 2019;15(3):179–83. 7. Concussion. [Film] Directed by: Peter Londesman. USA: Columbia Pictures; 2015. 8. Louis DN, Ohgaki H, Wiestler OD, et  al. The 2016 WHO classification of Tumours of the central nervous system. 4th ed. Lyon: International Agency for Research on Cancer; 2016. 9. Louis DN, Perry A, Reifenberger G, et al. The 2016 World Health Organization classification of tumors of the central nervous system: a summary. Acta Neuropathol. 2016;131:803–20. 10. Aldape K, Zadeh G, Mansouri S, et al. Glioblastoma: pathology, molecular mechanisms and markers. Acta Neuropathol. 2015;129:829–48. 11. Komori T. The 2016 WHO classification of tumors of the central nervous system: the major points of revision. Neurol Med Chir (Tokyo). 2017;57:301–11. 12. Soomro SH, Ting LR, Qing YY, et al. Molecular biology of glioblastoma: classification and mutational locations. JPMA. 2017;67(9):1410–4. 13. Verhaak RGW, Hoadley KA, Purdim E, et al. Integrated genomic analysis identified clinically relevant subtypes of glioblastoma characterized by abnormalities in PDGFRA, IDH1, EGFR and NF1. Cancer Cell. 2010;17:98–110. 14. Buerki RA, Horbinski CM, Kruser T, et  al. An overview of meningiomas. Future Oncol. 2018;14(21):2161–77. 15. Wiemels J, Wrensch M, Claus EB.  Epidemiology and etiology of meningiomas. J Neuro-­ Oncol. 2018;99(3):307–14. 16. Rogers L, Barani L, Chamberlain M, et al. Meningiomas: knowledge base, treatment outcome and uncertainties. A RANO Rev J Neurosurg. 2015;122(1):4–23.

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17. Melmed S. Pituitary-tumor endocrinopathies. New Engl J Med. 2020;382(10):937–50. 18. Di Ieva A, Rotondo F, Syro LV, Cuisimano MD, Kovacs K. Aggressive pituitary adenomas— diagnosis and emerging treatments. Nat Rev Endocrinol. 2014;10(7):423–35. 19. Scheithauer BW, Kurtkaya-Yapicier O, Kovacs K, Young WF Jr, Lloyd RV.  Pituitary carcinoma: a clinicopathologic review. Neurosurgery. 2005;56(5):1066–74. 20. Tanizaki Y, Jin L, Scheithauer BW, et al. P53 gene mutations in pituitary carcinomas. Endocr Pathol. 2007;18(4):217–22. 21. Asa SL, Casar-Borota U, Chanson C, et  al. From pituitary adenoma to pituitary neuroendocrine tumor (PitNET). An international pituitary pathology Club proposal. Endocr Relat Cancer. 2017;24(4):C5–8. 22. Ho K, Fleseriu M, Kaiser U, et al. Pituitary neoplasm nomenclature workshop: does adenoma stand the test of time? J Endocr Soc. 2021;5(3):bvaa205. 23. Sigurdson CJ, Bartz JC, Glatzel M. Cellular and molecular mechanisms of prion disease. Annu Rev Pathol Mech Dis. 2019;14:497–516. 24. Baiardi S, Ross M, Capellari S, et al. Recent advances in the histo-molecular pathology of human prion disease. Brain Pathol. 2019;29:278–300. 25. Basler K, Oesch B, Scott M, et al. Scrapie and cellular PrP isoform are encoded by the same chromosome gene. Cell. 1986;46:417–28. 26. Gibbs CJ Jr, Joy A, Heffner R, et al. Clinical and pathologic features and laboratory confirmation of Creutzfeldt-Jakob disease in a recipient of pituitary-derived human growth hormone. N Engl J Med. 1985;313:734–8. 27. Wroe SJ, Pal S, Siddique D, et  al. Clinical presentation and pre-mortem diagnosis of variant Creutzfeldt-Jacob disease associated with blood transfusion: a case report. Lancet. 2006;368:2061–7. 28. Mut M, Lopez MBS, Shaffrey M.  Lucien J Rubinstein enhancing contribution to neuro-­ oncology. Neurosurg Focus. 2005;18(4):e8. 29. Davis RL, Kepes JJ, Rorke LB, et al. The art or brain tumor classification—a tribute to Lucien J. Rubinstein (1925–1990). Brain Pathol. 1990;1:55–9. 30. Kepes JJ. In Memoriam. Lucien J Rubinstein 1924–1990. Acta Neuropathol. 1990;80:108–9. 31. Barnard RO. Obituary. Professor Lucien J Rubinstein. Neuroradiology. 1990;32:253–4. 32. Vandenberg S, Scheithauer B, Bignar D.  Rubinstein, Lucien J (1924–1990). Memoriam J Neuropath Expt Neurol. 1990;49(5):531–53. 33. Rubinstein LJ. Discussion on polar spongioblastoma. Acta Neurochir. 1964;11(Suppl10):126. 34. Kepes JJ, Rubinstein LJ, Eng LF. Pleomorphic xanthoastrocytoma. A distinct meningocerbral glioma of young subjects with relatively favorable prognosis: a study of 12 cases. Cancer. 1979;44(5):1839–52. 35. Perry A, Bernd W, Scheithauer MD.  Mentor, friend, prodigy. Arch Pathol Lab Med. 2012;136:350–1. 36. Giannini C.  In Memoriam: Bernd Walter Scheithauer (1946–2011). Acta Neuropathol. 2012;123:461–2. 37. Vandenberg S, Beatriz LM.  In Memoriam: Bernd Walter Scheithauer MD August 30, 1946-September 19, 2011. J Neuropathol Expt Neurol. 2012;71(11):1032–5. 38. Zorludemir S, Scheithauer BW, Hirose T, et al. Clear cell meningioma. A clinicopathologic study of a potentially aggressive variant of meningioma. Am J Surg Pathol. 1995;19(5):493–505. 39. Yan X, Marsh WR, Scheithauer BW. Dysembryoplastic neuroepithelial tumor and calcifying pseudoneoplasms of the neuraxis: a collision of two seizure-associated lesions. Clin Neurol. 2011;30(4):197–202. 40. Pomper MG, Passe TJ, Burger PC, et al. Chordoid glioma: a neoplasm unique to the hypothalamus and anterior third ventricle. AJNR Am J Neuroradiol. 2001;22(3):464–9. 41. Scheithauer BW, Kovacs K, Horvath E, et  al. Pituitary blastoma. Acta Neuropathol. 2008;116(6):657–66. 42. George DH, Scheithauer BW, Kovacs K, Horvath E, et al. Crooke cell adenoma of the pituitary: an aggressive variant of corticotroph adenoma. Am J Surg Pathol. 2003;27(10):1330–6.

Chapter 13

Endocrine Pathology

The area of surgical pathology designated as endocrine pathology involves the examination of endocrine tissues in multiple organs of the body for gross and microscopic analyses of abnormalities. These organs include the thyroid, parathyroid, adrenal cortex, adrenal medulla, pituitary, lung, gastrointestinal tract, and skin. Some of these organs represent pure endocrine tissues such as the thyroid and parathyroid glands. Others represent tissues with a limited number of endocrine cells combined with largely exocrine cells such as the gastrointestinal tract and lungs. The endocrine pathologist works closely with the endocrinologist, endocrine surgeon, and radiologist to analyze and diagnose specific endocrine lesions. Endocrine diseases seen by the pathologist include inflammatory, autoimmune, and neoplastic conditions including benign and malignant neoplasms. Endocrine pathologists also rely on abnormal hormone secretion to assist in the diagnosis of some endocrine disorders. Because many endocrine tissues secrete specific hormones, increased or decreased levels of hormones can provide clues about the functions of specific endocrine tissues. In addition to the ectopic production of hormones by specific cell types, some tissues produce hormones ectopically which makes the diagnosis of specific endocrine lesions more difficult. An example of the ectopic production of specific hormones would be ACTH production by small-cell neuroendocrine carcinoma of the lung [1]. Although the ACTH is not oversecreted by the anterior pituitary in this case, the effects on the adrenal cortex leading to Cushing syndrome is similar to that seen by overproduction of ACTH by the anterior pituitary gland. The endocrinologist, endocrine surgeon, and pathologist must distinguish between these various conditions to provide the most effective care for the patient. Disorders of endocrine glands/tissues can develop with only a single gland abnormality, which is most common. However, abnormalities of multiple endocrine glands can also occur. With some disorders there may be hyperfunctioning or hypofunctioning of multiple specific endocrine tissues. An example of multiple hyperfunctioning of specific endocrine tissues is Carney’s complex in which hyperplastic nodules of the adrenal cortex and pituitary tumors produce excessive hormones [2]. Other involved tissues © The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 R. V. Lloyd, Pathology: Historical and Contemporary Aspects, https://doi.org/10.1007/978-3-031-39554-3_13

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include the breast and skin with myxoid lesions involving these organs [2, 3]. The major defect in Carney’s complex is an inactivating mutation or large deletion of the PRKAR1 gene coding for the regulatory subunit of protein kinase A [2, 3]. An example of hypofunctioning disease processes is polyglandular autoimmune syndrome. The most important forms include the juvenile and adult types [4]. In the juvenile type (type 1), patients develop mucocutaneous candidiasis, Addison’s disease (adrenal cortical atrophy and hypofunction), and hypoparathyroidism as the major manifestation of this disorder. The juvenile form is caused by a mutation of the autoimmune regulatory gene [4]. In the adult form of the disease (type 2), patients develop diabetes mellitus, autoimmune thyroid disease, Addison’s disease, and other autoimmune disorders [4]. The endocrinologist or endocrine pathologist should suspect this disorder when a patient develops autoimmune diseases in two different endocrine organs or if the patient has a history of several autoimmune diseases [4, 5]. The thyroid gland is one of the most common endocrine tissues involved with type 2 polyglandular disorders and it may be manifested as Hashimoto thyroiditis or Graves disease [5].

Hashimoto’s Thyroiditis Chronic lymphocytic thyroiditis or Hashimoto thyroiditis was described in 1912 by a surgery resident in Japan, Dr. Hashimoto [6]. The disease is a progressive disorder of the thyroid gland caused by autoimmune destruction of the thyroid. Early in the disease the gland may be slightly enlarged and then with the passage of time the gland becomes fibrotic with varying degrees of nodularity. Microscopically, the thyroid shows an infiltration of lymphocytes and plasma cells as well as fibrosis (Fig. 13.1). There are variable numbers of metaplastic follicular cells with marked eosinophilia

Fig. 13.1  Thyroid tissue showing chronic lymphocytic thyroiditis (Hashimoto’s thyroiditis). Chronic inflammatory cells consisting of plasma cells and lymphocytes are the predominant cell types. A few residual follicles are present in the background (arrow)

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due to accumulation of cytoplasmic mitochondria. These cells are designated as Hurthle or oncocytic cells. In Hashimoto thyroiditis, the inflammatory process is confined to the thyroid. In contrast Riedel thyroiditis, which is sometimes confused with Hashimoto, the inflammatory process involves not only the thyroid, but the adjacent soft tissue and skeletal muscle in the perithyroidal area. Patients with Hashimoto thyroiditis usually have elevated titers of thyroid-specific antibodies. There is usually involvement of T and B inflammatory cells in Hashimoto thyroiditis [7, 8]. Although it has been suggested that patients with Hashimoto thyroiditis have an increased risk of developing papillary thyroid carcinoma [9], this has been controversial. However, it is recommended that patients with Hashimoto undergo close follow-up since this disease is commonly associated with thyroid lymphoma especially a specific type of lymphoma designated as mucosa-associated lymphoma [10].

Follicular Neoplasms of Thyroid Follicular Adenoma Follicular adenoma of the thyroid is a benign follicular-derived neoplasm that shows no evidence of invasive growth. The neoplasm is surrounded by a capsule that is usually thin (Fig. 13.2). In general, the thyroid capsule is frequently thin in benign thyroid tumors. Careful examination of the capsule for capsular or vascular invasion is done routinely when analyzing a follicular neoplasm under the microscope to exclude the possibility of a follicular carcinoma which is diagnosed by invasion of the capsule. If there is only cytological examination of a follicular-derived neoplasm of the thyroid without other materials such as tissue sections it not usually possible to separate follicular adenomas from carcinomas.

Fig. 13.2  Thyroid follicular adenoma. The thyroid tumor capsule (C) separates the benign neoplasm on the left from the nonneoplastic thyroid tissue on the upper right. The tumor is negative for vascular and capsular invasion

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Although it has been suggested that some molecular approaches can be used to separate follicular adenomas from follicular carcinomas such as loss of heterozygosity, and molecular profiling microarray analysis [11], these approaches are not used routinely in the diagnostic pathology laboratory. Some molecular changes overlap between follicular adenomas and follicular carcinomas such as RAS mutations that are present in up to 50% of follicular carcinomas. Adenomas also show RAS mutations in about 20–40% of cases [12, 13]. The finding of a clonal population of cells with RAS mutations does help to separate follicular adenomas from adenomatous or hyperplastic nodules. A helpful immunohistochemical marker that helps to separate follicular adenomas from carcinomas is the presence of HBME-1 expression which may be present in follicular carcinomas, but is less common in follicular adenomas [14]. However, a small percentage of adenomas can also express HBME-1, although there are significant differences in expression between adenomas and carcinomas [14].

Follicular Carcinomas Follicular carcinomas have a similar histological appearance to adenomas. The tumor capsule is usually thicker in carcinomas and there is some evidence of invasive growth into the capsule or into blood vessels in the capsule (Fig. 13.3). Both follicular adenomas and carcinomas can have different histological variants including an oncocytic or Hurthle cell variant in which the tumor cells are very eosinophilic, because they have abundant mitochondria in the cytoplasm. Follicular carcinomas can vary from minimally invasive in which there is only invasion through the capsule to widely invasive in which the tumor shows vascular invasion in many blood vessels within the capsule.

Fig. 13.3  Follicular carcinoma. The main tumor mass is on the lower left. Invasion into a vascular channel with tumor cells adherent to the blood vessel wall is present (arrow)

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Molecular findings in follicular carcinomas include translocation of PAX8-PPAR gamma which is present in about a third of follicular carcinomas. Rare cases of follicular adenomas and some other thyroid neoplasms such as the follicular variant of papillary thyroid carcinoma may also have this genetic rearrangement [12]. RAS mutations are also present in 40–50% of follicular carcinomas and TERT promoter mutations can be found in about 15% of follicular carcinomas [15]. A small percentage of follicular carcinomas may also have mutations in EIFIAX and DICER genes [15].

Papillary Thyroid Carcinoma Papillary thyroid carcinoma is the most common thyroid malignant neoplasm and represents 70–80% of thyroid cancers. A risk factor for the development of these malignancies includes prior exposure to radiation. These carcinomas may be solitary or multifocal. Incidental papillary microcarcinomas (less than 1 cm in diameter) are commonly found at autopsy and they may also be seen after thyroid surgery when the pathologist is examining the thyroid gland in the laboratory. Most of these carcinomas are papillary thyroid microcarcinomas [16]. Most of the microcarcinomas found at autopsy are indolent tumors without clinical significance, but a small number are capable of metastasizing to lymph nodes and to other organs. The prevalence of these small cancers at autopsy may range from 1% up to 30% of cases in reported series. The small tumors found at autopsy have been referred to as latent papillary thyroid microcarcinomas. It has been suggested that the papillary thyroid microcarcinomas found at autopsy are different from most surgically excised lesions [16]. There are many subtypes of papillary thyroid carcinomas. Some of these higher-­grade carcinomas behave more aggressively depending on the subtype of neoplasm. Diagnostic features of papillary thyroid carcinomas include the presence of papillae with fibrovascular cores, enlarged overlapping nuclei, prominent nuclear grooves, cytoplasmic invagination into nuclei, and nuclear clearing (Figs. 13.4 and 13.5). Calcification with psammoma bodies may also be present. Some variants of papillary thyroid carcinoma such as the follicular variant do not have papillae. Recent molecular studies have divided papillary thyroid carcinomas into two major groups depending on specific mutations. These include the BRAF group with BRAFV600E mutations which is present in many classical papillary thyroid carcinomas and the RAS group with HRAS, NRAS, and KRAS mutations. Many follicular variants of papillary thyroid carcinomas belong to the RAS group. Mutation in these two gene families is usually mutually exclusive [17]. Other genetic mutations in papillary thyroid carcinomas in greater than 5% of cases include TERT promoter (9.4%), TP53 (6%), NRAS (6%), and BRAF (61.7%). RET-PTC rearrangements are present in 6.8% of papillary thyroid carcinomas (15%).

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Fig. 13.4  Papillary thyroid carcinoma, classic type. The tumor cells show finger-like papillary projections into the adjacent spaces

Fig. 13.5  Papillary thyroid carcinoma, classic type with fingerlike papillary projections forming papillae (P). Cytological features including enlarged nuclei with nuclear overlap and focal nuclear clearing are present

Anaplastic Thyroid Carcinoma This subtype of thyroid carcinoma is highly lethal, and patients with this diagnosis rarely survive for more than a year after diagnosis, in spite of very aggressive treatment with surgery, chemotherapy, and radiation therapy. Many anaplastic carcinomas are derived from papillary or follicular carcinoma by dedifferentiation. The cancers are often large with areas of necrosis. Histologically the tumors are undifferentiated with large nuclei, prominent nucleoli, and spindle or epithelioid cells.

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Fig. 13.6  Anaplastic thyroid carcinoma. The tumor consists of disorganized enlarged cells with large nuclei and increased mitotic activity (arrow). Cells in this carcinoma can vary from round- to spindle-shaped. Patients with these carcinomas usually succumb to their disease in 1 year or less

Immunohistochemical staining for thyroglobulin and TTF-1 are usually negative indicating (Fig. 13.6) that the cancers are undifferentiated. They may be positive for keratin, PAX8, and p53. Some genetic mutations in anaplastic carcinomas include TP53, BRAF, TERT promoter, SWI/SNF, EIFIAX, NRAS, PAX8-PPARgamma, PI3KCA, and PTEN [15].

Medullary Thyroid Carcinoma Medullary thyroid carcinoma is a unique type of thyroid cancer that is derived from C cells or calcitonin-producing cells. This is a neuroendocrine carcinoma which produces polypeptide hormones including calcitonin and calcitonin gene-related peptide. Familial medullary thyroid carcinoma is usually associated with multiple endocrine neoplasia syndromes types 2A (II) and 2B(III). Patients with multiple endocrine neoplasia syndromes usually have tumors involving multiple endocrine organs such as the thyroid C cell, the adrenal medulla, and parathyroid glands. Histological examination of medullary thyroid carcinoma usually shows epithelioid and spindled cells (Fig. 13.7) with endocrine amyloid in the background in around 75% of cases. A procalcitonin molecule is responsible for this form of endocrine amyloid. Foci of amyloid in the upper and midportion of the thyroid are present in medullary thyroid carcinoma from familial and sporadic cases [18]. Immunohistochemical staining for chromogranin A and synaptophysin, INSM1, and for calcitonin are usually positive in theses cancers. Electron microscopic studies usually show dense core secretory granule which contain chromogranin, calcitonin, and other polypeptides.

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Fig. 13.7  Medullary thyroid carcinoma. The tumor capsule separates the neoplasm from the nonneoplastic thyroid follicles. The tumor cells are round- to spindle-­shaped. These tumors produce calcitonin and are positive for neuroendocrine markers including chromogranin A and synaptophysin

Mutations of the RET protooncogene is present in more than 65% of cases, while HRAS mutations is present in about 10% of cases [17, 18].

Adrenals The adrenal glands are bilateral glands composed of the adrenal cortex and medulla which are next to each other and show biochemical interactions. The gland adrenal cortex and medulla can be considered as two separate glands since they are composed of two different cell types and produce different hormones (Fig. 13.8). The adrenal cortex produces glucocorticoids, aldosterone, and sex steroid hormones. The adrenal medulla produces epinephrine and norepinephrine.

Adrenal Cortex Adrenal Cortical Hyperplasia The endocrine pathologist often has to distinguish between adrenal cortical hyperplasia, adenoma, and carcinoma. Adrenal cortical hyperplasia such as in primary hyperaldosteronism can lead to similar sign and symptoms of adrenal cortical neoplasms. Distinguishing between hyperplasia and adenomas in primary hyperaldosteronism is difficult, especially when more than one nodule is present in the adrenal

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Fig. 13.8 Normal adrenal gland tissue showing the adrenal cortex (C) which produces glucocorticoids such as cortisol and the adrenal medulla (M) which produces epinephrine and norepinephrine

cortex. There are many etiologies for adrenal cortical hyperplasia. These include micronodular hyperplasia, primary pigmented adrenocortical disease as part of Carney’s complex [19, 20]. Primary hyperplasia with hyperaldosteronism may be bilateral, which has a lot of implication for the management of this condition, so it is important for the pathologist to distinguish between these diseases. Secondary hyperplasia due to excessive ACTH production by the pituitary gland or from an ectopic source such as a lung carcinoma leading to Cushing disease or Cushing syndrome may be another etiology of adrenal cortical hyperplasia and is usually bilateral. The endocrine pathologist usually has to distinguish adrenal cortical adenomas from carcinomas on biopsy specimens or in complete resection of one adrenal gland. In addition, hyperplasia of the adrenal cortex associated with excessive production of aldosterone may be associated with bilateral adrenal cortical disease. There are many etiologies for adrenal cortical hyperplasia [19]. Some of these include micronodular adrenal cortical disease which includes primary pigmented adrenal cortical disease as part of Carney complex [19, 20]. Secondary hyperplasia may be related to excessive production of ACTH hormone by the pituitary gland leading to adrenal cortical hyperplasia and Cushing disease [19].

Adrenal Cortical Adenomas Adrenal cortical adenomas are benign tumors which may be functioning or nonfunctioning. Most adenomas are nonfunctioning or clinically silent and may be discovered incidentally during imaging studies or at autopsy. The tumor is usually

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Fig. 13.9  Adrenal cortical adenoma composed of cells with abundant eosinophilic cytoplasm and vesicular nuclei. Some of the tumors produce adrenal cortical hormones such as cortisol or aldosterone

yellow to yellow-brown, nodular, and circumscribed. Mitotic activity is uncommon and necrosis is usually absent (Fig.  13.9). Cortisol-producing adenomas may be associated with specific genetic abnormalities. Some of the genes include GNAS, PRAARIA, PRKACA, PRKACB, PDGIIA, and PDESB are associated with aberrant cyclic adenosine monophosphate kinase signaling. Aldosterone-producing tumors have been associated with aberrant signaling of KCN15, ATPIA1, ATP2B3, CACNIAD, CACNA1H, and CLCLN2 [21]. ARMC5 mutations are associated with primary bilateral macronodular hyperplasia with excessive cortisol or aldosterone production.

Adrenal Cortical Carcinoma Adrenal cortical carcinomas are usually large tumors with areas of necrosis. They may or may not be functional. Adrenal cortical carcinomas are highly lethal cancers. Depending on the hormones produced, they may be associated with Cushing, virilism, and less commonly with hyperaldosteronism. Carcinomas are invasive cancers that may invade the adrenal vein and inferior vena cava. The adrenal gland is also a site of metastatic tumors. Metastases to the adrenal is much more common than primary adrenal cortical carcinomas. For example, lung cancers frequently metastasize to the adrenal gland. Microscopically, adrenal cortical carcinomas have cells with large nuclei and small amounts of cytoplasm (Fig. 13.10). However, they may consist of spindled cells or large cell with multiple nuclei. A few syndromes are associated with adrenal cortical carcinomas including Li Fraumeni, multiple endocrine neoplasia type 1, Lynch syndrome, and Beckwith–Wiedemann syndrome [22]. Genetic mutations commonly seen in adrenal cortical carcinoma include TP53, MEN1, IGF2, IGF2R, p16/INK4A, TERT promoter, and RB1 [21]. Although

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Fig. 13.10  Adrenal cortical carcinoma composed of cells with enlarged nuclei and sparse amounts of cytoplasm. Two mitotic figures are present (arrows). Areas of necrosis are frequently seen in these carcinomas. These cancers may sometimes be functional and produce adrenal cortical hormones

most patients with adrenal cortical carcinomas have a poor prognosis, rare patients may survive 10 or more years [22].

Pheochromocytomas Pheochromocytomas are neural crest-derived chromaffin cell neoplasms arising in the adrenal medulla. The tumors are often associated with hypertension. Grossly the tumors are usually yellow-tan and hemorrhagic on cut section. Microscopic examination shows polygonal cells with abundant granular cytoplasm and the nuclei show a “salt-and-pepper” appearance characteristic of neuroendocrine cells and tumors (Fig. 13.11). The tumor cells are in clusters with a zellballen or cell nesting pattern. Sustentacular spindled cells are present around the periphery of the cell clusters. Immunohistochemical staining is positive for chromogranin A, synaptophysin, and INSM1. The sustentacular cells are positive for S100 protein and for SOX10. It is often difficult to predict which pheochromocytomas will metastasize. Pheochromocytomas are all considered potentially malignant [23, 24]. Pheochromocytomas may metastasize to regional lymph nodes, liver, lung, and bone. Pheochromocytomas may be associated with some familial syndromes including multiple endocrine neoplasia type 2A and 2B, von Hippel–Lindau syndrome, and neurofibromatosis. Molecular analyses have shown mutations of specific genes including HRAS, RET, ERAS1, and NF1 [25]. Correlates of pheochromocytomas with metastatic disease include the MAML3 fusion gene [25]. Other recent genetic alterations in pheochromocytomas have included THEM127 MAVPVS, ATRX, and TERT promoter [26, 27] (Fig. 13.12).

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Fig. 13.11  Pheochromocytoma. This tumor is derived from the adrenal medulla. The tumor cells have abundant basophilic cytoplasm and enlarged round nuclei. These tumors may produce epinephrine and norepinephrine and cause hypertension in the patient

Fig. 13.12  Paraganglioma or extra-adrenal pheochromocytoma. These tumors arise outside of the adrenal gland. They are composed of small cells with round nuclei and sparse amounts of eosinophilic cytoplasm. The tumor cells form cell nests (zellballen) (arrow)

Paragangliomas Paragangliomas or extra-adrenal paragangliomas/pheochromocytomas are neural crest-derived tumors that arise in the head and neck, chest, or abdomen. Unlike with pheochromocytomas in the adrenal glands, hypertension is present only in a small percentage of patients with these tumors. Paragangliomas may be associated with the sympathetic or parasympathetic nervous system. Epinephrine is usually not produced by most of these tumors, unlike the intra-adrenal tumors. This is related in

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part to the necessity of glucocorticoids for the synthesis of epinephrine from norepinephrine. Paragangliomas may show a zellballen or cell-nested pattern, but this is not always present. The cells in paragangliomas are usually smaller than those in pheochromocytomas, but the neuroendocrine type of nuclei is usually present. Immunohistochemical staining for chromogranin A, synaptophysin, and INSM1 is usually present. The sustentacular cells are also positive for S100 protein and SOX10. There is a higher rate of metastatic disease in paragangliomas compared to pheochromocytomas. Succinate dehydrogenase (SDHx) mutations are present in a small percentage of pheochromocytomas, but a much larger percentage of paragangliomas have mutations of the SDHx gene family which includes SDHA, SDHB, SDHC, SDHD, and SDHAF2. Patients with these mutations usually have multicentric tumors. Patients with SDHB mutations often have multicentric and metastatic tumors [27]. Metastatic paragangliomas can only be cured by surgery at this time [28]. Many pathologists have contributed to advances in endocrine pathology. The lives of two outstanding endocrine pathologist are summarized below.

Anthony GE Pearse (1916–2003) Anthony Guy Evenson Pearse was born in Kent, England in 1916. His father, Captain Radh Govinda Pearse, was an English army officer [29]. He attended Cambridge University for his undergraduate education at Trinity College. Dr. Pearse received his medical degree from St. Bartholemew’s Hospital in London. During World War II, he was a medical officer in the Royal Navy. He continued medical training after his naval service and decided to go into pathology. He received his pathology training at Hammersmith Hospital in London [29]. He subsequently received a teaching position and a faculty position at Royal Postgraduate Medical School [30]. He was a consultant pathologist at Hammersmith Hospital from 1951 to 1981 when he retired. Dr. Pearse married Elizabeth Himelhoch in 1947. They had one son and three daughters during their marriage. The early part of Dr. Pearse’s career was focused on histochemistry. He spent a great deal of time studying the microscopic components of cells and tissues. Since a lot of his research involved frozen sections of tissues, he created a special cryostat for working with frozen sections. He developed a microtome with a refrigerated chamber. This instrument had a major advantage of being able to maintain a constant temperature [29, 30]. Some of his research involved ultrastructural histochemistry using human as well as animal models. Dr. Pearse was the first person to show that calcitonin was produced by the C cells in the thyroid using immunofluorescent techniques with the assistance of one of the visiting pathologists in his laboratory from Italy, Dr. Giovanni Bussolati [31]. He did extensive studies on the pituitary gland using animal models as well as

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human tissues. He made many seminal observations about the pituitary, especially the gonadotroph cell [32]. In the 1960s, Dr. Pearse did a lot of studies of neuroendocrine cells [33, 34]. This led to the development of the amine precursor uptake and decarboxylation (APUD) theory. In some of his publications, he emphasized the common features of neuroendocrine cells. Some of these concepts had been developed by Professor Frederich Feyter in Germany. Many of the concepts were based on experimental data. However, a major speculative concept of Dr. Pearse was that all APUD cells were derived from the neural crest. This hypothesis was soon proven not to be true [35]. However, as the scientific literature accumulated and indicated that only a subset of neuroendocrine cells was of neural crest origin, Dr. Pearse revised his hypothesis to fit the emerging new data [34]. His revised hypothesis has been validated by many researchers and has served as a starting ground for many outstanding research work about neuroendocrine cells and tumors [35]. Dr. Pearse published more than 300 original manuscripts during his academic career. One of his major contributions was his textbook, Histochemistry Theoretical and Applied, first published in 1953. This major work went through several revisions and the fourth edition was expanded to three volumes. This major work has been translated into several language and remains a significant reference book in the field. In 1965, Dr Pearse received a personal chair in histochemistry and he also received a large scientific laboratory to continue his research work and to accommodate the many visitors that he had every year from many countries. His research on the APUD concept attracted major attention as investigators from many countries came to work under his guidance while others did experiments inspired by some of his original postulates. There were many outstanding scientists that trained with Dr. Pearse. Many of them such as Dr. Julia Polak and Dr. Susan Van Norden went on to become outstanding scientists as well. Over 50 of Pearse’s alumni went on to become professors or were professors when they visited his laboratory at the Hammersmith over the years. Dr. Pearse received many honors and awards. He was president of the Royal Microscopical Society; he received the Schleiden Medal from Germany, the Raymond Horton Smith prize from Cambridge University, and the Ernest Jung Award. He was a member of the founding editorial board of the Histochemical Journal and was on the editorial boards of several other journals. He died in 2003 of cardiovascular disease at the age of 86.

J. Aidan Carney (Born 1934) Dr. Aidan Carney was born in County Roscommon, Ireland in 1934 [35–37]. His father was a pharmacist, so Aidan had some knowledge of the medical world at a young age. He attended grade school in County Roscommon. He went on to high

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school at Clongowes Wood College, Naan County Kildare in Ireland [36]. Because of his interest in medicine, he enrolled in medical school and graduated from the University College of Dublin in 1959. He then did an internship at St. Vincent Hospital, Ireland. Because of his interest in the mechanisms of disease he decided to become a pathologist [37]. Dr. Carney migrated to the USA in 1962 and joined the Mayo Clinic, one of the most renown hospitals in the USA. While working at Mayo, Dr. Carney continued his formal studies and received a PhD from the University of Minnesota in 1969. His doctorate studies were in cardiac physiology [36]. He had become a consultant at Mayo Clinic in 1966 and was a full-time Mayo consultant while working on his PhD degree [37]. Dr. Carney married Claire Mary Carney in 1987 and they have been married for more than 35 years. Dr. Carney also received a doctorate degree from the Natural University of Ireland in 1978. He became a professor of pathology at Mayo Medical School in 1982. Dr. Carney’s interest in endocrine pathology and in genetics developed over many years. Because of his participation in the frozen section laboratory at Mayo, he was able to learn about many tumors. He had noticed that a type of tumor that was sometimes designated as papillary thyroid carcinoma because of some histological features also had unique histological characteristics. He undertook a study of these cases especially with follow-up studies of the patients with these tumors [38]. He designated these lesions as hyalinizing trabecular adenomas, because his follow-up studies showed that they were benign and so did not metastasize like true carcinomas. These tumors had unique features including striking hyalinization and fibrosis. He initially studied five cases from the Mayo Clinic and six cases from consultation cases sent to Mayo for a diagnosis. [38]. The tumors also had some features of medullary thyroid carcinoma, but amyloid was not present in any of the cases. These observations were supported by other pathologists and subsequent studies from many laboratories have confirmed that these are benign tumors although they have some features of papillary thyroid carcinoma and medullary thyroid carcinoma. This was a major advance in thyroid pathology, since some patients with diagnoses of cancer would now be diagnosed with a benign tumor that was not capable of metastasizing [38]. Recent molecular findings have supported these original observations, since hyalinizing trabecular adenomas have a unique molecular translocation that is not detected in papillary thyroid carcinoma (GLIS rearrangement) [39]. This has been most helpful for cytopathologists, since this lesion cannot be distinguished from papillary thyroid carcinoma by the cytopathologist without molecular studies. One of the earlier studies by Dr. Carney involved patients with multiple tumors involving the stomach, lungs, and paraganglionic tissues. These studies were reported in the renown New England Journal of Medicine in 1977 and the condition was designated as Carney’s triad [40]. The gastric lesions were recognized as being malignant and designated as leiomyosarcomas. With the evolution of terminologies, today they are designated as gastrointestinal stromal tumors. The lesions in the

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lungs of some patients were chondromas while the paragangliomas were present in many locations. In the original reports and in subsequent cases, very few of the same patients ever had all three tumors. Although only four female patients were reported in 1977, Dr. Carney had studied more than 150 patients with Carney triad [41, 42]. Dr. Carney and a major collaborator, Dr. Stratakis at the National Institutes of Health in Bethesda MD, went on to study a related group of patients with paragangliomas and gastrointestinal stromal tumors of the stomach. They designated these findings as the Carney–Stratakis syndrome (dyad) and reported that this dyad was inherited as an autosomal dominant trait [42]. The paragangliomas were multicentric, while the gastrointestinal tumors were multifocal in the same patients. Dr. Carney reported on another major observation in 1985 [2]. He described 40 patients with cardiac myxomas, skin pigmentation and nodular adrenocortical disease with Cushing syndrome in some patients, some patients also had myxoid breast lesions, testicular lesions, and pituitary adenomas. The maximum number of findings in a few patients were five [2]. Nine of the patients who had multifocal cardiac myxomas died of their disease. Dr Carney later showed that Carney complex was inherited as an autosomal dominant trait [43, 44]. A provisional diagnosis of Carney complex was made when two or more of the components were present. Carney complex was later shown to involve mutations of the protein kinase type 1subunit [44]. Dr. Carney considers that his most significant discoveries have involved Carney’s triad, Carney–Stratakis Dyad, and Carney complex [37]. Dr. Carney has received many honors and awards over the years of his illustrious career. Some of these have included the Donald C. Balfour Award for meritorious research from the Mayo Clinic and the Fellowship of Royal College of Physicians of Ireland; he has been named as an honorary member of the American Association of Endocrine Surgeons. He has received the Fred Steward Award from Memorial Sloan Kettering Cancer Center. He was presented many honorary lectures including the Peter Herman Lecture at the World Congress of Surgery. He was named as a fellow of pathology by the Royal College of Physicians of Ireland; he has given the Maude Abbott Lecture for the United States and Canadian Academy of Pathology and the William V. Gordies Memorial Lecture at Yale University in New Haven, CT among many others. Dr. Carney became an emeritus professor of pathology at Mayo Medical School in 1996. However, he continued his research work at the Mayo Clinic for another 22 years and he retired in 2018 [37]. During these 22 years, he has made many other important discoveries and has added to the understanding of Carney’s tTriad, Carney–Stratakis Dyad, and Carney complex [45, 46]. Dr. Carney and his wife, Claire Mary Carney, reside in Rochester, Minnesota, the home of the original Mayo Clinic. Over the years when he was not working on major discoveries Dr. Carney enjoyed gardening with his wife.

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References 1. Alexandraki KI, Grossman AB. The ectopic ACTH syndrome. Rev Endocr Metab Disorders. 2010;11(2):117–26. 2. Carney JA, Gordon H, Carpenter PC, et al. The complex of myxomas, spotty pigmentation and endocrine overactivity. Medicine (Baltimore). 1985;64:270–83. 3. Correa R, Salpea P, Stratakis CA.  Carney complex: an update. Eur J Endocrinol. 2015;173(4):85–97. 4. Komminoth RP. Polyglandular autoimmune syndrome: an overview. Pathologe. 2016;37:253–7. 5. Wemeau J-L, Proust-Lemoine E, Ryndak A, et al. Thyroid autoimmunity and polyglandular endocrine syndromes. Hormone. 2013;12(1):39–45. 6. Hiromatsu Y, Satoh H, Ainno N.  Hashimoto’s thyroiditis: history and future outlook. Hormones. 2013;12(1):12–8. 7. Rolli M, Angeletti D, Fiore M, et al. Hashimoto’s thyroiditis. An update on pathogenic mechanisms, diagnostic protocols, therapeutic strategies and potential malignant transformation. Autoimmun Rev. 2020;19(10):102649. https://doi.org/10.1016/J.autrev.2020.102649. 8. Weetman AP. An update on the pathogenesis of Hashimoto’s thyroiditis. J Endocrinol Invest. 2021;44(5):883–90. 9. Graceffa G, Pague R, Vieni S, et al. Association between Hashimoto’s thyroiditis and papillary thyroid carcinoma: a single center experience. Acta Endocrinol (Buchar). 2022;18(1):74–8. 10. Travaglino A, Pace M, Varricchio S. Hashimoto’s thyroiditis in primary thyroid non-Hodgkin’s lymphoma. A systematic review and meta-analysis. Am J Clin Pathol. 2020;153(2):156–64. 11. Carroll NM, Carty SE. Promising molecular techniques for discriminating among follicular thyroid neoplasms. Surg Oncol. 2006;15(2):59–64. 12. Nikiforov YE, Nikiforova MN. Molecular genetics and diagnosis of thyroid cancer. Nat Rev Endocrinol. 2011;7(10):569–80. 13. Esapa CT, Johnson SJ, Kendall-Taylor P, et al. Prevalence of RAS mutation in thyroid neoplasia. Clin Endocrinol (Oxf). 1999;50:529–35. 14. Iro Y, Yoshida H, Tomoda C. HBME-1 expression in follicular tumor of the thyroid: an investigation of whether it can be used as a marker to diagnose follicular carcinoma. Anticancer Res. 2005;25(1A):179–82. 15. Prete A, Borges de Souza P, Census S, et al. Update on fundamental mechanisms of thyroid cancer. Front Endocrinol. 2020;11:102. https://doi.org/10.3389/fendo.2020.00102. 16. Lee YS, Lim H, Chang H-S, et al. Papillary thyroid microcarcinoma different from latent papillary thyroid microcarcinoma at autopsy. J Korean Med Sci. 2014;29(5):676–9. 17. Cancer Genome Atlas Research Network. Integrated genomic characterization of papillary thyroid carcinoma. Cell. 2014;159(3):676–90. 18. Ceolin L, da Silva Duval NA, Benini AF, et al. Medullary thyroid carcinoma beyond surgery: advances, challenges and perspectives. Endocr Relat Cancer. 2019;26(9):R499–518. 19. Bourdeau I, Parisien-La-Salle JS, Lacroix A. Adrenocortical hyperplasia: a multifaceted disease. Best Practice Res Clin Endocrinol Metab. 2020;34(3):101386. https://doi.org/10.1016/ becon2020.101386. 20. Carney JA. The Carney complex (myxomas, spotty pigmentation, endocrine overactivity and schwannomas). Dermatol Clin. 1995;13:19–26. 21. Kamilaris CDC, Hannah-Shmouni F, Stratakis CA.  Adrenocortical tumorigenesis: lessons from genetics. Best Pract Res Clin Endocrinol Metab. 2020;34(3):101428. https://doi. org/10.1016/j.beem2020.101428. 22. Else T, Kirn AC, Saboolh A, et al. Adrenal cortical carcinoma. Endocr Rev. 2014;35(2):282–326. 23. Thompson LD.  Pheochromocytoma of the adrenal gland scaled score (PASS) to separate benign from malignant neoplasm: a clinicopathologic and immunophenotypic study of 100 cases. Am J Surg Pathol. 2002;26(5):551–66. 24. Kimura N, Takayanage R, Takizawa W, et al. Pathologic grading for predicting metastasis in pheochromocytoma and paraganglioma. Endocr Rel Cancer. 2014;21(3):405–14.

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25. Fishbein L, Leshchiner I, Walter V, et al. Comprehensive molecular characterization of pheochromocytomas and paragangliomas. Cancer Cell. 2017;31(2):181–93. 26. Wachtel H, Fishbein L.  Genetics of pheochromocytoma and paraganglioma. Curr Opin Endocrinol Diab Obes. 2021;28(3):283–90. 27. Fishbein L.  Pheochromocytoma and paraganglioma: genetics, diagnosis and treatment. Hematol Oncol Clin North Am. 2016;30(1):1135–50. 28. Favier J, Amor L, Gimenez-Roqueplo A-P.  Paraganglioma and pheochromocytoma: from genetics to personalized medicine. Nat Rev Endocrinol. 2014;11(2):101–11. 29. Wick MR. Pearse, Anthony G.E (1916-2003). In: van den Tweel JG, editor. Pioneers in pathology. In: van Krieken JHJM, series editor. Encyclopedia of pathology. Cham:Springer; 2017. p. 429–31 30. Van Noorden CJF. In memoriam Prof Dr. Anthony GE Pearse (1916-2003). Acta Histochem. 2004;106:255–6. 31. Bussolati G, Foster GV, Clark MB, et al. Immunofluorescent localization of calcitonin in medullary C-cell thyroid carcinoma using antibody to the pure porcine hormone. Virchows Arch B Cell Pathol. 1969;2(3):234–8. 32. Pearse AG. The application of cytochemistry to the localization of gonadotropin in the pituitary. J. Endocrinol. 1951;7(4):xlviii–l. 33. Pearse AGE.  Common cytochemical and ultrastructural characteristics of cells producing polypeptide hormones (the APUD series). And their relevance to thyroid and ultimobranchial C cells and calcitonin. Proc R Soc Lond B Biol Sci. 1968;170:71–80. 34. Pearse AGE, Takor T. Neuroendocrine embryology and the APUD concept. Clin Endocrinol. 1976;5(Suppl):229S–44S. 35. LeDourin N. The neural crest. Cambridge: Cambridge University Press; 1982. 36. GST Support International. J Aidan Carney biographical information. https://www.gistsupport.org/about-­gist/sdh-­deficient-­amp-­wildtype-­gist/carney-­triad-­summary/aidan-­carney/. Accessed 15 May 2023 37. Carney JA. Pathologist. Personal communication by email. 15 Feb 2022 38. Carney JA, Ryan J, Goellner JR. Hyalinizing trabecular adenoma of the thyroid gland. Am J Surg Pathol. 1987;11(8):583–92. 39. Nikiforova MN, Nikitski AV, Panebianco F, et al. GLIS rearrangement is a genomic hallmark of hyalinizing trabecular tumor of the thyroid gland. Thyroid. 2019;29(2):161–73. 40. Carney JA, Sheps SG, Go VL, et al. The triad of gastric leiomyosarcoma, functioning extra-­ adrenal paraganglioma and pulmonary chondroma. N Engl J Med. 1977;296(20):1517–8. 41. Carney JA. Carney triad. Front Horm Res. 2013;41:92–110. 42. Carney JA, Stratakis CA. Familial paraganglioma and gastric stromal sarcoma. A new syndrome distinct from the Carney’s triad. Am J Med Genet. 2002;108(2):132–9. 43. Carney JA, Hruska LS, Beauchamp GA. Dominant inheritance of the complex of myxomas, spotty pigmentation and endocrine overactivity. Mayo Clin Proc. 1986;61(3):165–72. 44. Kirschner CA, Carney JA, Pack SD, et al. Mutation of the gene encoding protein kinase A type 1-alpha regulatory subunit in patients with the Carney complex. Nat Genet. 2000;26(1):89–92. 45. Carney JA, Lyssikatos C, Seethala RR, et al. The spectrum of thyroid gland pathology in Carney complex. The importance of follicular carcinoma. Am J Surg Pathol. 2018;42(5):587–94. 46. Carney JA, Stratakis CA, Young WF Jr. Adrenal cortical adenoma: the fourth component of Carney triad and an association with subclinical Cushing syndrome. Am J Surg Pathol. 2013;37(8):1140–9.

Chapter 14

Hematopathology

Hematopathology is the study of diseases and disorders of the cells that make up the blood, bone marrow, spleen, and lymph nodes. The diagnoses of diseases such as lymphomas and leukemias are done by the hematopathologists. The history of leukemias extends back to more than 200  years. Splenitis acutus was described by Peter Cullen around 1811 [1]. Virchow described leukemia in 1847 based on his observations of the reversed white and red blood cell balance that he observed. He also described malignant lymphomas. Henry Fuller described the first leukemic patient based on microscopic observations. Abnormalities of lymph nodes with pathologic enlargement was observed around 1862 and follicular lymphoma, a low-­ grade, slow-growing variant of malignant lymphoma, was first recognized in 1925. It was initially thought to be a benign disorder, but was later shown with careful follow-up studies to be a low-grade malignancy [2]. Thomas Hodgkin reported on the clinical data and postmortem findings of seven cases of a disease that would be called Hodgkin disease a few decades later [3–5]. Surprisingly, these studies were done without microscopic examination, even though today one has to find the Reed–Sternberg cell in these lesions microscopically to confirm the diagnosis. Thirty years after Hodgkin’s report, Sir Samuel Wilks, a professor of pathology in London, proposed that the disease described by Hodgkin should be called Hodgkin’s disease. His studies were also done without the use of a microscope. It was Herbert Fox at the University of Pennsylvania who performed the first histological studies of Hodgkin’s disease in 1926. Based on his studies, he considered only three of the cases reported by Hodgkin to represent Hodgkin’s disease. The other cases included a case of tuberculosis, a case of syphilis, and one of systemic lymphosarcoma. Sternberg in 1902 described the occurrence of tuberculosis and Hodgkin’s disease in the same patient, so it is possible that they could have coexisted in one of Hodgkin’s cases. It was Dorothy Reed who performed careful clinicopathologic and morphologic studies of Hodgkin’s disease. She wrote in great detail about this disease and showed that it was different from tuberculosis [6]. Dorothy Reed (1874–1964) was homeschooled until she went to © The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 R. V. Lloyd, Pathology: Historical and Contemporary Aspects, https://doi.org/10.1007/978-3-031-39554-3_14

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Smith College in Massachusetts and then she attended Johns Hopkins medical school much to the surprise of her mother and other relatives. After graduation she did an internship under the mentorship of William Osler at Johns Hopkins then became a pathology fellow for 1 year under the tutelage of Dr. William Welch. It was during her fellowship year that she did her brilliant studies of Hodgkin disease and described the Reed–Sternberg cell (Fig. 14.1) [6]. In spite of her achievements, Dr. Reed could not get a teaching position at Johns Hopkins because of her sex. She decided to study pediatrics and worked at Babies Hospital in New York. She married one of her Hopkins’ classmates, Charles Mendenhall [6]. She and her husband then moved to Madison, Wisconsin, where she spent the rest of her life working in maternal and child health. In her brilliant studies, Reed observed the neoplastic cells in Hodgkin disease and she emphasized the contiguous nature of lymph nodes involved in the disease. She also noted that the disease often started in the cervical region. Dr. Ronald Dorfman, who was originally from South Africa and had worked at Washington University in St. Louis before going to Stanford Medical Center, was in the United Kingdom while on sabbatical from Stanford. He spent some time at Guy’s Hospital in London and reviewed the original autopsy materials studied by Hodgkin. Surprisingly, he was able to demonstrate Reed–Sternberg cells in some of the tumors by immunohistochemistry with CD15 antibody after 165 years of being in a fixative [7]. Fixation in a mercurial fixative probably helped to preserve the immunoreactivity, since specimens fixed in formalin often show decreased immunoreactivity after just a few weeks of fixation!

Fig. 14.1  Hodgkin lymphoma. The tumor contains a mixture of inflammatory cells in the background including lymphocytes, plasma cells, and eosinophils (red granular cytoplasm). The diagnostic Reed–Sternberg cells are often binucleated (arrow)

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Hematopathology has made a great deal of progress over many decades in establishing specific diagnoses with reliable specificity that can direct targeted therapy in the treatment of Hodgkin disease and non-Hodgkin lymphoma. This specialty was one of the first areas of pathology to adopt new technology such as immunohistochemistry, flow cytometry, and molecular genetics to assist in diagnosing specific diseases such as leukemias and lymphomas [8]. Today, the diagnosis of the many subtypes of leukemias and lymphomas relies on clinical information, histologic assessment, immunophenotypic characterization, and molecular alterations for the precise diagnoses, therapeutics, and prognoses of specific hematologic diseases [8]. Immunohistochemistry, with the development of monoclonal antibodies with specific cluster designations or clusters of differentiation (CD), has assisted in advancing the pathologic studies of these diseases. The cluster designations help to identify and investigate the cell surface protein molecules which provide targets for immunophenotyping normal and neoplastic cells. There are more than 350 CDs. They have many functions such as receptors, ligands, and other targets important for cell function. CDs are usually associated with immune functions. The various techniques used in the workup of hematopathologic cases have evolved over many decades. For example, B-cell immunoglobulin light chain restriction, developed many years ago, is a true clonal marker (Figs. 14.2 and 14.3). However, many B-cell malignancies do not express detectable immunoglobulin, so the utility of immunoglobulin light chain restriction for characterization of B-cell lesions can be somewhat limited [9]. In contrast to B-cell markers, the T-cell antigens which can be identified with monoclonal antibodies are not clonal markers, so other techniques have to be used to characterize clonal T cells to help in the diagnosis of malignant T-cell lesions (Fig.  14.4). Advances in molecular biologic techniques have been helpful in the diagnosis of B- and T-cell malignancies. Gene

Fig. 14.2  Large B-cell lymphoma in a lymph node. The neoplasm consists of sheets of tumor cells with large neoplastic B cells. The tumor cells are dyscohesive and this is a useful diagnostic feature

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Fig. 14.3  Large B-cell lymphoma from a lymph node. (Same tissue as in Fig. 14.2). The tumor cells are positive for CD20 which is a diagnostic marker for B cells. The positive staining is indicated by brown cytoplasmic positivity in the cytoplasm and cell membrane of the tumor

Fig. 14.4  Malignant lymphoma, T-cell type. The tumor is composed of sheets of neoplastic lymphoid cells. The empty spaces represent fat cells or adipocytes. The tumor cells have large nuclei and scant amounts of cytoplasm and have infiltrated between the fat cells. Immunohistochemical stains were positive for CD4 which is a T-cell marker

rearrangements have been useful in identifying chromosomal breakpoints and translocations corresponding to specific diseases with prognostic implications. The basic techniques of immunohistochemistry, flow cytometry, and molecular clonality assays can be used to diagnose specific types of lymphomas and leukemias [10]. Immunohistochemistry and flow cytometry are very powerful techniques and help in diagnosing most B- and T-cell lesions. However, sometimes these powerful approaches cannot help to distinguish benign and malignant lesions, so molecular clonality assays can be used [10]. Molecular clonality assays are tumor-specific and

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can help to sort out recurrence of a prior disease or the development of a new neoplasm. They can also be used to monitor minimal residual disease, which, as the name implies, would detect a few cells of the malignant process that would be difficult to identify with other techniques because of limited sensitivity [10]. Although lymphomas are more frequently encountered in lymph nodes, spleen, and bone marrow, it is important to realize that most tissues or organs in the body may develop a primary or secondary lymphomas. Thus, primary lymphomas may develop in disparate sites such as the eyes, skin, breast, uterus, thyroid, heart, and other tissues, to name a few uncommon sites. Rare cases of primary lymphomas may even develop in body fluids and are known as effusion lymphomas. Another source of lymphoproliferative disorders may occur in patients who have had solid organ transplants or hematopoietic stem cell transplants [11, 12]. Since these patients are usually treated with immunosuppressive drugs to avoid rejection of the transplants, it becomes a challenge for the clinician to treat the lymphoproliferative disorder while preserving the function of the allograft (transplanted tissue). New variants of lymphomas and leukemias are being discovered on a regular basis which is usually described in the latest publication of scientific manuscripts or other sources such as the World Health Organization Blue Books written by experts from many countries in the field [13, 14]. For example, breast implant-associated anaplastic large-cell lymphoma is a relatively recently described variant of lymphoma reported by several investigators [12]. This uncommon lymphoma variant is a peripheral T-cell lymphoma that develops in implants used for breast augmentation or for reconstruction that is done after a mastectomy procedure. The etiology is not known, but the disease may be related to chronic inflammation caused by textured devices and lead to malignant transformation of T cells [12]. Although some earlier studies suggested that small lymph node biopsies such as needle-core biopsies were less reliable than examining the entire lymph node, recent studies have documented the reliability of fine-needle aspiration biopsy and core-­ needle biopsies [15, 16]. Although a minimal size biopsy is recommended by the College of American Pathology, many small biopsies have diagnostic information, especially when a few guidelines have been followed [17]. These guidelines include using as much material from the biopsy as possible for histologic sections and submitting tissues for disaggregation and flow cytometry only when there is at least a 2.0 cm core biopsy length [17]. In most cases with small biopsies, the tools available in hematopathology such as immunohistochemical stains, polymerase chain reaction, clonality assessment and flow cytometry usually allow for an accurate diagnosis even with small specimens [17]. Another area of new development in hematopathology involves artificial intelligence and machine learning [18]. Three areas that have the potential for adaptation to machine learning and artificial intelligence in hematopathology include the following: 1. High volume, low complex questions such as low platelets or low levels of neutrophils in a sample. 2. Automated blood film reporting after digitizing all blood sample films coming into the laboratory. Such a system could do cross-checks of clinical records,

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biochemistry results, and information about drugs that could be provided by the pharmacist. 3. Modeling using large datasets that could be used in prediction and risk stratification. This could assist in improving prediction of how laboratory parameters will develop such as white blood cell counts in monoclonal gammopathy of undetermined significance or chronic myeloid leukemia and could be used for example to predict how often patients with these disorders would have to be seen [18]. Proof of principle studies using artificial intelligence with diffuse large B-cell lymphoma (DLBCL) are currently underway in studies of their cell of origin, that is, germinal-center-B-cell-like and activated B-cell-like DLBCL [19]. Such studies including clinical grade assays with targeted RNA sequencing and next-­ generation sequencing should assist in clinical outcome predictions [19]. Many outstanding hematopathologists have contributed to progress in this field. The next section will summarize the lives of two of these outstanding pathologists who have contributed greatly to progress in hematopathology.

Karl Lennert (1921–2012) Karl Lennert was born in Furth, Bavaria in 1921 where he attended school and graduated from high school. He attended medical school from 1939 at the University of Erlangen-Nurenburg and graduated in 1945 after the end of World War II. He became a physician in the Institute of Pathology in Erlangen. He then visited the Max Plank Institute for Biophysical Chemistry and studied histochemical methods for a few months. As a postdoctoral resident in pathology at the University of Erlangen, he developed a deep interest in hematopathology [20–22]. Some of his studies involved using a modified Giemsa stain to study aspiration biopsies of hematological specimens. He was able to observe for the first time that the germinal centers of lymph nodes contained centroblasts. This observation would be important later as he formulated his ideas about classification of non-Hodgkin lymphomas. Lennert later moved to Frankfurth and spent a great deal of his academic time studying lymph node diseases. He completed his habilitation which was focused on Hodgkin disease in 1952. His thesis also included the first description of “Lennert lymphoma,” which he mistakenly thought at that time was part of Hodgkin’s disease based on morphological features. The immunohistochemical, flow cytometric and molecular genetics tools were not available for an in-depth analysis. It was around this time that Lennert started collecting biopsy specimens which he was able to correlate with the patient’s clinical history and follow-up information over many years. He continued these types of studies for many decades during his illustrious and long career. He was appointed as an associate professor at the Institute of Pathology in Heidelberg in 1960. He became well known for his interest in and knowledge of lymphomas and received many consultation cases. In 1961, he published a monograph on the pathology of lymphadenitis which he had worked on for about 8 years.

Karl Lennert (1921–2012)

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The work was well received in academic circles. He was soon appointed as chairman of pathology at the University of Kiel in 1963. As chairman he continued his studies of lymph nodes and bone marrow with the assistant of many talented young pathologists. He started the Lymph Node Registry based on the model used in the USA at the Armed Forces Institute of Pathology in his early days as chairman. This led to the accumulation of more interesting and unusual cases which could be used for teaching as well as research. Around this time, the classification of non-Hodgkin lymphoma was rudimentary and controversial with the exception of Hodgkin disease based on the elegant studies of Lukes and Butler in the USA who had developed a practical classification of Hodgkin disease. The Hodgkin’s disease classification had allowed clinicians and radiologist to develop methods of treating patients with the various subtypes of these lymphomas and resulted in continuous improvement in survival for these cancers. Knowledge of immunology was rapidly advancing in the 1960s and 1970s and Lennert’s broad approach to the non-Hodgkin lymphoma utilizing morphological and functional studies led to major advances in understanding these diseases and in the classification of non-Hodgkin lymphomas. Lennert organized the European Lymphoma Club. These European hematopathologists led by Lennert used morphological as well as the emerging techniques of immunology in attempts to develop more relevant classification of lymphomas. This group worked on the classification of non-Hodgkin lymphoma based on morphology which was linked to correlation of histologically and cytologically defined tumor types and was named the Kiel classification [23]. This classification divided lymphomas into low-grade and high-grade malignancies and was a useful guide for physicians treating patients with lymphoma based on the morphological classification. Competing classification systems such as the Rapaport system and the working formulation in the USA had made some of the classifications of non-Hodgkin lymphoma very confusing for clinical treatment of patients with these diseases as well as in the pathological classification. The Kiel classification was a welcome advance in the late 1970s. The updated Kiel classification published in 1988 was able to separate non-Hodkgin lymphoma into B-cell and T-cell malignancies and was based on histological as well as immunohistochemical and molecular genetic findings [24]. Lukes and Collins in the USA had also independently developed their own classification of non-Hodgkin lymphoma using the same morphological and functional approach used for the Kiel classification as will be discussed below. After 1990, a compromise between European and American pathologists led to the Revised European-American Lymphoma (REAL) classification that maintained the insights gained from the Kiel classification and this helped to make the non-­Hodgkin lymphomas classification more acceptable. Lennert retired in 1989, but continued to be active in the field and publishing original manuscripts for many years. The grading of his high-grade and low-grade lymphoma classification was confirmed by the Ki-67 antibody which was also developed in Kiel, Germany [24]. Lennert received many honors for his singular contributions. He received several honorary doctorates from various European universities and many awards including the Fred W. Stewart Award from Memorial Sloan Kettering Cancer Center in 1992, The Rudolf

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Virchow Medal, and the Ernst Jung Prize. One of his students wrote after his passing that Karl Lennert was considered as one of the most important hematopathologists in history and was the founder of the European Association of Hematopathology and that he will be missed by many former students and friends after his passing.

Robert Lukes (1922–1999) Robert Lukes was born in Cleveland, Ohio in 1922. He attended medical school in Chicago at Loyola School of Medicine and then did an internship at Cleveland City Hospital. He received his pathology training at Crile VA Hospital [25, 26]. Lukes was in the Armed Forces during the Korean War and served as the chief of pathology at the First Medical Field Laboratory. It was while he was in the service during the Korean War that he performed a detailed study of epidemic hemorrhagic fever and published a classic paper on the subject [27]. In his study of 39 cases with fatal outcomes, he was able to correlate the clinical stages with the autopsy findings at these stages and to evaluate the role of shock in the disease. He observed that hemorrhage in the right atrium and infarction-like necrosis in the anterior pituitary supported earlier observations that capillary damage was the basic process involved in the disease as had been previously reported. However, he also noted that shock was not the only factor leading to pituitary necrosis, but thought that anoxemia from severe congestion was also an important contributing factor. After the war he worked at the Armed Forces Institute of Pathology between 1953 and 1961. It was during this period that he and a colleague, Dr. Butler, published a classic paper on Hodgkin disease [28]. In their study of 177 cases, they correlated the morphological features with the clinicopathologic features. They produced a new classification with excellent clinicopathologic and prognostic features. They separated Hodgkin disease into six groups and correlated the subtypes with favored locations such as nodular sclerosis being associated mainly with mediastinal involvement at presentation. They were able to show a relationship between histologic types, clinical stages, and survival [27, 28]. This new classification soon replaced the histological classification of Jackson and Parker in which the histological types were of limited effectiveness in prognosis, because subtypes such as the granuloma group of Jackson and Parker was very heterogeneous and represented 80–90% of the reported series and contained about 79% of the survivors after 15 years. The novel classification of Lukes and Butler was eventually adopted by major centers that were treating large numbers of patients with Hodgkin’s disease. Institutions such as Stanford Medical Center in California, the USA made tremendous progress as radiation therapist such as Dr. Kaplan and oncologist improved the survival of patients with Hodgkin disease using the Lukes and Butler method of defining groups of patients. Luke’s second major area of contribution to hematopathology was in studies of non-Hodgkin’s lymphoma. He collaborated with Dr. John Parker at the University of Southern California School of Medicine. Dr. Parker was studying the basic

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biology of transformed lymphocytes after immunologic stimulation. With the collaboration of Robert Collins at Vanderbilt University they were able to develop a functional classification for non-Hodgkin lymphoma. The Lukes and Collins classification was later confirmed by immunohistochemical and flow cytometric studies. With these techniques, they were able to analyze a large volume of normal and neoplastic B and T cells and their morphologic changes with differentiation [29]. The Lukes and Collins classification along with Lennert’s Kiel classification in Europe were similar conceptually, but used different terminologies, and they helped to make great advances in the classification and understanding of non-Hodgkin’s lymphomas [30]. Dr. Lukes and Dr. Lennert were amicable colleagues and their friendship helped to unite and advance the non-Hodgkin lymphoma classification that is currently used by the World Health Organization (WHO) today. Lukes retired from the University of Southern California Medical Center and the LA County Medical Center in 1984. He retired from the School of Medicine in 1986. He continued to work part time as a consultant at the University of California Irvine and later at the Scripps Clinic and Research Foundation in La Jolla, California. He received the distinguished Emeritus Award from the University of Southern California (USC) in 1986. The chief of hematology at USC, Dr. Alexandra Levine, was reported to have commented about Dr. Lukes: “In medicine you must see not just what the patient shows you, but what he or she does not.” She went on to say that “He was a giant of a man whose accomplishments will be remembered long after we are all gone” [26].

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10. Wang H-W, Raffeld M.  Molecular assessment of clonality in lymphoid neoplasia. Semin Hematol. 2019;56(1):37–45. 11. Jagadeesch D, Woda BA, Draper J, et al. Post transplant lymphoproliferative disorders, risk, classification and therapeutic recommendations. Curr Treat Options in Oncol. 2012;13(1):122–36. 12. Nelson JA, Dabic S, Mehrara BJ, et al. Breast implant-associated anaplastic large cell lymphoma incidence: determining and accuracy risk. Ann Surg. 2020;272(3):403–9. 13. Swerdlow SH, Campo E, Harris NL, et al. WHO classification of tumours of haematopoietic and lymphoid tissues. Revised 4th edition Lyon: IARC Press; 2017. 14. Lukes RJ, Collins RD.  Tumors of the hematopoietic system. Atlas of tumor pathology 2nd Series. Fascicle 28. Washington DC: Armed Forces Institute of Pathology; 1992. 15. Pfeiffer J, Kayser G, Ridder GJ. Sonography-assisted cutting needle biopsy in the head and neck for the diagnosis of lymphoma. Can it replace lymph node extirpation? Laryngoscope. 2007;119(4):689–95. 16. Jellout F-Z, Navarro M, Navale P, et al. Diagnosis of lymphoma using fine-needdle aspiration biopsy and core-needle biopsy: a single-institution experience. Acta Cytol. 2019;63:198–205. 17. Ranheim EA.  Pearls and pitfalls in the diagnostic workup of small lymph node biopsies. Modern Pathol. 2019;32(Suppl 1):538–43. 18. Sivapalaratam S. Artificial intelligence and machine learning in hematology. Br J Haematol. 2019;185(2):207–8. 19. Zijun Y, Xu-Monette ZH, et al. A refined cell-of-origin classifier with tangented NGS and artificial intelligence shows robust predictive value in DLBCL. Blood Adv. 2020;4(14):3391–404. 20. Mechler U, Klapper W, Muller-Hermelink HK, Lennert, K.  In: van den Tweel JG, editor. Pioneers in pathology. van Krieken JHJM, series editor. Encyclopedia of pathology. Switzerland:Springer International Publishing; 2017, p. 331–38. 21. Klapper W, Koch K, Mechler U, Borck C, Fuhry E, Siebert R. Lymphoma ‘type K’ in memory of Karl Lennert (1921–2012). Leukemia. 2013;27:519–21. 22. Pileri SA. Karl Lennert. Leukemia. 2013;27:517–8. 23. Lennert K, Feller AC. Histopathology of non-Hodgkin’s lymphoma (based on the updated Kiel classification). Berlin/Heidelberg/New York. 24. Gerdes J, Lennert K, Daltenbach F, et  al. Growth fractions in malignant non-Hodgkins lymphoma as determined in situ with the monoclonal antibody Ki-67. Hematol Oncol. 1981;2:365–71. 25. Taylor CR, Robert L In: van den Tweel JG, editor. Pioneers in pathology. van Krieken JHJM, series editor. Encyclopedia of pathology. Switzerland: Springer International Publishing; 2017, p. 348–50. 26. Byrne M, Robert L. Hodgin’s lymphoma pioneer, dies. Los Angeles: USC News; 1995. https:// news.usc.edu/110/Robert-­Lukes-­Hodgkin-­s-­lymphoma-­pioneer-­dies/ Accessed 20 Dec2022. 27. Lukes RJ. The pathology of thirty-nine fatal cases of epidemic hemorrhagic fever. Am J Med. 1954;16(5):639–50. 28. Lukes RJ, Butler JJ.  The pathology and nomenclature of Hodgkin’s disease. Cancer Res. 1966;26(6):1063–83. 29. Lukes RJ, Taylor CR, Parker JW, et al. A morphologic and immunologic surface marker study of 299 cases of non-Hodgkin’s lymphomas and related leukemias. Am J Pathol. 1978;90:461–85. 30. Lennert K, Collins RD, Lukes RJ. Concordance of the Kiel and Lukes-Collins classification of non-Hodgkins lymphoma. Histopathology. 1983;7(4):549–59.

Chapter 15

Introduction to Microbiology

Microbiology is one of the most active areas of practice in clinical pathology, because microbes remain a major cause of human diseases. Conditions ranging from sepsis due to bacterial infections to opportunistic infections in immunosuppressed patients come to the attention of the microbiology laboratory and challenges the skills of the microbiologists in solving these problems; some of these solutions make a major difference in the life or death of hospitalized patients. Although heart and blood vessel diseases along with cancer are the leading causes of death in adults in the USA today, historically infectious diseases had been the major cause of illnesses and death in the USA in earlier centuries. Infectious agents are classified into four major groups including (a) Viruses which are submicroscopic organisms that cannot exist independently without a host for replication. Viruses usually have either DNA or RNA as their nucleic acid and they represent the simplest forms of infectious agents. (b) Bacteria are the most numerous infectious agents. These single-cell organisms do not have a nucleus. They have both DNA and RNA and are mostly autonomous and free living and reproduce by binary fission. (c) Fungi are eukaryotes that can exist as single cell or are multicellular organisms which have a defined nucleus and cytoplasm. Yeast are single-cell fungi that reproduce by budding, while molds are more complex multicellular organisms that reproduce sexually and asexually. Dimorphic fungi have both yeast and mold phases [1]. (d) Parasites are a large group of complex microbes that range from single-cell organisms such as protozoa to more complex multicellular organisms. They range in size from microscopic to macroscopic and often obtain their nutrition at the expense of the host organisms [1]. Other rare microorganisms such as prions can also cause diseases. Prions are mainly composed of proteins; they do not have nucleic acids and cannot replicate in the conventional sense, but their abnormal proteins are associated with a replicative © The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 R. V. Lloyd, Pathology: Historical and Contemporary Aspects, https://doi.org/10.1007/978-3-031-39554-3_15

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cycle [1]. Prions interact with nerve cells leading to neurodegenerative brain diseases such as Jakob–Creutzfeldt disease in humans. Other organisms that are abundant in nature but are not associated with known human diseases include archaea. These organisms were once linked with bacteria since they are prokaryotes without cell nuclei and they could have evolved from bacteria. Although they are very abundant on earth and are present in the skin, mouth, gastrointestinal tract, and other sites in humans, they have not been associated with human diseases [2]. A wide spectrum of diagnostic techniques is used in the microbiology laboratory. Immunologic methods are some of the most powerful diagnostic techniques [3]. These methods rely on basic antigen-antibody interactions. Monoclonal antibodies, which have been developed against viruses, bacteria, fungal, and parasitic organisms are extremely powerful reagents for diagnostic microbiology by enzyme immunoassay methods and diagnostic antigen-antibody methods such as solid-­ phase immunoassay techniques and related methods. Another useful approach for the diagnosis of microbiological agents includes immunofluorescence techniques using direct or indirect approaches for antigen detection [3]. These techniques have simplified and accelerated diagnostic approaches in the laboratory. Molecular techniques have made major in-roads in diagnostic microbiology, because of the speed and efficiency that these approaches have brought to the laboratory [4]. In situ hybridization has been used in the laboratory for multiple purposes including the identification of infectious agents, molecular translocation, and gene amplification [4–7]. In situ hybridization has been used mainly for rapid identification of organisms in positive blood cultures [6], but it has also been used for identification of fungi and other organisms in tissue sections [5]. Polymerase chain reaction (PCR) has produced major diagnostic advances in the microbiology laboratory such as for the detection of organisms that could not be cultured as well as for fastidious and slow-growing organisms [4]. The use of automated and semiautomated PCR platforms and more rapid detection methods have increased the use of these assays and made them more widely applicable. DNA sequencing is now a regularly used molecular approach for the analysis of amplified products, but it is more complex, since it requires additional software and genetic databases [7]. Different types of mass spectrometry have been used to identify microorganisms in the clinical laboratory [8, 9]. A mass spectrometric profile of the ribosomal RNA (rRNA) proteins is useful for identification. MALDI-TOF mass spectrometry is often used in the clinical microbiology laboratory [3]. MALDI-TOF is considered to be a cost-effective major advance for the identification of many organisms in the diagnostic laboratory [3].

Vaccinations Although the story of the vaccine used by Edward Jenner obtained from cowpox pustules to provide protection against smallpox in 1796 is well known, there is some historical suggestion that other groups such as the Chinese used smallpox inoculation or

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variolation to prevent smallpox disease many years earlier. Jenner’s work in 1796 was a major step forward and with more research developments and generation of vaccines against viruses and bacteria led to the elimination of some infectious diseases such as smallpox which was eventually eliminated as a disease of humankind in 1980. Louis Pasteur made a major advance in vaccination in 1885 by developing a vaccine for rabies in humans and in animals [10, 11]. Since that time vaccines and antitoxins had been developed against tetanus, diphtheria, anthrax, cholera, plague, typhoid, tuberculosis, and other infectious diseases by the 1930s. Subsequently, vaccines against childhood and other diseases ranging from polio to measles and mumps have been developed and have reduced illnesses and deaths from many infectious diseases. Vaccines have ranged from live attenuated forms developed by Pasteur and his colleagues [10] to tuberculosis bacilli by Calmette-Guérin in 1927. Vaccine preparation ranges from killed whole organisms to purified proteins or polysaccharides to recently genetically engineered vaccines [9]. Some organisms remain resistant to the development of effective vaccines such as human immunodeficiency virus (HIV) [12]. Nevertheless, major advances have been made in recent times with novel vaccines such as the one for malaria. Another major advance is the development of messenger RNA-based vaccines such as the ones directed against COVID-19 [13]. Many microbiologists have contributed to significant advances in this field. The biography of one of the most outstanding microbiologists of all times is summarized below.

Louis Pasteur (1822–1895) Louis Pasteur is probably one of the best-known scientists to the general public. He is often considered the “Father of Microbiology.” He was born in France on December 27, 1822. Although he did not receive training as a physician, some of the scientific advances in public health and in medical sciences that he made were simply extraordinary [14–17]. Pasteur started grade school in 1831 and was reported to be an average student. Although he was interested in learning, his major interests were in fishing and art. He attended secondary school in the College d’Arbois and in 1839 he began to study philosophy at the College Royal at Bescancon where he earned his Bachelor of Letters degree in 1840. He continued his studies in the sciences and special mathematics, but failed his examination in 1846. He later earned his Bachelor of Science in Mathematics degree in 1842. Later that year he took the entrance exam for the École Normale Supérieure, but his scores were not high enough for him to enroll. In 1843 he took the exam again, passed with good scores, and entered the École Normale Supérieure and graduated in 1845. He was appointed as a professor of physics in 1846. He then worked with the chemist Antoine Jerome Balard as a laboratory assistant. He submitted two theses in physics and chemistry. He served as a professor of physics at Lycées de Dijon and was subsequently made professor of chemistry at the University of Strasbourg. In 1852, he became the chair

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of chemistry at that same institution. After his appointment at Strasbourg, he married Marie Laurent and they had five children. As was common in this era, only two of the children survived into adulthood. Pasteur was promoted to dean of the faculty of sciences at the University of Lille in 1854. In 1858 he became the director of scientific studies at the École Normale Supérieure, a position that he held until 1867. He then became professor of geology, physics, and chemistry at the École Nationale Superieure des Beaux-Arts in 1863, but resigned in 1867 to become chair of organic chemistry at the Sorbonne. He went on to establish the Pasteur Institute in 1867 and was its director until 1895. It was in 1859 when Darwin published his masterpiece on evolution, The Origin of Species, that Pasteur performed experiments to disprove the concept of spontaneous generation of life. This theory suggested that air could cause spontaneous generation of living organisms in liquid. Pasteur was able to show that by eliminating mercury from experiments in which sterile water was used to generate microbes, that he could avoid contamination of the liquid and air and prevent what appeared to be spontaneous generation. In another set of experiments when he placed boiled liquid in a flask (killing all of the organisms) and allowed hot air to enter the flask, he found that organisms did not grow after the flask was closed. His experiments with a Swan-neck flask with sterilization of the humidity of the tube did not allow germs to get pass the bend in the Swan-neck flask. He was able to prevent development of organisms with these approaches also because the flasks remained sterile indefinitely [14, 15]. Pasteur performed experiments to show how the growth of microorganisms was mainly responsible for spoilage of wine, beer, and milk and he went on to develop the process of pasteurization by heating liquids between 60 and 100 °C to kill bacteria and molds which eliminated spoilage of these commercially important beverages [14, 15]. In some of his earlier work in chemistry, his studies of tartaric acid, he was able to show that the optical activity was related to the shape of the crystals and that asymmetry of the molecules of tartrate was responsible for distortion of the light providing a rational explanation for isomerism [16, 17]. Although he made important developments with animal vaccines for diseases such as chicken cholera and swine erysipelas, it was his work on rabies and anthrax that had the greatest impact on vaccine development for humans. The rabies vaccine was developed by Emile Roux, a colleague of Pasteur, who had tested it in dogs before it was finally used in humans. In 1885, Joseph Meister, a 9-year-old boy, who had been mauled by a rabid dog was treated by Pasteur with13 inoculations over a period of 11 days. Pasteur used viruses that were attenuated for a shortened period of time for each inoculation. Several months later, the youngster remained rabies-­ free and in good health. Pasteur’s notebooks suggested that Meister was not the first human that he had treated with the vaccine. However, he went on to treat 350 more patients by 1886 and only one person had developed rabies [18]. Pasteur’s development of a vaccine for anthrax was somewhat more controversial. In 1881, Pasteur had announced that he had successfully vaccinated sheep against anthrax. A public experiment was conducted in May 1881 at Pouilly-le-Fort in which a group of sheep and some cattle were divided into two groups to test

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Pasteur’s vaccine. One half of the animals were given the vaccine on two occasions, while the other half were not treated with the vaccine. All of the animals were injected with fresh virulent culture of anthrax bacillus. The results were analyzed in front of a large group of spectators. All of the vaccinated cows, sheep, and goats survived, while the unvaccinated animals became very ill and many died. Although Pasteur indicated that he used a “live vaccine,” other reports indicated that Pasteur had used a vaccine prepared by the method of Henry Toussaint. Toussaint was a veterinarian who had been a follower and admirer of Pasteur. He had developed the attenuated vaccine against anthrax using a chemical method that Pasteur allegedly did not believe in [19]. Pasteur is considered the “Father of Microbiology” for his many contributions to the field. He received many awards for his accomplishments. He received awards from the French Academy of Science and was elected to the French National Academy of Medicine. He also received awards from other European universities. The Institute Pasteur and the Université Louis-Pasteur were both named in his honor.

References 1. Procop GW, Church DL, Hall GS, Janda WM, Koneman EW, Schreckenberger PC, Woods GL. Introduction to microbiology. In: Procop GW, Church DL, Hall GS, Janda WM, Koneman EW, Schreckenberger PC, Woods GL, editors. Koneman’s color atlas and textbook of diagnostic microbiology. 7th ed. Philadelphia: Wolters Kluwer; 2017. p. 1–65. 2. Petitjean C, Deschamps P, Lopez-Garcia P, et  al. Rooting the domain archaea by phylogenomic analysis supports the foundation of the new kingdom Proteoarchaeota. Genome Biol Evol. 2014;7(1):191–204. 3. Procop GW, Church DL, Hall GS, Janda WM, Koneman EW, Schreckenberger PC, Woods GL. Diagnosis by immunologic methods. In: Procop GW, Church DL, Hall GS, Janda WM, Koneman EW, Schreckenberger PC, Woods GL, editors. Koneman’s color atlas and textbook of diagnostic microbiology. 7th ed. Philadelphia: Wolters Kluwer; 2017. p. 111–36. 4. Procop GW, Church DL, Hall GS, Janda WM, Koneman EW, Schreckenberger PC, Woods GL.  Molecular Microbiology. In: Procop GW, Church DL, Hall GS, Janda WM, Koneman EW, Schreckenberger PC, Woods GL, editors. Koneman’s color atlas and textbook of diagnostic microbiology. 7th ed. Philadelphia: Wolters Kluwer; 2017. p. 137–72. 5. McNicol AM, Farquhar MA. In situ hybridization and its diagnostic applications in pathology. J Pathol. 1997;182:250–61. 6. Hayden RT, Qian X, Roberts CD, et al. In situ hybridization for the identification of yeast-like organisms in tissue sections. Diag. Mol. Pathol. 2001;10:15–23. 7. Janssen GJ, Mooibroeck M, Idema J, et al. Rapid identification of bacteria in blood cultures by using fluorescently labeled oligonucleotide probes. J Clin Microbiol. 2000;38:814–7. 8. Xu J, Millar BC, Moore JE, et al. Employment of broad range 16s rRNA PCR to detect aetiologic agents from clinical specimens in patients with acute meningitis: rapid separation of 16s rRNA PCR amplification without the need for cloning. J Appl Microbiol. 2003;94:197–206. 9. Krishnamurthy T, Ross PL.  Rapid identification of bacteria by direct matrix-assisted laser desorption/ionization mass spectrometric analysis of whole cells. Rapid Commun Mass Spectrom. 1996;10:1992–6. 10. Plotkin S. History of vaccination. Proc Natl Acad Sci. 2014;111(34):12283–7. 11. Lombard M, Pastoret P-P, Moulin AM. A brief history of vaccines and vaccinations. Rev Sci Tech. 2007;26(1):29–48.

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12. Gray GE, Laher F, Lazarus E, et al. Approaches to preventative and therapeutic HIV vaccines. Curr Opin Virol. 2016;17:104–9. 13. Pascos S. Messenger RNA-based vaccines. Expert Opin Biol Ther. 2004;4(8):1285–94. 14. Feinstein S. Louis Pasteur: the father of microbiology. New York City: Enslow Publishers Inc; 2008. p. 1–128. 15. Bordenave G, et al. Microbes Infect. 2003;5:553–60. 16. Berche P.  Louis Pasteur from crystals of life to vaccination. Clin Microbiol Infect. 2012;18(Suppl 5):1–6. 17. Flack HD.  Louis Pasteur’s discovery of molecular clonality and spontaneous resolution in 1848 with a complete review of his crystallographic and chemical work. Acta Crystallogr A. 2009;65:371–89. 18. Jackson AC. Rabies: scientific basis of the disease and its management. 3rd ed. Amsterdam: Academic Press; 2013. 19. Chevallier-Jussiau N.  Henry Toussaint and Louis Pasteur. Rivalry over a vaccine. Hist Sci Med. 2010;44(1):55–64.

Chapter 16

Virology

Viruses are infectious agents that contain proteins and nucleic acids. The nucleic acids are usually either RNA or DNA, but not both. Viruses are dependent on other organisms for replication; some scientists have suggested that they are not truly living organisms [1]. They are submicroscopic in size measuring from a few nanometers to a few hundred nanometers in maximum length. Their shapes can vary from simple, helical, and icosahedral to more complex forms. Viruses can be visualized with the electron microscope with or without negative staining, so it was not until the late 1930s that scientist were able to visualize viruses after the development of the electron microscope. There are probably millions of types of viruses, but only about 9000 species have been identified to date. Viruses are thought to be one of the most abundant biological species on earth [2, 3]. Although viruses can infect animals, plants, fungi, and bacteria, diagnostic viral microbiology is focused on human diseases caused by infectious viruses. There are debates among biologists as to whether viruses constitute a true-life form. In favor of them being a true-life form is the observation that they have nucleic acids, either RNA or DNA; that they reproduce by generating new viruses; and that they can evolve through natural selection. However, they lack some of the features of true living forms such as cell structures and organelles. They are also totally dependent on their host for reproduction [2, 3]. Since viruses consist of DNA or RNA, they can encode the proteins that are an integral portion of their structure with the help of the host that they invade. They are surrounded by a capsid or protein coat which surrounds the nucleic acids. Some viruses have an envelope made up of lipids that surround the viral particles. Most medically important DNA viruses are double stranded, while most medically important RNA viruses are single stranded. DNA viruses are generally assembled in the nucleus of host cells, while RNA viruses are assembled in the cytoplasm of the host cell.

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Historically, the earliest means of identifying viruses in the microbiology lab was by the inoculation of animals or embryonic eggs. This was followed by cell culture methods to propagate the virus [1]. Electron microscopy was also used to identify virus by morphology with negative staining using heavy metals. Today, most of the earlier methods have been complemented by sophisticated techniques including next-generation nucleic acid sequencing [1]. The light microscope can be used to detect viral inclusions in infected cells. Detection of intracytoplasmic or intranuclear inclusions can be aided with antibodies directed against the specific virus as part of an immunohistochemical protocol. This approach has been used for a variety of viruses such as Herpes simplex, Varicella zoster, cytomegalovirus, adenovirus, human papilloma virus, as well as for BK and JC viruses. In situ hybridization techniques targeting nucleic acids rather than proteins are an alternative laboratory protocol that can be used to identify specific viruses in tissue sections. It is used less commonly than immunohistochemistry, because antibodies are more readily available commercially compared to in situ hybridization probes. Electron microscopy can be used to identify some well-­ characterized viruses such as Herpes simplex which has distinct diagnostic morphological features. Immunologic methods can also be used to identify viruses in the microbiology laboratory by detecting viral antigens. Direct immunofluorescence with highly specific antigens can be used to identify some viruses such as respiratory viruses, herpes viruses, human immunodeficiency virus, and many others [1]. Molecular techniques are the latest method used to characterize viruses today. Molecular methods can range from nucleic acid hybridization to polymerase chain reaction and genomic sequencing [1].

Some Common and Uncommon Viral Infections Orthomyxovirus These single-stranded RNA viruses include the influenza A, B, and C viruses. Influenza viruses produce a wide variety of respiratory infections which are characterized by sudden onset of fever, chills, headache, and dry cough with subsequent higher fevers, chills, and malaise. Influenza viral infections occur mainly in the winter months and mainly with influenza A and B, since influenza C is uncommon in humans. The main antigen is hemagglutinin which is responsible for attachment to the respiratory epithelium. Hemagglutinin is also an important target for the influenza virus vaccines that are offered annually to decrease illness and death from influenza viral infections. The antigen changes constantly due to antigenic drift and this often leads to recurring epidemics and pandemics [1].

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Paramyxovirus This single-stranded RNA virus is the etiology of many historical viral infections. Mumps virus infections lead to inflammation of the parotid salivary glands and to mild aseptic meningitis. The virus may also affect the pancreas, gonads, and less commonly the joints. The availability of a vaccine for mumps has decreased the incidence of infection, although there remains a few outbreaks each year [1].

Measles Virus (Rubeola) Although there are vaccines for measles, there are still occasional outbreaks of measles infections. Infected patients may have a diagnostic rash of antigen and antibody complexes. A molecular diagnostic test is frequently used to confirm the diagnosis [4].

Respiratory Syncytial Virus Respiratory syncytial virus is associated with recurrent respiratory infections, mostly in infants and children. They may occasionally occur in adults. Infections spread by larger particles droplets and this is a major cause of nosocomial infections (infections occurring in the hospital).

Picornavirus These viruses include rhinovirus, the cause of the common cold, and enteroviruses such as poliovirus and coxsackievirus. Immunologic diagnosis of rhinovirus is not practical, because there are so many serotypes and rhinovirus infection is usually not life-threatening [1]. Enteroviruses are associated with myocarditis and epicarditis. Infection with enteroviruses also produce febrile diseases with a rash.

Gastroenteritis Viruses Viral respiratory disease is the most common cause of morbidity in the USA and this is followed by viral gastroenteritis. Viral gastroenteritis is estimated to cause about 11% of cases of gastroenteritis per year [1]. Fortunately, mortality is minimal for gastroenteritis. Affected patients usually have vomiting or diarrhea. However,

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bloody diarrhea is rare. Symptoms should last less than 10 days and if longer another etiology should be investigated [1, 5].

Uncommon Viral Infections Rhabdovirus There are some RNA viruses that are associated with rare diseases in humans such as rabies. Rhabdoviruses like the one causing rabies are distributed worldwide [1, 6]. The diagnosis is made by detection of viral antigens or nucleic acids in clinical specimens from humans. Immunofluorescence detection is commonly used, while cultures are uncommon. Rabies is endemic in wild animals. Human exposure is rare and mainly occurs from bats and hunting dogs. Rare cases of transmission in humans associated with organ transplantation including corneal transplant have been reported [6].

Retrovirus These RNA viruses transcribe RNA into DNA using reverse transcriptase enzyme. These viruses are the cause of T-cell lymphocytic lymphomas, but they have a larger role in human history as the cause of acquired immunodeficiency syndrome (AIDS) and human immunodeficiency disease (HIV). Human immunodeficiency virus was first described in the USA in 1981. The etiology was first linked to drug injections in drug users and gay men. The virus was isolated in 1983. HIV-1 is distributed worldwide, while HIV-2 is encountered mainly in sub-Saharan Africa. HIV-1 targets the CD4+ lymphocytes. The virus may also be sequestered in macrophages [7]. After infection, patients develop a transient febrile illness, lymphadenopathy, and pharyngitis. They may also develop a diffuse rash. The diagnosis can be confirmed by serological detection of the p24 antigen or by detecting viral nucleic acids. Antibodies usually develop weeks to months after infection and detecting these is very useful in supporting the diagnosis. The onset of chronic illness is usually linked to increases in the amount of virus in the blood as well as in the mononuclear cells in the body. HIV is a pandemic, having killed more than 39 million people by 2013. About 1.5 million people died from HIV-related causes in 2013 and there were 25 million people living with HIV [1]. Individuals from sub-Saharan Africa have been most affected with HIV. HIV-­ infected persons may develop a wide spectrum of opportunistic infections because of their immunosuppression such as candidiasis, bacillary angiomatosis, oral hairy leukoplakia, Herpes zoster, Mycobacterium avium complex, pneumocystis pneumonia, progressive multifocal leukoencephalopathy, and others [1]. They may also develop malignancies including invasive cervical cancer, lymphomas such as

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Burkett’s lymphoma and Kaposi’s sarcoma, a vascular cancer of soft tissues [1]. Retroviral treatment has been relatively successful in controlling HIV infections.

Coronavirus Classical coronaviruses cause the common cold [1]. For studies in the laboratory, organ culture can be used for isolating coronavirus causing the common cold, but this is rarely if ever done. In 2003, a novel coronavirus was described. This was associated with a new respiratory virus in China. It had been isolated from the Chinese ferret badgers and was designated as severe acute respiratory syndrome (SARS). It was an airborne virus that could be spread by small droplets of saliva similar to influenza virus [8]. Patients usually developed leukopenia and lymphopenia and were infectious for about 10  days from the start of the symptoms. Most affected individuals were adults between 25 and 70 years of age. However, in rare cases it could also affect children. SARS had a fatality rate of 10% and there were 8096 documented cases from 26 different countries [8]. Another coronavirus, Middle East respiratory syndrome coronavirus (MERS-COV) [9], was subsequently reported. This second novel coronavirus was described in Saudi Arabia in 2012 and was endemic in countries around the Arabian Peninsula. This coronavirus was associated with a higher death rate, about 36.7%, and had spread to 19 countries. MERS was characterized by fever, cough, and dyspnea. Patients with MERS also developed leukopenia and lymphopenia [9].

SARS-CoV-2 (COV-19) Another coronavirus was discovered in Wuhan, Hubei Province, China in December 2019, SARS-CoV-2 (COV-19). Infection with this virus was associated with acute atypical respiratory disease. The disease was termed “COVID-19,” indicating the year that it was first encountered. It was found to be transmissible between people [10]. Elder patients appeared to be more susceptible to severe disease, while children seemed to have milder symptoms. The infection evolved into a pandemic in a few months. It was interesting in that affected individuals were contagious early on when they were asymptomatic [11, 12]. The virus was rapidly sequenced in China and the sequence was deposited into GenBank and made public on December 26, 2019. This allowed for the rapid development of testing for the virus and to start development of vaccines against COVID-19  in a relatively short period of a few months. In addition, there was rapid development of diagnostic tests in various countries. SARS-CoV-2 shares 79% sequence identity with SARS-CoV, the virus that caused a major outbreak in 2002–2003 [11]. The first cluster of cases with COVID-19 was identified by the South China Seafood Market, a “wet market” which sold a variety of live or freshly killed animals including poultry, bats, and

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snakes. COVID-19 was thought to have originated from a bat host, since there is an 88–96% sequence similarity to bat coronavirus [12]. Some people have suggested that the virus could have escaped from a nearby experimental virology laboratory in Wuhan, but this has not been documented. Patients with COVID-19 usually have fever, cough, dyspnea, and chest tightness and in many patients the initial illness is mild. However, as more patients needed to be hospitalized, the median age in one series was 49 [13]. The major symptoms included fever, cough, dyspnea and myalgia or fatigue. In an early series about 23% of patients were admitted to the intensive care unit and the mortality was 11% [13]. The most severely affected patients were older men with comorbidities such as obesity, diabetes, hypertension, or heart disease. About half of the early cases were associated with the seafood market [11]. As of March 2021, the pandemic had affected over 100 million people in over 210 countries and 2 million people had died of COVID-19 [12]. Among patients showing symptoms of COVID-19, 80% of patients had a mild illness, 14% had a severe illness, and 5% developed critical illnesses that needed intensive care treatment or mechanical ventilation assistance. The overall fatality rate from COVID-19 has been around 2% which is lower than SARS (10%) and MERS (37%). In spite of the lower mortality rate of this coronavirus, the high infection rate has resulted in many more fatalities than SARS and MERS combined [12]. There is also a high level of genetic variability and virus adaptability, so it is expected that SARS-CoV-2 variants such as alpha, beta, delta, and future variants will continue to emerge because of selective pressures. Early autopsy reports from patients dying of COVID-19 showed extensive inflammation in the pulmonary small airways and alveolar damage similar to patients dying of SARS and MERS [14]. Electron microscopic studies showed large numbers of viral particles in alveolar epithelial cells [14]. The lungs had decreased volumes and showed consolidation. Microscopic examination showed increased numbers of macrophages in the lungs, serous and fibrinous exudates, hemorrhage, diffuse alveolar lesions hyaline formation, and interstitial fibrosis. Fungal hyphae were noted in the lungs of some patients.

 he 1918 Influenza Pandemic and Comparison with the 2019 T COVID-19 Pandemic The 1918 pandemic was one of the worst pandemics in the world with respect to the number of lives lost. It occurred in three waves which included spring 1918, autumn 1918, and winter 1918–1919 [15]. It has been estimated that the pandemic infected over one third of the world’s population and killed about 50 million people [16, 17]. This pandemic was caused by influenza virus H1N1 and was the largest influenza pandemic on record. Three additional influenza pandemics have been recorded since then including the 1957 “Asian influenza pandemic,” the 1968 “Hong Kong” pandemic, and the 2009 “swine flu” pandemic. All three of these were relatively mild compared to the 1918 pandemic.

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The 1918 pandemic has been referred to erroneously as the “Spanish flu.” One reason for this misnomer was that because Spain was a neutral country during World War I, the press in that country printed a great deal of news about the pandemic. In contrast other countries in Europe that were at war like Germany, France, and the United Kingdom printed very little information about the pandemic, because they did not want to worry the soldiers in battle, so they suppressed the news about the pandemic in order not to discourage the fighting soldiers. The first case of influenza was reported in March 1918 in Kansas, the USA, in a military camp and could have been transported to Europe by American troops to battlefields in France and other places [18]. The geographic origin of the pandemic remains uncertain. The possibility of reassortment, which occurs when two influenza virus strains coinfect the same cell leading to the emergence of a new reassortment virus which contains a new constellation of genes from both strains such as the human and avian strains is one possibility. This reassortment occurred in the 1957 and 1968 pandemics [15]. The 2009 influenza pandemic occurred from reassortment of avian, human, and swine influenza viruses [15]. It has been suggested that the 1918 pandemic could have been directly introduced into the human population without reassortment from a single unidentified host [15]. However, the identity of the host has not been established. During the first wave of the pandemic, the infections were mild and mortality rates were relatively low. In the second wave in the fall of 1918, the rates were much higher with more mortality and virulence. The subsequent third wave was not as virulent as the second wave [15]. About 500 million people were infected during the pandemic with a case fatality rate of more than 2.5% [16]. Clinical and autopsy studies suggested that excessive influenza deaths (i.e., the deaths above background levels expected for influenza) were associated with two overlapping clinicopathologic features [16]. These included an acute aggressive bronchopneumonia with epithelial and microvascular necrosis, hemorrhage, and edema with varying histopathologic features [16]. At autopsy, some areas of the lungs were infected with bacteria such as staphylococcus pneumonia and Hemophilus influenza, while other areas not involved with bacteria. The second syndrome included acute respiratory distress-like syndromes (ARDS) with accumulation of watery bloody exudates in the lungs and bronchi [17–19]. Molecular analyses of the virus causing the 1918 influenza infection was performed more than 70 years after the pandemic. Fortunately, lung tissues were preserved in pathology laboratories including formalin-fixed paraffin-embedded materials that could be used for analysis of nucleic acids [20]. RNA analysis of a victim of the pandemic lung tissue was analyzed by sequencing fragments of viral RNA from the coding region of the influenza virus showed H1N1 influenza virus that infected humans and swine and not the avian subgroup [20]. Host factors involved with the influenza virus pandemic included the age of patients; surprisingly the average age of affected patients was much younger than would have been predicted with the 15–30 years of age more frequently infected than patients older than 30 years of age. It was believed that older patients probably had cross protection from antibodies acquired earlier in life from prior infections

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with influenza. CD8+ T cells were also noted to provide protection against severe disease in the 1957 and 2009 pandemics.

Comparison of 1918 and 2019 Pandemics Although the viruses involved in the 1918 and 2019 pandemics were different, it is thought that cytokine storm was important in the massive responses of both with increased mortality in both pandemics. In cytokine, storm inflammatory cells in the lungs and other sites produce excessive cytokines such as tumor necrosis factor which leads to massive recruitment of immune cells to the lungs and results in organ damage and death of inflammatory cells. Although the pathophysiology of cytokine storm is not clearly elucidated, it is thought that factors contributing to comorbidities such as diabetes, obesity, and hypertension contribute to this massive response. Some of the factors influencing poor outcomes in both pandemics were somewhat similar. For example, (A) Maritime quarantine or strict quarantine was shown to be important in the 1918 pandemic and it has been in the 2019 pandemic. Evidence from 1918 showed that mortality differences were influenced by strict quarantines. For example, there was a marked difference between mortality in American Samoa and Western Samoa, islands that were separated by around 100 km. There was strict enforcement of quarantine in American Samoa compared to Western Samoa where there was no quarantine. About a quarter of the population from Western Samoa died from the pandemic compared to a lot less in American Samoa [15]. In addition to social distancing, handwashing was also important in the 1918 pandemic as it currently is in the 2019 pandemic. The type of face masks used in the 1918 pandemic were usually made of gauze which were not properly made and properly fitted, so these were usually not quite as effective in reducing infection. In contrast, for the 2019 pandemic a wide variety of masks were available ranging from cloth masks which were least effective to surgical masks and N-95 masks which were usually properly fitted and very effective. These masks along with social distancing helped to reduce transmissibility by reducing respiratory particles and were most effective when compliance was very high [21, 22]. Two unique features of the 2019 pandemic that was not reported in the previous pandemic was the chronic COVID syndrome in which patients continued to have signs and symptoms of infection with the virus many months after the original infection [23] and multisystem inflammatory syndrome in children and adolescents in which pediatric patients developed multiorgan involvement with COVID-19 many weeks and months after the original infections [24]. Although vaccines for some infectious diseases had been available for more than 100 years in 1918, this was not the case for the 1918 pandemic. In contrast, vaccines became available relatively early in the 2019 pandemic. This was led largely by the rapid advances in technology and science during the ensuing 100 years. The rapid sequencing of the COVID-2019 genome and the wide availability of this data facilitated the quick advances in vaccine production [24]. At one point, there were over

Viruses and Cancer

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40 vaccines undergoing clinical evaluation and ten of these were in Phase III clinical trials. Three of these had ended Phase III trials with positive results [25]. The free availability of basic science data had led to the creation of vaccines founded on innovative platforms. The vaccines were directed toward well-defined targets and included mRNA-based vaccines, vaccines based on DNA targets and SARS CoV-2 protein targets. Many of these vaccines were developed in a relatively short period of time compared to historical vaccine development. Several of the vaccines proved to be very effective in rapidly immunizing populations in many countries.

Viruses and Cancer Viruses have been shown to cause cancer in humans and other animal species. Cancer-causing viruses include both DNA and RNA viruses, so it is not restricted to one subtype of viruses. Viruses causing cancers in humans include human papilloma virus (HPV) associated with cancers of the cervix, oropharynx, anus, and penis; hepatitis viruses including hepatitis B and hepatitis C associated with cancer of the liver; Epstein–Barr virus associated with cancer of the nasopharynx, Burkett’s lymphoma, and B-cell lymphoproliferative disorders; human lymphotropic virus leading to adult T-cell leukemia, herpesvirus associated with Kaposi’s sarcoma, especially in patients with HIV; and Merkel cell polyoma virus associated with a rare form of skin cancer with neuroendocrine differentiation called Merkel cell carcinoma. Cervical cancer caused by human papilloma virus (HPV) is diagnosed in around 500,000 individuals around the world each year and results in 300,000 deaths annually [25]. High-risk HPV subtypes are the cause of the disease in most cases. The two main subtypes of HPV include HPV 16 and 18, but there are another 12 high-­ risk HPV types that may be related to the etiology of this cancer in rare cases [26, 27]. More than 90% of the cervical cancers occur in low- and middle-income countries [26], so cervical pap smear screening is an excellent method to screen and detect this disease early in its development. Oropharyngeal squamous cell cancer is also associated with high-risk HPV subtypes. High-risk HPV 16 is the most common subtype and accounts for 80% of cases [28, 29]. Unlike cervical cancer, which is the most common in low- and middle-­income countries, oropharyngeal carcinoma related to HPV is most common in developed countries including the United Kingdom, France, and the USA [28, 29]. In addition to detection of HPV 16 and 18, E6 and E7 mRNA viral genes, immunohistochemical detection of p16 in at least 70% of the cancer cells is another way of diagnosing HPV 16 and 18 in the surgical pathology laboratory [30]. HPV vaccination has also been effective in reducing the prevalence of HPV infections, since its initiation in Australia in 2006 followed by Europe and the USA. After the first 6 years of Gardasil vaccination in the USA, the prevalence of HPV infection among girls 14–24 years of age had decreased significantly [31].

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References 1. Procop GW, Church DL, Hall GS, Janda WM, Koneman EW, Schreckenberger PC, Woods GL.  Diagnosis of infections caused by viruses, chlamydia/Chlamydophila, rickettsia, and related organisms. In: Procop GW, Church DL, Hall GS, Janda WM, Koneman EW, Schreckenberger PC, Woods GL, editors. Koneman’s color atlas and textbook of diagnostic microbiology. 7th ed. Philadelphia: Wolters Kluwer; 2017. p. 1500–86. 2. Cann AJ. Principles of molecular virology. 5th ed. London: Academic Press; 2011. 3. Calisher CCH, Horzinek MC, editors. 100 years of virology: the birth and growth of a discipline1999. New York: Springer; 1999. 4. Rota PA, Liffick SL, Rota JS, et al. Molecular epidemiology of measles viruses in the United States 1997–2001. Emerg Infect Dis. 2002;8:902–8. 5. Dolin R, Treanor JJ, Madore HP.  Novel agents of viral enteritis in humans. J Infect Dis. 1987;155:355–76. 6. Houff SA, Burton RC, Wilson RW, et al. Human to human transmission of rabies virus by corneal transplant. N Engl J Med. 1979;300:603–4. 7. Cohen OJ, et al. Pathogenesis and medical aspects of HIV-1infection. In: Knipe DM, Howley PM, editors. Fields virology. 4th ed. Philadelphia: Lippincott; 2001. p. 2043–94. 8. Skowroronski DM, Astelll C, Barnham RC, et al. Severe acute respiratory syndrome (SARS) a year in review. Ann. Rev. Med. 2005;56:157–81. 9. Rha B, Rudd J, Feiten D, et al. Update on the epidemiology of Middle East respiratory syndrome coronavirus (MERS-Cor) infection and guidance to the public, clinicians and public health authorities. January 2015. Morb. Mortal. Weekly Rep. 2015;64:61–2. 10. Yuki K, Fujiogi M, Koutsogiannaki S. COVID-19. Pathophysiology: a review. Clin Immunol. 2020;215:108427. 11. Lake MA. What we know so far: COVID-19 current clinical knowledge and research. Clin. Med. (Lond.). 2020;20(2):124–7. 12. Wang C, Wang Z, Wang G, et al. COVID-19 in early 2021. Current status and looking forward. Signal Transduct Target Ther. 2021;6(1):114. 13. Huang C, Wang Y, Li X, et al. Clinical features of patients infected with 2019 novel coronavirus in Wuhan. China Lancet. 2020;395(10223):497–506. 14. Yao XH, Luo T, Shi Y, et al. A cohort autopsy study defines COVID-19 systemic pathogenesis. Cell Res. 2021;31(8):836–46. 15. Short KP, Kedzierska K, van de Sandt KE. Back to the future: lessons learned from the 1918 influenza pandemic. Front. Cell Infect. Microbiol. 2018;8:343. 16. Morens DM, Fauci AS. The 1918 influenza pandemic: insights for the 21st century. J. Inf. Dis. 2007;195:1018–28. 17. Jester B, Uyeki TM, Jernigan DB, et al. Historical and clinical aspects of the 1918 H1N1 pandemic in the United States. Virology. 2004;527:32–7. 18. Barry JM. The site of origin of the 1918 influenza pandemic and its public health implications. J Transl Med. 2004;2(1):3. 19. Niall AS, Johnson NP, Mueller J. Updating the accounts: global mortality of the 1918–1919 “Spanish” flu influenza pandemic. Bull Hist Med. 2002;76:105–15. 20. Taubenbeger JK, Reid AH, Krafft AE, et al. Initial genetic characteristics of the 1918 “Spanish” influenza virus. Science. 1997;275:1793–6. 21. Du Z, Xu X, Wang L, et al. Effects of proactive spatial distancing on COVID-19 outbreaks in 58 cities China. Emerg Infect Dis. 2020;26(9):2267–9. 22. Howard J, Huang A, Li Z, et al. An evidence review of the face masks against COVID-19. PNAS. 2021;118(4):1–12. 23. Baig AM. Chronic COVID syndrome: need for an appropriate medical terminology for long-­ COVID and COVID-long-haulers. J Med Virol. 2020;93(5):2555–6.

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24. Alsohime F, Temsah M-H, Al-Nemri AM, et  al. COVID-19 infection, prevalence in pediatric population: etiology, clinical presentation and outcome. J Infect Public Health. 2020;13(12):1791–6. 25. Forni G, Mantovani A. COVID-19 vaccines: where we stand and challenges ahead. Cell Death Differ. 2021;28:626–39. 26. Cohen PA, Jhingran A, Oaknin A, et al. Cervical cancer. Lancet. 2019;393(10167):169–82. 27. Burd EM. Human papillomavirus and cervical cancer. Clin. Microbiol. Rev. 2003;16:1–17. 28. Budu VA, Decuseara T, Balica NC, et al. The role of HPV infection in oropharyngeal cancer. Romanian J Morphol Embryol. 2019;60(3):769–73. 29. Marur S, D’Souza G, Westra WH, et al. HPV-associated head and neck cancer: a virus-related cancer epidemic. Lancet Oncol. 2010;11(8):781–9. 30. Westra WH. Detection of human papillomavirus (HPV) in clinical samples: evolving methods and strategies for the accurate determination of HPV status of head and neck carcinomas. Oral Oncol. 2014;50(9):771–9. 31. Markovitz LE, Liu G, Hariri S, et al. Prevalence of HPV after introduction of the vaccination program in the United States. Pediatrics. 2016;137(3):e20151968.

Chapter 17

Bacteriology

Bacteria are some of the oldest living organisms on earth. These prokaryotic organisms have been on earth for around 3.5 million years [1]. Bacteria are abundantly present in the human body. Although it was previously estimated that the ratio of bacteria to human cells in the body was around 10 to 1, recent estimates have suggested that there are probably similar numbers of bacteria and human cells which would indicate that there are around 3  ×  1013 cells and bacteria [2]. The highest number of bacteria are in the colon (1014) followed by dental plaques 1(012), ileum, saliva, and skin with lower numbers in the stomach (107) and upper small intestine [3]. There are about 1000 bacterial species living in the human gut and most people share a core of 50–100 bacterial species and a core microbiome of 6000 functional gene groups [3]. Thus, the human gut microbiome is very important for many body functions in health and in diseases [4, 5]. Bacteria have distinct shapes that are used in their classification. These include bacilli, cocci, and spirilli. They range in size from 0.5 to 2.0 microns [6]. Prokaryotic organisms like bacteria have a single chromosome or nuceleoside that is not encased in a nuclear membrane. The subcellular organelles of eukaryotes probably evolved from prokaryotic organisms. Bacteria can be separated by staining into two major groups, Gram-positive and Gram-negative organisms. The gram stain distinguishes bacteria by their cell wall structures. Gram-positive bacterial cell wall is composed of peptidoglycans. The cell wall also contains multiple proteins and polysaccharides and polymers of rabitol and glycerol (teichoic acids). The cell wall of Gram-negative bacteria is thinner than that of that of Gram-positive organisms, but is more complex. The acid-fast bacteria such as Mycobacteria have abundant lipid make up about 60% of the dry weight of the organism. In addition to gram stains and acid-fast stains, some other methods used to characterize bacteria in the clinical microbiology laboratory include A. Cell morphology such as the cell size and shape of bacteria. © The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 R. V. Lloyd, Pathology: Historical and Contemporary Aspects, https://doi.org/10.1007/978-3-031-39554-3_17

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B. Motility in wet preparation when studied under the microscope. C. Rate of growth of the bacteria in the laboratory such as normal growth in 1–2 days versus slow growth requiring a few days as with Mycobacteria. D. Requirement of oxygen for growth—aerobic organisms versus anaerobic organisms not requiring oxygen and facultative organisms that can grow in the presence or absence of oxygen. E. Optimal temperature for growth—usually 35–37 °C. F. Molecular genetic analyses representing more novel methods to characterize bacteria such as polymerase chain reaction, in situ hybridization, and DNA based-sequencing. G. Proteomic analysis instead of the traditional biochemical analyses such as MALDI-TOF which is available commercially. Immunizations are available for a small group of bacteria that infect humans. Some of these vaccinations are given to pediatric patients, while others such as Streptococcus pneumoniae are given to older adults or immunosuppressed patients. Other immunizations include Corynebacterium diphtheriae for diphtheria, Clostridium tetani for tetanus, Bordetella pertussis for whooping cough, Haemophilus influenzae for meningitis, Streptococcus pneumoniae for pneumonia and meningitis, Neisseria meningitidis for meningitis, Salmonella typhi for typhoid, Mycobacterium tuberculosis for TB, Bacillus anthracis for anthrax, Yersinia pestis for bubonic plague, and Vibrio cholerae for cholera. The latter two organisms have been historically responsible for devastating pandemics. A few examples of bacterial infections having a major effect on humans are discussed below.

Tuberculosis Tuberculosis (TB) remains one of the world’s deadliest infectious diseases with around 9.6 million cases and 1.5 million deaths in 2014 [8]. TB had been detected in ancient Egypt, Pre-Columbian America, and Neolithic Europe. Polymerase chain reaction analysis of mummies has detected M. tuberculosis in mummified tissues and bones. Countries with the highest number of cases today include India, China, and South Africa. The lowest rates are present in the USA (around 10,000 new cases in 2013) [7]. There is a much higher percentage of cases in foreign-born individuals in the USA. Recently acquired infections of TB may follow the classical slowly progressive disease course of secondary disease or they may be rapidly progressive with military TB-type spread. This is especially true for immunosuppressed patients such as those with AIDS. Respiratory precautions are needed in all cases of TB such as the use of positive pressure rooms for the patients. Persons taking care of patients as well as laboratory personnel who handle the clinical specimens from these patients are all at risk for infection. Universal precautions and handling specimens in laminar flow hoods are definitely needed [8].

Infection with Spirochetes

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Fig. 17.1  Histochemical stain for tuberculous bacteria using the Ziehl–Neelsen stain. The red staining bodies (arrow) represent positive staining for mycobacteria

For collection of specimens for testing in the hospital, TB can be recovered from respiratory specimens, bronchial biopsies, urine, feces, blood, or cerebrospinal fluid. Deep-needle aspiration biopsies of most organs may be another source of specimens for laboratory analysis. Acid-fast staining relies on the high lipid content of the cell wall binding to fuchsin dye which is not removed or destroyed by acid alcohol (Fig.  17.1). The staining pattern in addition to the size and shape of the organism are important diagnostic pattern on microscopic examination [6]. Molecular analysis with nucleic acid testing for MTB is very sensitive and allows for earlier diagnosis and decreased periods of infectiousness before the start of drug treatment. DNA sequencing can be used to accurately classify the MTB. Gas-liquid chromatography for analysis of long-chain fatty acids has also been used to characterize Mycobacteria. In addition, high-performance liquid chromatography with fluorescence detection has also been used to detect Mycobacteria. In addition to MTB, Mycobacterium bovis from cows may also infect humans as well as bovine species. Because multidrug resistance to MTB may develop rapidly, it is usually recommended that prompt and accurate susceptibility testing be performed on the first isolate of Mycobcterial TB that is recovered from the patient. It is also recommended that repeat sensitivity testing be performed after 3 months of therapy [6].

Infection with Spirochetes Treponema pallidum species, the etiologic agent of syphilis, has been a major cause of sexually transmitted disease for many centuries [9]. Spirochetes are long Gram-­ negative bacteria with helical coiling measuring 0.1–3 microns in diameter and 5–120 microns in length. They are very motile and have a corkscrew appearance.

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Syphilis was in the Americas for a long period of time, but did not appear in the Old World until around 1500. It has been assumed that it was brought from the Americas to the Old World by Columbus and his crew [10]. Syphilis has made a major comeback in the latter part of the twentieth century partly due to coinfection with HIV [11]. Historically, bone changes in skeletons thought to be due to syphilis had been detected in New World skeletons [12], but similar lesions had also been reported in pre-Columbian skeletons from the Old World [12]. Syphilis may be transmitted by various means including direct inoculation into the vascular system by shared needles or by transfusions. However, sexual contact with an infected person remains the most common way of contacting the disease [9]. Men are more likely to be diagnosed during the primary stage, while women are more often diagnosed in the latent phase later on. Congenital syphilis can develop by transmission of treponemes to the infant via the placenta [9]. The incubation period for syphilis is 3–90 days with an average of 3 weeks. Patients with T. pallidum and HIV are more likely to present with secondary syphilis. These individuals have an increased risk of developing neurosyphilis. Syphilis has been called “the great imitator” because of its varied and complex nature of its clinical manifestations and for the many other diseases that it may imitate. All positive cases of syphilis should be reported to public health for contact tracing and record keeping [9]. Rapid commercial testing is available for syphilis. However, several traditional tests are also available such as the fluorescent treponemal absorption assay and the microagglutination assay [9]. Notably, molecular testing has become more popular for detecting spirochetes in tissues and body fluids [13].

Lyme Disease Lyme disease is caused by the spirochete Borrelia burgdorferi. B. burgdorferi is transmitted from infected Ixodes ticks to humans. Other animals that may be involved in its transmission include deer, cows, rabbits, and squirrels depending on the time of the year and season [9]. Lyme disease is a multisystem inflammatory disease involving the skin, joints, heart, and central nervous system. Early descriptions in the USA in the 1970s in Connecticut were associated with an epidemic of arthritis [14]. Lyme arthritis was usually preceded by a distinct skin rash. Lyme disease is the most common vector-borne infection in the USA.  It may also be acquired congenitally, although it has not been associated with blood transfusions [15]. Laboratory diagnosis is made by isolation of B. burgdorfi from a clinical specimen or by detection of diagnostic levels of IgM or IgG antibodies to B. burgdorferi in the serum or cerebrospinal fluid [9]. Lyme disease is the most commonly reported vector-borne disease in the USA as well as in Europe. In the USA, most cases are in the North East and North Central regions of the country. However, one can encounter Lyme disease in almost every state in the USA. The first two disease states of

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Lyme disease occur within weeks or months after Borrelia infection. The third stage can occur several months or years later. When the skin lesion is not present, Lyme disease can be very difficult to diagnose because the symptoms are not specific; they may involve malaise, headaches, musculoskeletal pain, fatigue, and nonspecific neurological symptoms such as sleep disturbances. Laboratory diagnosis includes serological testing for specific antibodies directed against Borrelia antigens. Diagnosis may also be made by culture of the spirochete or demonstration of the spirochete in tissues with immunologic or molecular techniques. Diagnosis can also be made by microscopic detection of the organism or by molecular analysis of the species-specific nucleic acids [9].

Yersinia- and Vibrio Cholerae-Associated Historical Pandemics Organisms associated with Yersiniae species causing human plague and Vibrio cholerae as the agent for Asiatic cholera causing severe diarrhea have affected humans for centuries [16]. Yersinia consists of three species including Yersinia pestis, Yersinia pseudotuberculosis, and Yersinia enterocolitica. These are the main pathogens among the 17 species of Yersinia. Yersinia pestis or plague is an infectious disease from ancient times. It has a flea-rodent-flea life cycle and causes bubonic plague in humans. Yersinia pestis is endemic in some rodents including rats, ground squirrels, prairie dogs, mice, and rabbits. Geographically, there is an urban plague which is maintained in the urban rat population and sylvatic plague which is endemic in 17 western states in the USA. Transmission is usually from rodent to rodent or rodent to humans by fleas. In the USA, most human cases are in the Southwestern New Mexico to Colorado and Utah or in the Pacific Coast in California, Oregon, and parts of Nevada. Cats can carry the fleas into households and pass it on directly to humans. Travelers may become infected and bring the disease to areas where it is not endemic as a peripatetic plague. Clinical forms of plague include bubonic, septicemic, and pneumonic plague. The latter two forms are usually secondary to bubonic plague which is manifested by swelling of lymph nodes in the inguinal, cervical, and axillary regions. The bacteria can disperse hematogenously to other tissues leading to endotoxic shock and dark discoloration of the arms and legs. Laboratory diagnosis of the aspirates from lymph nodes usually show Gram-­ negative bacteria. Wright–Giemsa stains of blood smears show the characteristic bipolar staining of Yersinia. The colonies are usually slow growing, but a rapid diagnostic test with monoclonal antibodies can detect a protein specific for Yersinia pestis and can yield results in about 15 min [17, 18]. Yersinia pestis has been the bacterial infection associated with highest death rates in history [18, 19]. These plagues have included the Plague of Justinian from 541–544 AD caused by Yersinia pestis associated with fleas and wild rodents. It was named after the Roman Emperor from the sixth century. This plague resulted in more than 100 million deaths in the Roman Empire with many of these in

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Constantinople [18]. Microbial DNA isolated from dental pulp has been helpful in documenting the organism. The bacteria are trapped in the teeth and dental pulp early on in the infection during the course of bacteremia and can be isolated from teeth of preserved victims [18]. The second plague pandemic was the Black Death (1347–1351) pandemic which originated in East Asia and then spread to Central Asia and to Europe. This plague led to the death of between 7 and 28 million Europeans. This represented 30–40% of the European population at that time. The third major pandemic caused by Yersinia started in Yunnan, China and more than 10 million people died between 1896 and 1918 just when the viral pandemic due to influenza was beginning. Subsequently, several epidemics developed in many countries until the 1950s when the pandemic ended. Antibiotics such as streptomycin and doxycycline have been effective against this plague. Vibrio cholerae has been the cause of Asiatic cholera in historical pandemics. The infection is usually associated with severe diarrheal disease. Although there are 35 Vibrio species (mainly marine vibrio species), only 11 species are associated with human diseases. The Americas have been free of cholera historically, but the disease spread to the New World in 1991 after cholera became endemic. Laboratory studies have shown that in Vibrio cholerae, the agent causing epidemic and pandemic cholera in humans, there are differences in the cell wall composition and this is helpful in serotyping. All groups have a flagellar antigen termed the H antigen and all pandemic forms agglutinate to a single antiserum designated as O1. The El TOR strain, which was isolated in Egypt, is thought to be responsible for most of the current epidemic outbreaks. The O1 has been reported to be hardier and more capable of surviving in the environment. Vibrio cholerae produces a toxin that interrupts normal intraluminal exchange of water and electrolytes. The toxigenic strains produce a toxin that bind to the receptors on epithelial cells and activate adenyl cyclase leading to hypersecretion of salt and water with the characteristic “rice water” diarrhea of cholera. There has been seven or eight pandemics of cholera in recorded history [18, 19]. The first recorded epidemic started in 1817 and was from contaminated water, as were the second through the fifth pandemics between 1827 and 1896. The sixth pandemic was from 1899 to 1923. The seventh pandemic started in Indonesia and has been ongoing [18, 19]. Cholera infection is associated with acute watery diarrhea. The disease is confirmed by detection of Vibrio cholerae in stool samples by culture or by molecular testing. A rapid diagnostic test is also available. Patients are treated with oral rehydration or intravenous fluids and antibiotics such as doxycycline and tetracycline. Today, cholera outbreaks are associated with natural disasters, wars, and refugee camps with poor hygienic conditions. For example, the epidemic in Haiti in 2010–2012 occurred after the earthquake of January 2010 when over 100,000 people perished from the earthquake [20].

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References 1. DiGiulvo M.  The universal ancestor and the ancestor of bacteria were hyperthermophiles. J. Mol. Evol. 2003;57(6):721–30. 2. Sender R, Fuchs S, Milo R. Revised estimates for the number of human and bacteria cells in the body. PLoS Biol. 2016;14(8):e1002533. 3. Zhu B, Wang X, Li L. Human gut microbiome: the second genome of human body. Prot. Cell. 2010;1:718–25. 4. Arumgam M, Raes J, Pelletier E.  Enteroypes of the human gut microbiome. Nature. 2011;473:174–80. 5. Can IPD.  Human gut microbiome: hopes, threats and promises. Gut. 2018;67(9):1716–25. https://doi.org/10.1136/gutjnl-­2018316723. 6. Procop GW, Church DL, Hall GS, Janda WM, Koneman EW, Schreckenberger PC, Woods GL. Medical bacteriology: taxonomy, mophology, physiology, and virulence. In: Procop GW, Church DL, Hall GS, Janda WM, Koneman EW, Schreckenberger PC, Woods GL, editors. Koneman’s color atlas and textbook of diagnostic microbiology. 7th ed. Philadelphia: Wolters Kluwer; 2017. p. 173–212. 7. Procop GW, Church DL, Hall GS, Janda WM, Koneman EW, Schreckenberger PC, Woods GL.  Mycobacteria. In: Procop GW, Church DL, Hall GS, Janda WM, Koneman EW, Schreckenberger PC, Woods GL, editors. Koneman’s color atlas and textbook of diagnostic microbiology. 7th ed. Philadelphia: Wolters Kluwer; 2017. p. 1219–68. 8. Donoghue HD. Paleomicrobiology of human tuberculosis. Microbiol. Spectr. 2016;4(4):10. 9. Procop GW, Church DL, Hall GS, Janda WM, Koneman EW, Schreckenberger PC, Woods GL. The spirochetal infections. In: Procop GW, Church DL, Hall GS, Janda WM, Koneman EW, Schreckenberger PC, Woods GL, editors. Koneman’s color atlas and textbook of diagnostic microbiology. 7th ed. Philadelphia: Wolters Kluwer; 2017. p. 1269–321. 10. Crosby AW Jr. The early history of syphilis: a reappraisal. Am Anthropol. 1969;71(2):218–27. 11. Eaton M.  Syphilis and HIV: old and new faces aligned against us. Curr Infect Dis Rep. 2009;11:157–62. 12. Rothschild BM, Calderon FL, Coppa A, et  al. First European exposure to syphilis. The Dominican Republic at the time of Columbian contact. Clin Infect Dis. 2000;31:936–41. 13. Centurion-Lara A, Castro C, Schaffer JM, et al. Detection of Treponema pallidum by a sensitive reverse transcriptase PCR. J Clin Microbiol. 1997;35:1348–52. 14. Steens AC, Malawista SE, Snydman DD, et  al. Lyme arthritis: an epidemic of oligoarticular arthritis in children and adults in three consecutive communities. Arthritis Rheum. 1997;29:7–17. 15. Shapiro ED. Lyme disease. Adv Exp Med Biol. 2008;609:185–95. 16. Procop GW, Church DL, Hall GS, Janda WM, Koneman EW, Schreckenberger PC, Woods GL. The enterobacteriaceae. In: Procop GW, Church DL, Hall GS, Janda WM, Koneman EW, Schreckenberger PC, Woods GL, editors. Koneman’s color atlas and textbook of diagnostic microbiology. 7th ed. Philadelphia: Wolters Kluwer; 2017. p. 213–315. 17. Procop GW, Church DL, Hall GS, Janda WM, Koneman EW, Schreckenberger PC, Woods GL. Curved gram negative bacilli and oxidase-positive fermenters. In: Procop GW, Church DL, Hall GS, Janda WM, Koneman EW, Schreckenberger PC, Woods GL, editors. Koneman’s color atlas and textbook of diagnostic microbiology. 7th ed. Philadelphia: Wolters Kluwer; 2017. p. 432–71. 18. Chanteau S, Rahralison I, Ralafiarisoa L, et al. Development and testing of a rapid diagnostic test for bubonic and pneumonic plague. Lancet. 2003;361(9533):211–6. 19. Piret J, Boivin G. Pandemics throughout history. Front Microbiol. 2021;11:631736. 20. Hoiby N. Pandemics: past, present and future. APMIS. 2021;129(7):352–71.

Chapter 18

Mycology and Parasitology

Fungal Infections Fungal infections include a wide variety of diseases caused by fungi. A wide spectrum of fungi can lead to fungal infections in humans. Fungi that exist as single cells and reproduce by budding are known as yeasts. Molds are fungi that exist as multiple cells and form a filamentous mycelium. Fungi reproduce though the production of spores which can be derived sexually or asexually [1]. A team approach is ideal for the diagnosis of fungal infections. This team consist of the microbiologists who is the laboratory expert in culturing and identifying the fungus; the clinician is familiar with the patient’s history, signs, and symptoms and these may lead them to suspect a fungal infection in the appropriate clinical setting. The surgical pathologist is skilled in recognizing fungal organisms in tissue sections or cytological preparations based on the morphological appearances. The clinician should notify the microbiologist and the surgical pathologist when a fungal infection is suspected based on the clinical information when a biopsy or other specimen type is submitted to the pathology laboratory. Fungal infections are most common in infants and elderly patients as well as in patients that are immunosuppressed. These may include patients with HIV infection, those receiving organ transplants, patients with cancers, or others that are immunosuppressed because of autoimmune diseases such as lupus erythematosus or other collagen vascular diseases. Patients on high-dose corticosteroids that can suppress the immune system are also more susceptible to fungal infection. Travel to specific areas in which some fungi are endemic is also important in the work-up of patients with possible fungal infection. Another source of fungal infections that should be kept in mind are opportunistic infections, that is, presence of some fungi that usually do not cause infections, but can become infectious when conditions in the patient’s health or in the fungus changes and the organism becomes more virulent. © The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 R. V. Lloyd, Pathology: Historical and Contemporary Aspects, https://doi.org/10.1007/978-3-031-39554-3_18

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Fungal identification in the laboratory relies on recognizing colony morphology and specific microscopic features as well as on biochemical testing. Mass spectrometry (MALDI-TOF) has been used in recent years to identify some fungi in the clinical laboratory. Molecular approaches such as DNA sequencing in special regions (D2) is also part of recent laboratory approaches used to identify fungi [2]. In the laboratory, fungal specimens should be identified as quickly as possible, especially with the yeast forms. The use of wet mounts and smears are the preferred methods of morphological identification, since direct microscopic examination is rapid and this may be the first direct evidence to document a fungal infection [1]. However, the laboratory worker should be experienced in fungal morphology to avoid making gross errors. For incubation of fungi, a 30 °C oven is used and the earlier practice of incubating a second specimen at 35 °C for yeast form recovery in dimorphic fungi is rarely used today, because it is not cost-effective and more sophisticated molecular techniques are now available [1]. A few of the common infectious fungi encountered in the laboratory are summarized below:

Candida Species Candida species is the most common clinical fungal infection. Candida infections are often seen in patients with cancer such as leukemias and other malignancies in which there is a decreased white blood cell count. In older patient, there is increased morbidity and mortality associated with Candidiasis such as with mucocutaneous candidiasis. Neonates that are critically ill are also at increased risk for Candidiasis from nosocomial infections [1–4]. Other variables such as suppression of the normal bacterial flora, pregnancy, and diabetes mellitus can all lead to increased Candida infections. Various methods can be used to identify the specific Candida species such as germ tube testing, use of chromogenic media, cornmeal agar, and biochemical testing [1]. If germ tube testing is positive, a presumed diagnosis of Candida albicans can be made [1]. In this test a filamentous extension from a yeast cell that is half the width and three to four times the length of the parent cell is produced. The cornmeal agar test is used to determine if pseudohyphae are present. The presence of pseudohyphae and blastoconidia can lead to characterization of the fungus as Candida species.

Histoplasma Species Histoplasma capsulatum is abundant in the soil in some areas of North America. It is frequently found in Ohio, Mississippi, and the Missouri River Valley. Histoplasma capsulatum is a common cause of systemic fungal diseases in the USA.  The

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mycelial form of Histoplasma capsulatum can be easily encountered in moist nitrogen-­rich soil produced by bird droppings or bats [1]. In the hospital setting, immunosuppressed patients are quite vulnerable to infections from Histoplasma capsulatum and related species [5]. The mold form of Histoplasma capsulatum is very slow growing in the laboratory, requiring up to 30 days or so for growth. Under the microscope, the yeast form measures 2–4 microns in diameter. Cut tissue sections examined under the microscope or stained smears can be used to readily identify the organism. Histoplasma capsulatum can also be seen ingested within macrophage on microscopic examination. Patients infected with Histoplasma species may have fevers, headaches, chest pain, coughing, and chills. An occasional patient may develop pneumonia and mediastinal lymphadenopathy. Patients suffering from AIDS may develop disseminated histoplasmosis and succumb to this condition [6]. Although culturing the organism had been the former method of making the diagnosis in suspected cases, it is now common to use molecular methods with nucleic acid probes to make the diagnosis. Broad range fungal PCR with DNA sequencing can also be used to confirm the diagnosis [7].

Aspergillus Species Aspergillus species are widely distributed in nature because they are ubiquitous. They are frequently recovered in the clinical laboratory [1]. Aspergillus species is found in decaying vegetation in the soil. The most common mode of infection is from inhalation of dust contaminated with Aspergillus fungi in humans. This can lead to infection of the sinuses, or to bronchopulmonary disease. Susceptible patients include those that are immunosuppressed, patients with bone marrow and organ transplants, as well as patients with leukemias and other malignancies [8]. Aspergillus fumigatus is the most common cause of critical infections. In the laboratory, Aspergillus species grow in culture relatively rapidly within 3–5  days. When viewed under the microscope Aspergillus species measures 6 microns in diameter and has septate hyphae with 45-degree branching which helps to separate them form Zygomeces such as mucor under the microscope. Aspergillus forms a fungus ball consisting of amorphous poorly staining hyphae in a preexisting cavity such as the sinuses. Bronchopulmonary aspergillosis may be caused by several different organisms including aerobic actinomycetes [1]. Aspergillus organisms have a propensity to invade blood vessels, so even if a patient has localized disease, it may spread to other areas of the body. In patients with bone marrow transplants the mortality rate from Aspergillus infection is extremely high, usually around 90%. Use of amphotericin B to treat these and other patients with disseminated Aspergillosis is usually effective in only about half of the cases [9]. Aspergillus can be diagnosed by the galactomannan assay which is a sandwich enzyme-°linked immunosorbent test available in clinical reference laboratories [10]. The assay monitors circulating galactomannan [1].

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Blastomyces Blastomyces, like histoplasma is a dimorphic fungus. Blastomyces exist in mold form in the environment at room temperature and in yeast form in the human body at 37 °C. Blastaomyces dermatitides is endemic in some parts of the USA including parts of Mississippi in the South and in the Midwest including Wisconsin and Illinois. It can also be found in some animals such as dogs with a high incidence of infections. The infection usually begins in the lungs with a flu-like syndrome. Patients may have a dry cough, fever, weight loss, night sweats, myalgia, and pleuritic pain [11]. Extrapulmonary infection may involve the bone, genitourinary track, kidney, and brain. Blastomycosis may also involve the skin and other areas of the body with squamous epithelium such as the larynx. Because of the stimulation of proliferation of the squamous epithelium, it may sometimes be mistaken for squamous cell carcinoma of the skin or laryngeal mucosa, so care must be taken in the examination of tissues from these sites in patients with a history of blastomycosis or coming from areas endemic with blastomycosis. Non-culture techniques to diagnose blastomycosis include serologic antibody detection, immunodiffusion, and complement fixation [1]. Blastomyces urinary antigen may also be used as an ancillary diagnostic test [1]. The organisms can readily be recognized in tissue sections or smears under the microscope because of their relatively large size of 10–15 μm and distinct broad-base budding [1].

Parasitology Parasites have existed with humans since early antiquity [12]. Calcified Schistosoma haematobium has been found in Ancient Egypt from around 1200 to 1090 BC in mummies [13]. Severe hookworm infection was found in China in ancient times [12]. It was once thought that spontaneous generation was the explanation for the origin of parasites. In the USA, the Centers for Disease Control (CDC) has estimated that among intestinal parasites, Entamoeba histolytica was present in 0.6% of all stool specimens examined. Trichuris trichiura ova was present in 2.7% of stool samples [12]. The worldwide prevalence of some parasitic diseases includes 900 million people with hookworm and 100–270 million people with malaria [12]. Most parasitic diseases are contracted by ingestion of contaminated food or water or thorough the bite of an arthropod vector [12–14]. Untreated water including ice water and non-pasteurized fresh milk is another potential source of infection. Heated water is relatively safe. Undercooked meats, including raw or smoked fish, can also lead to infections with flukes and tapeworms. The use of insect repellents and using protective clothing to avoid insect bites in regions known to be infested with parasite-transmitting insects is always recommended. Also taking prophylactic medication such as chloroquine when traveling in malaria-infested areas is also recommended [1].

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Individuals affected by intestinal parasites most commonly present with diarrhea which may vary from bloody to watery or purulent depending on the specific parasitic infection. Some parasitic infections are associated with increased eosinophilic counts in the blood, sputum, or stools [12]. Specimen collection for certain parasites include fecal collection. For intestinal parasites, one fecal specimen collection over three consecutive days is usually sufficient to establish a diagnosis. On examination of the stool specimen, areas with blood or mucin from the specimen are most likely to lead to a higher probability of making a diagnosis of the parasite after microscopic examination. For definitive identification of parasites in stool, permanently stained smears should be used [1]. Two examples of parasitic infections and the approach to diagnosing in the clinical microbiology laboratory are summarized below.

Tapeworms Tapeworms are cestodes that produce eggs such as Taenia solium and Taenia saginata for pork and beef tapeworms. They have been recognized from biblical times. Humans acquire the infections from eating poorly cooked pork or beef. Cystocercosis, which is the extraintestinal encysted larvae from T. solium in various organs such as the brain after the ingestion of gravid eggs in food or contaminated with feces can occur. Symptoms depend on the location of the cysts. If these are in the cerebral cortex of the brain, patients may have seizures or specific neurological symptoms [15]. Cystocercosis has been reported in 2–3% of autopsy cases in Mexico City and accounts for 25% of all intracranial masses found on computerized tomography scans [16]. Laboratory diagnosis can be made by observing the distinct eggs with the microscope or in fecal materials. To identify the specific species adult worms are needed [12]. Serological diagnosis by enzyme immunoassay can be done [17]. A molecular test with DNA probes that can identify the species to differentiate the eggs of T. solium from T. saginata is currently available [18]. The DNA test is more reliable than using only egg morphology for diagnosis.

Malaria Malaria is a good example of a parasite that involves both the blood and tissue. The life cycle of malarial parasites is more complex than intestinal parasites with involvement of sexual and asexual stages of the life cycle. An arthropod vector is used for the transmission of the sexual phase of the life cycle.

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The worldwide incidence of malaria is extensive with 207 million infections in 2010 leading to 627,000 deaths. Most of the deaths have been in Africa. In the USA, most cases of malaria are reported in travelers, foreign-born individuals, and immigrants exposed to the infection in other countries. There were 1925 cases in the USA reported to the CDC in 2011 [19]. Individuals with malaria have a fever as the main finding. In addition, flu-like syndromes with headache, fatigue, and muscle aching may also occur when the merozoites leave the liver cells and invade the red blood cells, patients develop a cycle of fevers. The three species of malaria with tertian fever include P. vivax, P. ovale and P. malariae. P. falciparum is associated with quartan fever spikes. A fever episode is associated with “cold periods” of about an hour followed by “hot periods” with the fever reaching 105–106 °F for a few hours. Between febrile episodes patients are usually asymptomatic. Central nervous system complications occur with P. falciparum. The infected red blood cells become sticky because of changes in the membranes of the cells and this can lead to capillary blockage, infarction, and hemorrhage. P. falciparum is the most aggressive variant of malaria and is more often associated with death of the patients. Laboratory diagnosis is made by identification of the malarial parasite in red blood cells using thick and thin blood smears. Babesia infection, caused by a tick vector, may be confused with malarial infection on blood films by inexperienced laboratory technologists. Chloroquine prophylaxis should be used before traveling to areas endemic for malaria. Mosquirix, a vaccine for malaria and the first vaccine ever for a parasite, was reported in 2021 by the World Health Organization [20].

References 1. Procop GW, Church DL, Hall GS, Janda WM, Koneman EW, Schreckenberger PC, Woods GL.  Mycology. In: Procop GW, Church DL, Hall GS, Janda WM, Koneman EW, Schreckenberger PC, Woods GL, editors. Koneman’s color atlas and textbook of diagnostic microbiology. 7th ed. Philadelphia: Wolters Kluwer; 2017. p. 1322–416. 2. Westblade LF, Jennemann R, Branda JA, et  al. Multicenter study evaluating the Vitek MS system for identification of medically important yeast. J Clin Microbiol. 2013;51:2267–72. 3. Jarvics WR. Epidemiology of nosocomial fungal infections with emphasis on Candida species. Clin Inf Dis. 1995;20:1526–36. 4. McCullough MJ, Ross RC, Reade PC. Candida albicans: a review of the history, taxonomy, epidemiology virulence attributes and methods of strain differentiation. Int J Oral Maxillofac Surg. 1996;25:130–44. 5. Minamoto GY, Rosenberg AS. Fungal infections in patients with acquired immunodeficiency syndrome. Med Clin North Am. 1997;81(2):381–409. 6. Huang CT, McGrarty T, Cooper S, et al. Disseminated histoplasmosis in the acquired immunodeficiency syndrome. Report of 5 cases from a non-endemic area. Arch Intern Med. 1987;147:1181–4. 7. Wheat LJ, Kauffman CA. Histoplasmosis. Infect Dis Clin N Am. 2003;17:1–19. 8. Vonberg RP, Gastmeier N.  Nosocomial aspergillosis in outbreak settings. J Hosp Infect. 2006;63(3):246–54.

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9. Denning DW, Stevens DA. Antifungal and surgical treatment of invasive aspergillosis review of 2121 published cases. Rev Infect Dis. 1990;12:1147–201. 10. Mennink-Kersten MA, Donnelly JP, Verweij PE. Detection of circulating galactomannan for the diagnosis and management of invasive aspergillosis. Lancet Infect Dis. 2004;4:349–57. 11. Reeder PA, Neel HB 3rd. Blastomycosis in otolaryngology. Review of a large series. Laryngoscope. 1993;103:53–8. 12. Procop GW, Church DL, Hall GS, Janda WM, Koneman EW, Schreckenberger PC, Woods GL. Parasitology. In: Procop GW, Church DL, Hall GS, Janda WM, Koneman EW, Schreckenberger PC, Woods GL, editors. Koneman’s color atlas and textbook of diagnostic microbiology. 7th ed. Philadelphia: Wolters Kluwer; 2017. p. 1417–99. 13. Ruffer MA. Note on the presence of “Bilharzia haematobia” in Egyptian mummies of the 20th dynasty (1250–1000 BC). BMJ. 1910;1(2557):16. 14. Welch TP. Risk of giardiasis from consumption of wilderness water in North America: a systematic review of epidemiologic data. Int J Infect Dis. 2000;4:100–3. 15. McCormick GF, Zee CS, Heiden J. Cysticercosis cerebri: review of 127 cases. Arch Neurol. 1982;39:534–9. 16. Polly SM. Nerocystercosis. Inf Dis Newsl. 1986;5:89–91. 17. Sloan I, Schneider S, Rosenblatt J. Evaluation of enzyme-linked immunoassay for serological diagnosis of cysticercosis. J Clin Microbiol. 1995;33:3124–8. 18. Chapman A, Vallejo V, Mossie KG, et  al. Isolation and characterization of species-specific DNA probes from Taenia solium and Taenia saginata and their use in an egg detection assay. J Clin Microbiol. 1995;33:1283–8. 19. Cullen KA, Arguin PM.  Malaria surveillance-United States 2011. MMWR Surveill Summ. 2011;62:1–18. 20. Zavala F. RTS,S: the first malaria vaccine. J Clin Invest. 2022;132(1):e156588.

Chapter 19

Clinical Chemistry

Clinical chemistry is a broad area of pathology covered in traditional clinical pathology or laboratory medicine. Clinical chemistry or clinical biochemistry is a specialized area of clinical pathology that involves the quantitative analysis of bodily fluids for diagnostic and therapeutic purposes [1, 2]. Clinical chemistry focuses on specific analytical procedures that allows for accurate measurements of their concentrations in the body. The field of modern clinical chemistry is very broad and covers many areas such as the evaluation of renal function, bone metabolism, clinical enzymology, evaluation of liver function, endocrine, and reproductive function. It also includes analysis of vitamins and trace elements. The clinical chemist measures fluid in the body including the blood and urine and other body fluids. These measurements produce accurate data that can be used by many physicians in various specialties of medicine. Training to become a clinical chemist can involve various routes. One method is to follow the traditional training in pathology and then subspecialize in clinical chemistry. Another route is to obtain a doctorate degree in biochemistry or a related field and then specialize in clinical chemistry. At the completion of clinical chemistry training, the student must pass a clinical chemistry board examination to become board-certified. The examination tests the mastery and knowledge of clinical chemistry including, problem solving, analytical instrumentation and methodology interpretation, as well as statistical analysis, toxicology, regulatory and management practices and molecular genetics before certification is awarded. Clinical chemistry had a long history before it became a scientific discipline. The examination of urine specimens from patients was practiced in antiquity [3, 4]. Clinical laboratories were used by clinicians to prepare chemical medications since the end of the sixteenth century [3]. It was around the nineteenth century that clinical laboratories were used for analysis of body fluids [3]. In 1781 a French Physician, Antoine Francois de Foire, began setting up clinical laboratories in hospitals. He and others used the analyses to determine the nature of various diseases. A clinician by the name of Johann Christian Reid set up a pathological chemistry institute. © The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 R. V. Lloyd, Pathology: Historical and Contemporary Aspects, https://doi.org/10.1007/978-3-031-39554-3_19

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A major advance occurred in 1827 when another physician, Richard Bright, defined a disease which was later named after him. Bright’s disease was associated with renal failure and patients had increased albumin in their urine and dropsy associated with kidney failure [3]. Chemical analyses provided evidence of disease such as in the detection of sugar in the blood or urine in diabetic patients. When patients had renal diseases, physicians were able to detect blood urea nitrogen and urinary proteins associated with renal failure [3]. These clinical tests were performed by pharmacists and chemists with experience in analytical methods. The Germanic school of medicine had a great deal of influence in the development of clinical chemistry. Their laboratories were sometimes incorporated into universities as early as the late nineteenth century. Clinical laboratories continued to be developed at the start of the twentieth century. Abraham Flexner, who was sponsored by the Carnegie Foundation, studied and reported on the practice of clinical chemistry by European physicians. Rudolph Virchow in the 1860s established a section for physiological and pathological chemistry. In the early 1900, clinical chemistry began expanding to the USA. William Osler was one of the pioneers in this field in the USA and he established a clinical laboratory at Johns Hopkins Medical Hospital. Dr. Donald Van Slyke at the Rockefeller Institute was appointed the clinical chemist at two institutions. He became known as the father of modern clinical chemistry with the publication of his clinical chemistry textbook. Major advances were made in clinical chemistry in the twentieth century. Some of these included advances in instrumental techniques such as spectrophotometry, electrochemical techniques, zone electrophoresis, and radioimmunoassays in 1959. The use of monoclonal antibodies which started in the 1970s as well as the use of computers in the laboratory in the 1960s and the polymerase chain reaction in the laboratory in the 1980s contributed to advances in clinical chemistry. Development of new techniques also helped to advance clinical chemistry. Some of these included clinical enzymology, blood gas analysis, enzyme immunoassays, lipid and lipoprotein testing, and more recently molecular diagnostic techniques [4]. Other techniques and technologies included continuous flow auto analyzers, centrifugal analyzers, and auto analyzers such as the sequential multiple analyzer with computer (SMAC) which was introduced in the 1970s. The SMAC also helped to speed up the development of clinical chemistry in handling millions of automated tests [3, 4]. Other major advances in clinical chemistry between 1967 and 2017 included testing for hemoglobin A1c in patients with diabetes, prostate-specific antigen in screening for prostate cancer, and free light chains of immunoglobulin in monitoring for multiple myeloma, natriuretic peptides for management of heart failure and many others [5]. Other procedural improvements included the increased use of immunoassays, replacement of radioactive labeling by various nonradioactive immunoassays, the development of monoclonal antibodies for specific and sensitive immunoassays, and high-performance liquid chromatography [3, 5]. Other significant advances included salivary assays for steroid hormones, and dry blood spots for subsequent analyses. In gastroenterology, fecal calprotectin has been regularly used for the diagnosis of inflammatory bowel diseases and related diseases involving the intestines [6]. Another general advance is that point-of-care

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analyses have become more practical for some analytes. Point-of-care analysis allows for laboratory testing in places where there are no other alternatives. Some point-of-care analyses include chip-based miniaturized portable and self-containing systems which allow for the assay of different analytes [6]. The use of clinical laboratory tests has continued to increase. An estimated 60–80% of clinical decisions are made based on laboratory test results which highlights the importance of laboratory tests [2]. When errors in sample testing such as blood analyses are examined, most of the errors are pre-analytical (76.3%) followed by postanalytical (21.6%) while the lowest percentages are analytical (2.1%) as indicated in one large study [2]. Since pre-analytical errors are so high, it has become a challenge to decrease the percentage of these errors. Some of the tested changes include improving transportation of samples, practices such as the use of vacuum tubes system and using certified regional transporters [5]. Other suggested approaches which are very challenging include modifying the behavior of nurses, doctors, and other individuals who are responsible for preparing the patients for phlebotomy and other pre-analytical protocols that are outside of the central clinical laboratory [5].

A Few Specific Areas of Analyses in Clinical Chemistry Cardiac Troponin Troponins consist of a complex of three proteins that are present in the filament of striated muscles. These include tropomyosin-binding subunit (TnTo), inhibitory subunit (TnI), and calcium-binding subunit (TnC) [7]. Although troponins are present in cardiac and skeletal muscles, in the appropriate clinical setting troponin almost always refers to cardiac-specific troponin, because measurement of the nonclinical forms have no clinical applications [7]. Although other enzymes such as lactate dehydrogenase, creatine phosphokinase, and serum glutamic oxaloacetic transaminase were used to monitor myocardial infarctions in the past, they are produced by other tissues such as liver and testis, so their use for myocardial infarction is not specific and so they are not frequently used to monitor myocardial infarctions. Tropomyosin-binding subunit is a tissue marker with very high sensitivity and specificity [8]. The tropomyosin in cardiac tissue is mostly bound to the muscle fibers and they are released over a 1–2-week period after myocardial injury. The plasma levels of these proteins fall slowly after myocardial infarction, even though they are small proteins. The calcium-binding subunit can be detected within an hour after a myocardial infarct and reaches a peak around 24 h. There is also a rapid renal clearance. Circulating levels of the subunit declines to base line levels between 5 and 10 days later and this is dependent on the size of the infarction [8]. Elevation in serum troponin may also be present in other diseases such as pericarditis, myocarditis, pulmonary embolism, renal failure, and in some other critical illnesses [8].

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Glycosylated Hemoglobin Diabetes mellitus affects about 450 million people worldwide and its incidence has been increasing. Type 2 diabetes mellitus make up about 90% of the cases in the USA [9]. Risk factors for diabetes include obesity, a sedentary lifestyle, first-degree relatives with diabetes mellitus, and advanced age. Ethnicity is also a contributing factor with increased risks in African Americans, Latinos, Native Americans Asians, and some other groups [9]. Good glycemic control is associated with delay onset of some of the complications of diabetes mellitus including microvascular disease, neuropathy, kidney disease, and retinopathy [9]. Glycosylated hemoglobin (HbA1c) is the hemoglobin that is irreversibly glycosylate at one or both N-terminal valines of the beta chain of hemoglobin. HbA1c testing provides valuable clinical information about glucose levels during the previous 2–4 months. HbA1c was discovered in the 1960s and its use as a marker for glycemic control has become invaluable over the past few decades. It is now considered the gold standard for diabetes survey, since clinical use began in the 1970s [10]. The glycation reaction, which is nonenzymatic, became an important monitoring laboratory test for patients with diabetes mellitus in the 1980s [10]. HbA1c is higher in men than in women and it increases in patients with fatty liver, weight gain, age, and systolic blood pressure. Protective factors for increase in HbA1c include high-density lipoproteins, cholesterol, uric acid, and hemoglobin [11, 12]. The use of HbA1c testing requires strict quality management including accreditation of the laboratory, a dedicated internal control design, participation in proficiency testing, and continued assessment of pre- and postanalytical aspects of the test [11]. HbA1c levels are affected by blood loss, blood transfusion, and conditions that affect the age and survival of red blood cells such as hemolysis and pregnancy [9]. HbA1c may also be affected by body weight in groups without diabetes mellitus [12].

Evaluation of Liver Function in Clinical Chemistry The liver is a large complex organ that forms a significant component of the gastrointestinal system. It has many functions including protein synthesis, glucose metabolism as well as the metabolism of other carbohydrates, glycogen synthesis and degradation, amino acid and nucleic acid metabolism, and lipoprotein synthesis [13]. Other functions include drug metabolism, and degradation of numerous proteins including insulin. Most coagulation proteins as well as albumin are synthesized in the liver [13]. Analysis of serum analytes can provide important information about liver function [13]. The location of many enzymes in the liver is often in specific regions of cells, so the pattern of liver injury is usually associated with certain types of enzymatic changes [13]. Some enzymes are present in the cytoplasm such as alanine

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aminotransferase (ALT) and lactate dehydrogenase (LDH). Other enzymes may be present in mitochondria such as aspartate aminotransferase (AST). Others may be localized to canalicular spaces such as gamma glutamyl transferase (GGT) and would be associated with obstructive processes. An enzyme like alkaline phosphatase (ALP) is located in canalicular spaces, but is not specific for the liver. Obstruction of bile ducts is associated with elevation of ALP. The enzyme gamma glutamyl transferase regulates transport of amino acids across cell membranes, so it is also associated with chronic obstruction.

Alcoholic Liver Disease Alcoholic liver disease is prevalent in many countries including the USA [13–15]. It is usually caused by chronic ingestion of alcohol. There is a spectrum in disease progression associated with alcoholic liver disease [14]. Early on in the disease, there is fatty liver or steatosis with accumulation of triglycerides in hepatocytes. Some subjects may develop hepatic inflammation with hepatocyte injury associated with fat accumulation or alcoholic steatohepatitis. This condition can progress to fibrosis and cirrhosis of the liver when there is chronic liver injury. Some patients may develop alcoholic hepatitis which is usually associated with a poor prognosis [14, 15]. A subset of patients with alcoholic liver disease may go on to develop hepatocellular carcinoma in a background of cirrhosis and this carcinoma is associated with a high mortality [14]. Inflammation is often associated with alcoholic steatohepatitis, liver fibrosis, and hepatocellular carcinoma [14, 15]. Elevation of serum gamma glutamyl transferase is common. Although this is a marker of bile ducts, it may also be present in other tissues. Gamma glutamyl transferase may be markedly elevated up to a few thousand units per liter. If there is elevation of gamma glutamyl transferase without elevation of serum transaminase (a marker of liver cellular integrity), the combined sensitivity and specificity for alcohol-associated hepatic inflammation is greater than 70% [14, 15]. Elevated gamma glutamyl transferase may also be present in other diseases such as cholestatic liver disease, cardiac insufficiency, and drug-induced liver injury. Newer markers such as caspase-cleaved cytokeratin fragment M30 and M65 are more sensitive than transaminases and often suggest apoptotic death of hepatocytes [14]. Alcoholic liver disease may also occur as the initial evidence of alcoholic liver disease that is clinically silent [15].

Intraoperative Parathyroid Hormone Assessment Primary hyperparathyroidism is an endocrine disorder involving calcium metabolism associated with hypercalcemia. In the USA, the annual incidence is 21.6 per 100,000 persons [16–18]. The most common form of this disease is parathyroid

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adenoma which occurs in 80% of cases. In this disorder, one parathyroid gland is enlarged while the other glands are normal. Parathyroid hyperplasia is usually associated with elevation of most or all of the parathyroid glands, while parathyroid carcinoma is a very rare disease associated with only one gland. Patients with parathyroid adenomas and other parathyroid disorders often have elevated serum parathyroid hormone and calcium. If there is long-standing disease, patients may present with kidney stones and bone loss. Surgery is the principal method of treatment. A sestamibi scan is often used to localize the enlarged parathyroid gland in patients with hyperparathyroidism [16–18]. Unilateral neck exploration with excision of the enlarged parathyroid gland is often performed. After removal of the abnormal gland, the surgeon waits 10–20 min and then obtains blood for post-removal parathyroid hormone levels. If only one gland was involved in the hyperparathyroidism, one should see a 50–75% decrease of the parathyroid hormone levels below the preoperative values or the parathyroid hormone levels should show significant changes toward normal if the patient previously had significantly elevated parathyroid hormone levels. The marked decrease in parathyroid hormone levels allows the surgeon to conclude that all abnormal parathyroid tissues have been removed. If the parathyroid hormone levels do not decrease as anticipated, the surgeon usually continues with the operation and may even have to explore the other side of the neck, the thymus, or search for an intrathyroidal abnormal parathyroid gland. Intraoperative parathyroid hormone monitoring is usually used for patients undergoing surgery for primary hyperparathyroidism or for recurrent disease [16]. The method of analyzing for parathyroid hormone levels include using blood collected in EDTA or a red top tube for serum. The sample should be maintained at a cold temperature to avoid degradation and submitted for rapid parathyroid hormone testing. To do the rapid testing, some of the parameters for the usual parathyroid hormone assay are modified including using a higher incubation temperature with continuous shaking during the incubation and modification of the sample and reagent volumes [16]. The reaction is more rapid and more expensive, but less sensitive than the conventional assay. There is usually a good correlation with the standard assay [16]. A few decades ago, the surgical pathologists had to perform multiple frozen sections to identify abnormal parathyroid gland tissues and this was a very labor-­ intensive procedure with multiple specimens examined. However, frozen sections of parathyroids are rarely done during parathyroid surgery today, except to identify a tissue as parathyroid or to evaluate the rare parathyroid carcinoma, so that the surgeon can perform a thyroid lobectomy during the same operation if the frozen section shows a carcinoma.

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References 1. Bock JL, Bluth MH, Pincus MR. Clinical chemistry. In: McPherson RA, Pincus MR, editors. Henry’s clinical diagnosis and management by laboratory methods. 24th ed. Philadelphia: Elsevier Inc; 2022. p. 182–465. 2. Teshome M, Worede A, Asmelash D. Total clinical chemistry laboratory errors and evaluation of the analytical quality control using sigma metric for routine clinical chemistry tests. J Multidiscip Healthc. 2021;14:125–36. 3. Buttner J. Clinical chemistry as a scientific discipline. Historical perspective. Clin Chim Acta. 1994;232(1–2):1–9. 4. Kricka LJ, Savory J. International year of chemistry 2011. A guide to the history of clinical chemistry. Clin Chem. 2011;57:1118–26. 5. Delanghe JR.  The achievements of clinical chemistry testing:1967–2017. Clin Biochem. 2017;50(4–5):165–7. 6. Walsham NE, Sherwood RA.  Fecal calprotectin in inflammatory bowel disease. Clin Exp Gastroenterol. 2016;9:21–9. 7. Theodorsson E. Quality assurance in clinical chemistry: a touch of statistics and a lot of common sense. J Med Biochem. 2016;35(2):103–12. 8. Bock J.  Cardiac injury, atherosclerosis and thrombotic disease. In: McPherson RA, Pincus MR, editors. Henry’s clinical diagnosis and management by laboratory methods. 24th ed. Philadelphia: Elsevier Inc; 2022. p. 267–75. 9. Mojica A, Weinstock RS.  Carbohydrates. In: McPherson RA, Pincus MR, editors. Henry’s clinical diagnosis and management by laboratory methods. 24th ed. Philadelphia: Elsevier Inc; 2022. p. 225–43. 10. Gillery P. A history of the HbA1c through clinical chemistry and laboratory medicine. Clin Chem Lab Med. 2013;51(1):65–74. 11. Weykamp C.  HbA1c: a review of analytical and clinical aspects. Ann Lab Med. 2013;33(6):393–400. 12. Kim KJ, Choi J, Bae JH, et al. Time to reach target glycosylated hemoglobin remains associated with long-term durable glycemic control and risk of diabetic complications in patients with newly diagnosed type 2 diabetes mellitus: a 6-year observational study. Diabetes Metab J. 2021;45:368–78. 13. Daniels L, Khalili M, Goldstein E, et  al. Evaluation of liver function. In: McPherson RA, Pincus MR, editors. Henry’s clinical diagnosis and management by laboratory methods. 24th ed. Philadelphia: Elsevier Inc; 2022. Chapter 22. p. 314–30. 14. Seitz HK, Bataller R, Cortez-Pinto H, et  al. Alcoholic liver disease. Nat Rev Dis Primers. 2018;4:16. 15. Mueller S, Seitz HK, Rausch V.  Non-invasive diagnosis of alcoholic liver disease. World J Gastroenterol. 2014;20(40):14626–41. 16. Klemm KM, Klein MJ, Zhang Y. Biochemical markers of bone metabolism. In: McPherson RA, Pincus MR, editors. Henry’s clinical diagnosis and management by laboratory methods. 24th ed. Philadelphia: Elsevier Inc; 2022. p. 208–24. 17. Bilezikian JP, Bandeira L, Khan A, et al. Hyperparathyroidism. Lancet. 2018;391:168–78. 18. Patel IN, Caso R.  Intraoperative parathyroid hormone monitoring: optimal utilization. Surg Oncol Clin. 2016;25:91–101.

Chapter 20

Transfusion Medicine and Hemostasis

Transfusion medicine is a broad area of clinical pathology that includes blood transfusion, preparation and analysis of blood components, blood cell serology, and blood transfusion therapy [1, 2]. The operational basis of transfusion medicine includes blood centers for blood components and transfusion services which collect blood from donors and distribute them. These services provide medical support for blood transfusions and also perform compatibility testing before a transfusion is performed. This specialty also provides blood components for patients when requested by other physicians [1, 2]. In the USA, blood centers and transfusion medicine services are regulated by the US Food and Drug Administration (FDA). Blood centers must be registered with the FDA. Centers that make blood components are licensed for distribution in interstate commerce [1]. Blood donors donate blood on a voluntary basis and donors are not paid for donations in the USA. Blood donors have to meet some minimal requirements: they have to be at least 16 years old, they must weigh at least 110 pounds, and have a minimal hemoglobin concentration. They should also wait for an appropriate period of time since previously donating blood [1]. Historically, blood transfusions have occurred for several centuries [1–3]. Pope Innocent VIII may have been given the first blood transfusion when his personal doctor had him drink the blood of three young boys; however, he died subsequently. One of the earliest blood transfusions from animals to humans was done by a physician to King Louis XIV of France who transfused sheep blood into a 15-year-old boy. Although this particular young man survived the transfusion, many subsequent attempts at transfusions led to the death of the patients [3]. Dr. Karl Landsteiner from Austria discovered the ABO blood groups in 1901 and he subsequently won the Nobel Prize in physiology or medicine in 1930 for his pioneering discoveries. Dr. Landsteiner and his group also discovered the Rh blood groups in 1939. It was during World War I that the use of sodium citrate as an anticoagulant along with glucose was developed and this allowed storage of blood for a limited period © The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 R. V. Lloyd, Pathology: Historical and Contemporary Aspects, https://doi.org/10.1007/978-3-031-39554-3_20

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of time. Methods of fractionating blood was developed by Dr. Cohn in the early 1940s, and this led to the preparation of fibrinogen, albumin, and gamma globulin. Rh immune globulin was first used commercially in 1967 and was rapidly adopted for treating Rh hemolytic disease [4]. A major advance occurred in the 1950s when Dr. Cohn and his team developed donor apheresis which allowed for the collection of platelets and red blood cells from the same donor [2]. The use of apheresis donor led to collection of larger volumes of blood components from a single donor [1].

Preparation of Blood Components Red blood cells are prepared from whole blood by centrifugation and removal of plasma as well as platelets. Dextrose and adenine are usually added to the preparation and the prepared red blood cells are stable up to 35 days at temperatures between 0 and 1 °C. Platelet concentrates are also prepared from whole blood and are stored at room temperature to preserve post-transfusion survival [1]. Leukocytes are preserved by apharesis and stored at room temperature, but for a duration of only 24 h [1, 2].

Therapeutic Uses of Blood Components Red blood cells are used to treat acute and chronic anemia. Platelets are used to treat bleeding due to thrombocytopenia or dysfunctional platelets. Plasma transfusion can be done for multiple conditions including coagulation factor deficiency, disseminated intravascular coagulation, and thrombotic thrombocytopenic purpura [1]. Transfusion reactions may occur during or shortly after a patient receives a transfusion. In the case of ongoing transfusion, the procedure should be stopped immediately and should be reported to the bank or transfusion service [1–3]. There are many potential transfusion-transmitted diseases, but these are usually detected by screening and testing the specimens after collection and processing. These diseases have been markedly reduced in incidence by careful blood collection [1]. Some of these diseases include hepatitis from hepatitis C virus, cytomegalovirus, parvovirus B-10, and human immunodeficiency virus. Nonviral diseases such as malaria are rarely reported in the USA and is usually associated with travel in tropical regions of the world. Rare cases of spongiform encephalopathy or prion disease may occur and there is no standard way of testing donated blood for prion disease [1].

Therapeutic Plasma Exchange (Apheresis) Therapeutic apheresis is used to remove pathogenic substances that may be present in plasma or pathogenic cells from the plasma [5, 6]. This can involve removal of disease-causing substances from the blood or exchange of deficient substances by

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using plasma from healthy donors such as fluid replacement [5]. It can also be used for treating uncommon diseases ranging from autoimmune diseases to coagulation factor inhibitors, thrombotic thrombocytopenic purpura, and fulminant Wilson’s disease (associated with copper disorders). Apheresis involves separation of whole blood into component fractions with removal of a specific fraction and returning the remaining components to the patient [5]. The separation can be done by centrifugation or by membrane filtration. Apheresis techniques need high blood flow states, so peripheral venous access or double lumen dialysis/apheresis catheters are usually used in adults. Anticoagulant solutions usually needed to keep the flow through devices such as plastic tubing that are used in apheresis. Many laboratories use citrate or heparin during the procedure [5]. Potential complications of apheresis include allergic reactions which may be associated with the use of plasma replacement fluids. Citrate toxicity is another potential complication and may lead to mild toxicity [5]. Some patients may also develop hypotension or acetyl cholinesterase inhibitor reactions [5]. Therapeutic plasma exchange can also be used as a treatment of diseases associated with a toxic substance in plasma. Removal of the toxic substances usually helps with the resolution of the disease. It is used mainly for antibody removal, but other substance such as drugs and low-density lipoproteins can also be targets. During the procedure whole blood from the patient goes through the apheresis device and then separation and removes plasma while other components are returned to the patient along with replacement fluid. Examples of diseases treated in whole plasma apheresis include Guillain–Barré syndrome, Goodpasture’s syndrome, focal segmental glomerulosclerosis, myasthenia gravis, and many others [7]. Therapeutic erythrocytapheresis and red blood cell exchange can be used for treating a range of diseases such as ABO incompatibility, hereditary hemochromatosis, and sickle-cell anemia [7]. Therapeutic thrombocytapheresis or plateletpheresis, platelets can be used for prevention or treatment of hemorrhage [8, 9]. With leukocytapheresis white blood cells are selectively removed in patients with disorders such as hyperleukocytosis and inflammatory diseases [8–11].

 xamples of Specific Diseases Requiring Transfusion E Medical Intervention Hemophilia A and B Both hemophilia A and B are congenital recessive X-linked disorders due to a deficiency of clotting factors including Factor VIII (hemophilia A) or Factor IX (hemophilia B). These deficiencies lead to spontaneous bleeding after trauma. Bleeding usually occurs in joints such as the ankles, elbows, and knees and this can lead to joint diseases [12–14]. If the patient bleeds in the brain or into internal organs, this can be life-threatening. Although life expectancy for patients with hemophilia during the 1960s was around 30 years of age, today patients with these diseases can

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have a normal life expectancy and good quality of life with the use of prophylactic therapy including replacement of the missing factor(s) [14]. Hemophilia affects 1/5000 to 1/10,000 males and there is no ethnic predilection [13]. Factor VIII and Factor IX genes are both located on the X chromosome, so males are usually affected. Females with a mutation on one of their X chromosomes would be carriers. However, in rare cases females can also be affected such as if they have Turner’s syndrome [13]. If the child is a daughter of an affected male and a carrier female, they can also inherit the disease. About one third of hemophilia A cases arise from spontaneous mutations. The transfusion specialist can evaluate these patients to make a diagnosis when a patient with a bleeding history is evaluated for partial prothrombin time, prothrombin time, and platelet counts. The diagnosis can be supported by an assay for FVIII and FIX. About half the cases of hemophilia A is due to a partial inversion of the FVIII gene up to and including the intron [12]. Currently, all of the FVIII and FIX replacement concentrates are free of viruses such as HIV, hepatitis B, and hepatitis C. Rare infectious viruses such as parvovirus B19 may still be transmitted in spite of ultrafiltration [13]. Comprehensive hemophilia treatment centers are available to care for patients with hemophilia A and B. They are staffed by hematologists, orthopedists, physical therapists, nurses, genetic counselors, psychologists, and social workers who care for patients with these bleeding disorders [12, 13]. Patients taken care of by such centers usually have a longer life expectancy [12–14]. Gene therapy for hemophilia as a possible cure for the disease has targeted endogenous expression of FV111 or F1X.  In this treatment, a functional gene is used to replace the patient’s defective gene [15]. The approach that has been used successfully with adeno-associated viral vectors. In patients with this disorder, use of this vector for FIX replacement of the clotting factor has remained stable for 7 years in follow-up studies and have resulted in a decreased in spontaneous bleeding and FIX protein usage without any reported toxicities [16].

Sickle-Cell Disease Sickle-cell disease is a hereditary hemoglobinopathy that is caused by genetic mutations in the hemoglobin gene [16]. A wide range of individuals are affected in addition to African Americans and individuals in Africa. People of Hispanic, Middle Eastern, South Asians, Mediterranean, and Caribbean descent are also affected by this disease [16]. In the USA, there are about 100,000 individuals with sickle-cell disease and around 300,000 babies are born with sickle-cell anemia per year [17]. Because of the mutation in the hemoglobin gene, deoxygenated sickle-cell hemoglobin forms polymers within the red blood cells. This leads to distortion of the red blood cell shape and increased blood viscosity with intravascular hemolysis and development of anemia. Patients may develop recurrent severe pain, strokes, and acute chest pain [16]. Some organs are more vulnerable to occlusion especially if there is slow flow through the sinusoids. These include the spleen, liver, bone marrow, and penis [17–19].

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Life expectancy with sickle-cell disease has improved significantly during the past few years. The median survival for patients is around 60 years [18]. The basic defect in sickle-cell anemia is a genetic mutation in one amino acid in the sickle hemoglobin with a nonsense mutation (Glu 6 for Val). Fetal hemoglobin interferes with polymerization of hemoglobin S and does not form part of the hemoglobin S polymer. Although fetal hemoglobin makes up less than 1% of the total hemoglobin in healthy adults, the levels of hemoglobin F are higher in most adults with sickle-­ cell disease [17]. Infection is a major factor in the outcome of patients with sickle-cell disease. With the loss of the spleen due to auto-infarction, there is increased susceptibility to bacterial infections. In Africa, malaria probably contributes to an increase in mortality [17]. Sickle-cell disease occurs most commonly with homozygous hemoglobin S mutation, but it can also occur by heterozygosity of hemoglobin S and hemoglobin C (alpha and beta thalassemia.). Thalassemia is considered to be the most commonly inherited gene mutation in the world and there are an estimated 1000 patients with beta thalassemia major in the USA. It results from mutation of the beta globin gene in beta thalassemia and the alpha globin gene in alpha thalassemia [16]. Laboratory testing for hemoglobin abnormalities may be done in the transfusion medicine or the hematopathology laboratory. Samples are usually examined by electrophoresis and isoelectric focusing in the laboratory. Electrophoresis is most commonly done in cellulose acetate with an alkaline pH. This procedure can readily separate the hemoglobin variants. Isoelectric focusing can be used to separate hemoglobin based on their isoelectric points along a pH gradient and usually provides excellent resolution. Other tests that can be used to analyze for sickle-cell disease include the metabisulfite slide test and the sickle solubility test [17]. Hemoglobin S is present in about 5% of African Americans and in around 45% of the population in some areas of West Africa. Hemoglobin S trait usually is not associated with specific symptoms or bleeding abnormalities [18, 19]. However, extremely low oxygen tension could initiate sickling and rare cases of splenic infarction when flying at high altitudes in unpressurized airplanes have been reported [18]. An exciting new area that is being explored for treatment of sickle-cell disease is the use of CRISPR/Cas9 gene-editing for potential cure of sickle-cell disease [20]. Correction of the gene mutation in beta hemoglobin and the induction of fetal hemoglobin to reverse sickling can be done [19]. This approach uses hematopoietic stem cells treated with CRISPR/Cas9 technology and autologous transplantation. This exciting approach uses gene-edited stem cells to attempt a cure of sickle-cell disease [18].

von Willebrand Disease von Willebrand disease is caused by a reduction in plasma von Willebrand factor (VWF). It is the most commonly inherited bleeding disorder [21–24]. It is usually inherited as an autosomal dominant disorder and is associated with excessive

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bleeding after surgery or from a traumatic insult. Patients usually have mucocutaneous bleeding and it often affects the quality of life [21–23]. There are three major types of VWD with Type I representing the most common type (around 75% of cases) [24]. In Type I VWD, there is a partial quantitative deficiency of plasma VWF. Type 2 includes patients with qualitative defects that impairs one or more aspects of VWF function and represent about 25% of cases of this disease. Type 3 is a very rare form in which there is quantitative VWF deficiency, and plasma VWF is almost completely absent [24]. Type I VWF is caused by a missense mutation resulting in decreased amounts of VWF protein levels and decreased functions of platelets. In Type 2 VWD, the antigen is borderline to normal while one of the functions of VWF is affected. Four subtypes of Type 2 VWD are also present. Type 2A is the most common with absent or decreased VWF activity [22]. In Type 3 VWD, there is a complete absence of VWF. Type 3 VWD may be confused with hemophilia, but can be distinguished by appropriate laboratory tests [21]. The diagnosis of VWD is established with the clinical history of bleeding or having family members diagnosed with VWD. Dental bleeding, nose bleeds, and purpuric lesions are the most common bleeding symptoms in children. In the laboratory workup specific testing for VWF is needed, since plasma thromboplastin time, thrombin time, fibrinogen, and complete blood counts are usually normal. The VWF antigen is usually measured immunologically by enzyme-linked immunosorbent assay or latex immunoassay. Acquired von Willebrand’s syndrome is a rare condition which should be suspected if a patient has abnormal VWD and no history of excessive bleeding. About 300 cases of this rare condition have been reported in the literature. In some cases, autoantibodies specific for the VWF which forms immune complexes are then eliminated from the circulation [24, 25]. Genetic testing for VWD is not commonly used in the clinical laboratory, in spite of the fact that the VWF gene was cloned many years ago. Recently, next-generation sequencing has been utilized to detect various mutations, but these findings have not advanced the treatment of the disease [23, 24].

References 1. Elkins MB, Davenport RD, Mintz PD.  Transfusion medicine. In: McPherson RA, Pincus MR, editors. Henry’s clinical diagnosis and management by laboratory methods. 24th ed. Philadelphia: Elsevier Inc; 2022. p. 771–87. 2. Hillyer CD, Shaz BH. Introduction to blood banking and transfusion medicine. In: Shaz BH, Hillyer CD, Gil MR, editors. Transfusion medicine and hemostasis. Clinical and laboratory aspects. 3rd ed. Amsterdam: Elsevier; 2019. p. 3–6. 3. Duffy J. History of medicine. In: A scandalously short introduction. 3rd ed. Toronto: University of Toronto Press; 1999. p. 171. 4. Shaz BH, Hillyer CD. Transfusion medicine as a profession: evolution over the past 50 years. Transfusion. 2010;50:2536–41.

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5. DiGuardo MA, Bobr A, Winters JL. Hemapheresis. In: McPherson RA, Pincus MR, editors. Henry’s clinical diagnosis and management by laboratory methods. 24th ed. Philadelphia: Elsevier Inc; 2022. p. 788–816. 6. Godbey EA, Schwartz J, Pham HP. Overview of therapeutic apheresis. In: Shaz BH, Hillyer CD, Gil MR, editors. Transfusion medicine and hemostasis. Clinical and laboratory aspects. 3rd ed. Amsterdam: Elsevier; 2019. p. 455–67. 7. Godbey EA, Schwartz J, Pham HP. Therapeutic plasma exchange. In: Shaz BH, Hillyer CD, Gil MR, editors. Transfusion medicine and hemostasis. Clinical and laboratory aspects. 3rd ed. Amsterdam: Elsevier; 2019. p. 469–76. 8. Staley EM, Schwartz J, Pham HP. Therapeutic erythrocytapheresis and red cell exchange. In: Shaz BH, Hillyer CD, Gil MR, editors. Transfusion medicine and hemostasis. Clinical and laboratory aspects. 3rd ed. Amsterdam: Elsevier; 2019. p. 477–80. 9. Simmons SC, Schwartz J, Pham HP. Therapeutic thrombocytapheresis. In: Shaz BH, Hillyer CD, Gil MR, editors. Transfusion medicine and hemostasis. Clinical and laboratory aspects. 3rd ed. Amsterdam: Elsevier; 2019. p. 481–2. 10. Staley EM, Schwartz J, Pham HP. Therapeutic leukocytapheresis and adsorptive cytapheresis. In: Shaz BH, Hillyer CD, Gil MR, editors. Transfusion medicine and hemostasis. Clinical and laboratory aspects. 3rd ed. Amsterdam: Elsevier; 2019. p. 483–6. 11. Sarode R, Kessler CM. Coagulation and fibrinolysis. In: McPherson RA, Pincus MR, editors. Henry’s clinical diagnosis and management by laboratory methods. 24th ed. Philadelphia: Elsevier Inc; 2022. p. 828–46. 12. Saini S, Dunn AL.  Hemophilia A.  In: Shaz BH, Hillyer CD, Gil MR, editors. Transfusion medicine and hemostasis. Clinical and laboratory aspects. 3rd ed. Amsterdam: Elsevier; 2019. p. 677–84. 13. Saini S, Dunn AL.  Hemophilia B.  In: Shaz BH, Hillyer CD, Gil MR, editors. Transfusion medicine and hemostasis. Clinical and laboratory aspects. 3rd ed. Amsterdam: Elsevier; 2019. p. 685–90. 14. Berntorp E, Fischer K, Hart DP et al. Haemophilia. Nat Rev Dis Primers. 2021;7(1). https:// doi.org/10.1038/s41572-­021-­00278-­x. 15. Nathami AC.  Gene therapy for hemophilia. Hematology Am Soc Hematol Educ Program. 2019;1:1–8. 16. Tanhehco YC, Shi PA. Transfusion management of patients with sickle cell disease and thalassemia. In: Shaz BH, Hillyer CD, Gil MR, editors. Transfusion medicine and hemostasis. Clinical and laboratory aspects. 3rd ed. Amsterdam: Elsevier; 2019. p. 325–35. 17. Piel FB, Steinberg MN, Rees DC. Sickle cell disease. N Engl J Med. 2017;376:1561–73. 18. Elghetany MT, Banki K.  Erythrocyte disorders. In: McPherson RA, Pincus MR, editors. Henry’s clinical diagnosis and management by laboratory methods. 24th ed. Philadelphia: Elsevier Inc; 2022. p. 588–635. 19. Rees DC, Williams TN, Gladwin MT. Sickle cell disease. Lancet. 2010;376(9757):2018–31. 20. Park SH, Bao G. CRISPR/Cas9 gene editing for curing sickle cell disease. Transfus Apher Sci. 2021;60(1):103060. 21. Gil MR. von Willebrand disease. In: Shaz BH, Hillyer CD, Gil MR, editors. Transfusion medicine and hemostasis. Clinical and laboratory aspects. 3rd ed. Amsterdam: Elsevier; 2019. p. 669–76. 22. Rao AK, Songdej N, Di Paola JA. Platelet disorders and von Willebrand disease. In: McPherson RA, Pincus MR, editors. Henry’s clinical diagnosis and management by laboratory methods. 24th ed. Philadelphia: Elsevier Inc; 2022. p. 847–69. 23. Leebeek FWG, Eirenboom JCJ. von Willebrand’s disease. N Engl J Med. 2016;375:2067–80. 24. Fogarty H, Doherty D, O’Donnell JS.  New developments in von Willebrand disease. B J Hematol. 2020;191(3):329–39. 25. Miller CH. Laboratory diagnosis of acquired von Willebrand’s syndrome. In: Shaz BH, Hillyer CD, Gil MR, editors. Transfusion medicine and hemostasis. Clinical and laboratory aspects. 3rd ed. Amsterdam: Elsevier; 2019. p. 807–8.

Chapter 21

Outstanding Women in Pathology

The history of women and their contributions to Western medicine is relatively new. During the past century and a half, women had to overcome many obstacles including the intransigencies of men in leadership positions in medicine who decided which individuals would be accepted into medical school based mainly on the person’s sex. Elizabeth Blackwell (1821–1910), the first woman to graduate from medical school in the USA, had to fight her way to get into Geneva Medical College (Norton College of Medicine) in New York in 1849 and her sister, Emily, followed her into a medical career a few years later and had to endure the same hardships to obtain a medical education [1]. Together these sisters became pioneers in the practice of medicine by women and they went on to establish a rigorous medical school for women in New York [1]. In the specialty of pathology, one of the earliest practitioners in this field in the USA was Myrtelle May Canavan (1879–1953). Dr. Canavan attended Michigan State Agricultural College, the University of Michigan and Women’s Medical College of Pennsylvania and received her MD degree in 1905 [2]. She went on to specialize in neuropathology and became a resident pathologist at Boston State Hospital in 1910. She studied the brain of her mentor, Dr. Elmer Southard, as well as the brains of both of his parents. One of the major contributions in medicine that she made was the study and first report of a disease that came to be known as Canavan disease in 1931. She reported for the first time about a case of a young child who died at 16 months of age. At autopsy, the child’s brain was degenerated with a soft spongy section that had turned white. Dr. Canavan concluded from her studies that it was a degenerative condition of the central nervous system containing microcapillaries with myelin vacuolization and giant mitochondria [3]. Dr. Caravan was an assistant curator and then curator of the Warren Anatomical Museum at Harvard Medical School and added more than 1500 specimens to the museum’s collection. However, she was never appointed to the faculty at Harvard Medical School in spite of her outstanding contributions!

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Maude Elizabeth Seymour Abbott (1869–1940) Dr. Maude Abbott was born in St Andrew’s East, Quebec, Canada in 1869. Her mother contracted tuberculosis and died when Maude was less than 1 year old, so she and her sister went to live with their maternal grandmother. She was homeschooled until she was 15  years old. She then attended McGill University as an undergraduate in 1886 and graduated 4 years later as the first female from the arts faculty. Although she was interested in studying medicine at McGill University, she was refused admission. She attended the University of Bishop’s College. After graduation she spent time in London, Zurich, Heidelberg, and Vienna. She decided to spend 2 years in Vienna studying clinical medicine and pathology. She worked on the wards of the Royal Victoria Hospital. She later developed an interest in cardiology and cardiac pathology and she wrote a paper on functional heart murmurs which was published in the Montreal Medical Journal in 1899. This research was based on the study of 2780 case records that were analyzed for cardiac murmurs without other evidence of heart disease. She was also appointed to the Medical Museum and was responsible for categorizing the entire pathology collection of Dr. William Osler who was a major contributor to the museum as a pathologist to the Montreal General Hospital. Dr. Abbott and Dr. Osler became friends and he encouraged her to work at the Museum indicating that it was a major source of medical school teaching. Dr. Osler had asked her to write a chapter on congenital heart disease in 1905 and praised her extensively for her outstanding chapter that was published in Osler and McCrae’s Modern Medicine. Her approach to writing was data-driven, systematic, and methodical and this was greatly appreciated by Osler [4]. Her most significant contribution to the scientific literature was her Atlas of Congenital Cardiac Diseases published in 1936. In this widely recognized work, she classified the cardiac anomalies pathologically and clinically using around 1000 cases. This outstanding work increased her visibility in the academic world and she soon became the most authoritative person on congenital heart diseases [5, 6]. Dr. Abbott retired in 1936. She received many honors and awards including an honorary doctorate from McGill University. Her meticulous work on congenital heart disease was an important contribution to the treatment of the tetralogy of Fallot and led to consultations from Dr. Helen Taussig, one of the giants in the surgical treatment of congenital heart disease. She was awarded the Canadian Medal Hall of Fame in 1994. Dr. Abbott was one of the founding organizers of the International Association of Medical Museums. Today, one of the most prestigious lectures at the United States and Canadian Academy of Pathology is named the Maude Abbott Lecture.

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Dorothy Reed (1874–1964) Dorothy Reed was born in Columbus, Ohio in 1874. She was homeschooled and then studied at Smith College in Massachusetts. After graduation she decided to study medicine. Although her parents were not happy with this decision, she successfully applied to Johns Hopkins University Medical School, and was admitted to the fourth medical school class. She was one of the few women in her class [7, 8]. After graduating from medical school she did her internship at Johns Hopkins under Dr. Osler, who was also a mentor for other pioneering women in medicine such as Maude Abbott. Dr. Reed became a pathology fellow under Dr. William Welch, an outstanding pathologist at Hopkins. It was during that year that she did her brilliant research on Hodgkin disease. She also assisted with autopsies and helped to teach bacteriology during her fellowship year. Her studies on Hodgkin disease were meticulous and led to a great deal of insights about the disease which had been previously thought to be a variant of tuberculosis. Her study of eight cases showed that five of the patients were negative for tuberculosis, reinforcing her conclusions [8, 9]. She concluded in her report that the pathological agent was unknown, but that the disease had no direct relationship to tuberculosis. Although her paper was largely ignored shortly after publication, Dr. McCallum mentioned her work in his book, Textbook of Pathology, which brought scientific recognition [10] for her outstanding study. At the end of her fellowship, Dr. Reed tried to obtain a faculty position at Johns Hopkins, but was discouraged by her mentor, because women were not being hired as faculty members at that time. She was also reported to have had an unsuccessful relationship with a faculty person at Hopkins. So, for these reasons she decided to leave the institution and even changed her specialty interest. She decided to specialize in maternal and child health after training in pediatrics at Babies Hospital in New  York City. In 1906 she married an old friend, Charles Mendenhall, and the family moved to Madison, Wisconsin where he taught physics at the University of Wisconsin in Madison, WI and they started a family. Her first child died during infancy and this motivated Dr. Reed to study the causes of infant mortality. She compared infant mortality in the USA and Denmark in 1936 and concluded that mortality in the USA was higher because the USA had more interference in the natural process of childbirth. She recommended that the USA follow the Danish model. The Dorothy Reed Mendenhall Scholarship was established by Johns Hopkins in her honor by her family in 1957 to help deserving female medical students. Smith College established the Sabin-Reed Hall in 1965 to honor Dr. Reed and Dr. Sabin who were both alumnae at this institution. The first infant welfare clinic was established by Dr. Reed in Madison, WI. It was through Dr. Reed’s efforts that Madison, WI was recognized as the city with the lowest infant mortality in the USA when Dr. Reed worked there [8]. Dr. Reed was considered to be an outstanding observer in pathology as well as an outstanding scientist. She died in 1964.

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Dorothy Russell (1895–1983) Dorothy Russell was born in Sydney, Australia in 1895. She became an orphan at age 10 [11] and went to live with relatives in Cambridge, England. She spent the rest of her life in England. She attended Groton College and graduated with a first in natural sciences. This was during World War I and women were being encouraged to train as doctors, since many of the men were away fighting in the war [12]. She started medical training at London Hospital Medical College in 1919. Dr. Hubert M. Turnbull, the chair of morbid anatomy, had a great deal of influence on Dorothy and she decided to specialize in pathology. She was a very good student and won the Sutton Prize in pathology during medical school. After graduating in 1923, she was appointed as an assistant in the Bernhard Baron Institute of Pathology under Dr. Turnbull. She wrote a thesis on Bright’s disease and was awarded the MD degree in 1930. Dr. Russell’s interest in neuropathology was influenced by Dr. Hugh Cairns who had trained with Dr. Harvey Cushing in Boston. She went to Montreal, Canada to work with Dr. Wilder Penafield at the Montreal Neurological Institute where she acquired skills in various techniques including metallic staining. These techniques would serve her well for her future research after returning to England. She returned to London in 1930 and worked in neuropathology. In 1929 she had joined Dr. Cairns, one of her earlier mentors in Oxford, and later became the neuropathologist at the Radcliff Infirmary. She became part of the scientific staff at the Medical Research Council at the London Hospital in 1933. In 1946, after World War II had ended, she was named professor of morbid anatomy in London and became the Director of the Institute of Pathology, at the London Hospital having succeeded Dr. Turnbull in this position. Dr. Russell had several areas of interest which ranged from basic science to diagnostic neuropathology. She developed tissue culture techniques to study brain tumors and became a leader in diagnostic and research techniques in neuro-oncology. Dr. Russell was a pioneer in the use of smear techniques to diagnose brain tumors. She was the first person to describe pinealomas and microgliomatosis in neuropathology [13]. She also did pioneering studies on the human pituitary gland [14]. One of her major achievements was in the writing of the classic textbook, Pathology of Tumors of the Nervous System, which she wrote with Dr. Lucien Rubinstein. The first edition was published in 1959. The textbook was used for diagnostic as well as research in neuropathology. The two authors wrote four other editions together and Dr. Rubinstein wrote the fifth edition after the death of Dr. Russell. In addition to her studies in neuro-oncology, Dr. Russell performed studies on hydrocephalus, effects of radiation in the brain, and in several other areas of neuropathology. Dr. Russell received many honors for her contributions to pathology. She served as president of the Association of Clinical Pathologist and was an honorary member of the Pathological Society of Great Britain. She was awarded honorary doctorate

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degrees from several universities. She was also the first woman to be the head of a department in a London medical school [12]. Dr. Rubinstein considered Russell to be a “woman of stately dignity who was truly unselfish.” Dr. Russell retired in 1960 and became an emeritus professor. She died in 1983 at the age of 88 after a brief illness.

Emma Sadler Moss (1898–1970) Emma Moss was one of the earliest female leaders in clinical pathology and pathology administration. She was born in Pearlington, Mississippi in 1898. She graduated from college in 1919 at the end of World War I with a degree in bacteriology and worked for 10  years as a laboratory technologist. After her husband died of tuberculosis in 1929, she decided to become a medical doctor. She attended the University of Alabama School of Medicine in 1930. Because that university was only a 2-year medical school, she transferred to Louisiana State University and completed her medical training in 1935. She did an internship and residency in pathology at the Charity Hospital in New Orleans, Louisiana. She was an outstanding student and was elected to Alpha Omega Alpha Honorary Society. She completed board certification in anatomical and clinical pathology as well as in microbiology [15]. During her residency, she demonstrated that she was a highly organized and efficient resident and was appointed the acting director of the department of pathology at Charity Hospital. She was also appointed as a faculty member of Louisiana State University School of Medicine, a great honor for someone so young. She was subsequently appointed as the director of pathology at Louisiana State University School of Medicine in 1940 and was in this position until 1970 three decades later which was a testimony to her organizational and administrative skills. She also held academic appointments in the department of pathology and microbiology and was appointed as clinical professor of pathology at Louisiana State University Medical School in 1951. Because of her demonstrated outstanding leadership and administrative skills, she was appointed as the acting head of the department of pathology from 1945 to 1946. There was a lot of disarray in the department at that time and there were not sufficient faculty involved in teaching the medical students. Dr. Moss was able to organize a curriculum with outstanding visiting lecturers from other institutions to present lectures in the pathology course that year and this was very successful [15]. As a testimonial to her administrative skill, it should be noted that the department of pathology at Charity Hospital was one of the largest in the USA with over 1800 autopsies per year in a 3000-bed hospital. The hospital also had more than 750,000 clinical laboratory tests per year. Dr. Moss was able to direct this large pathology laboratory for several decades. She also had many challenges with her personal health. These ranged from premature birth as an infant to hepatic necrosis and breast

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carcinoma with lymph node metastasis as an adult [16]. Dr. Moss was able to maintain a smoothly running hospital in spite of her personal health problems. Dr. Moss was a pioneer in many ways. She established the first medical technology training program which required a bachelor’s degree for admission. This enabled the program to enroll students with a strong background in the sciences and in other areas. Over her many years as an administrator and teacher she had trained over 555 medical technologists and 150 pathology residents during her long career [16]. With her strong background in bacteriology and microbiology she and a colleague wrote the Atlas of Medical Microbiology in 1953 which was well received by students. Dr. Moss received many awards during her career including the Gold Medal Award. She was an active member of the American Society of Clinical Pathology and became the first female president of this prestigious organization in 1955. Dr. Moss died on April 30, 1970. This was somewhat ironic, because her demise was on the very same day that she was supposed to retire after continuous service for more than 36 years [15]. Louisiana State University School of Medicine established the Emma Sadler Moss Lectureship in her honor in 1968. Her dedication to teaching, service, and pathology administration and her efforts as a pioneering female pathologist have been part of her great legacy.

Sophie Spitz (1910–1956) Dr. Sophie Spitz had a brilliant but short career in pathology because of her early demise. She was born in Nashville Tennessee in 1910. She also had two younger twin brothers. The family moved to Alabama and then to Texas, but they moved back to Tennessee when she was 14 years old [17]. She had an uncle who was a pathologist and this undoubtedly influenced her career choice. She received her bachelor’s degree from Vanderbilt University in 1929, the year of the Wall Street Crash in the USA.  She graduated from Vanderbilt University Medical School in 1932. Although she was also interested in surgery, this was a difficult profession for women to get into at that time. She decided to train in pathology and completed her training in 1936. She was appointed as an assistant pathologist in New York and was then promoted and worked until 1953, the year of her demise. During World War II, she undertook active military service at the Army Institute of Pathology between 1943 and 1945. She was an assistant attending pathologist at Memorial Hospital in New York City from 1941 to 1956. She was also an assistant professor of pathology at Cornell University Medical College in New  York City between 1952 and 1956. Dr. Spitz was also affiliated with the New York Infirmary and was director of laboratories at this institution. Dr. Spitz was married to Dr. Arthur Allen, another famous pathologist in 1942. Dr. Spitz developed an interest in tropical medicine while she was in the Army Institute of Pathology and wrote an atlas on pathology of tropical diseases. She also published studies on malaria and rickettsial diseases as well as on the carcinogenic

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effects of benzidine. Her major research work that she is best remembered for is her studies of juvenile melanomas. She studied 12 cases with follow-up information while she was at Memorial Hospital. She showed that 11 of the cases behaved in a benign manner with only one malignant lesion in the group. This landmark study was published in the American Journal of Pathology in 1948 [18]. The tumors became known as Spitz nevi after her demise. Because juvenile melanomas were treated like malignant melanomas before her timely report, her findings had a major impact in pathology and in the surgical treatment of juvenile melanomas. Because of her original observations, surgeons could be more conservative in the treatment of these lesions. Dr. Spitz died of metastatic colon cancer at the young age of 46. Because her father also died at a young age and he had multiple polyps of the colon, this has raised the possibility of familial adenomatous polyposis syndrome in the Spitz family, although this was not documented.

Dr. Alexandra Piringer-Kuchinka (1912–2004) Dr. Piringer-Kuchinka is best known for her description of toxoplasmic lymphadenitis. She was born in Vienna, Austria in 1912. She graduated from high school with honors in 1930 and attended medical school at the University of Vienna. While she was still a student she served as a part-time instructor at the Institute of Anatomy of Vienna University. After graduating from medical school in 1936, she trained as a pathologist and worked in pathology at the Vienna General Polyclinic and Associated Hospitals. She also did studies for a PhD degree with Professor Friedrich Feyter in 1947. Her research studies were in gastrointestinal stromal tumors [19]. Dr. Pringer-Kuchinka had outstanding diagnostic skills in surgical pathology and served as an important consultant for her colleagues at the medical center. She is best known for her original description of toxoplasmic lympadenitis which was published in 1958, having described the original features in 1952 [20]. She was considered as the “first lady” in pathology in Europe by some of her colleagues [19]. Dr. Pringer-Kuchinka was the first woman to serve as the head of The Austrian Society of Pathology and she did this three times! She also served as the president of the European Society of Pathology (1971–1975). She died in 2004 after having had an illustrious career in pathology.

Lotte Strauss (1913–1985) Dr. Lotte Straus was born in Nuremberg, Germany in 1913, just before the start of World War I. She began her medical studies in Germany, and completed them in Italy in 1937. Because of changes in the prewar political climate in Italy before the start of World War II, she migrated to New York and studied microbiology at Beth

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Israel Hospital [21]. She switched to pathology under the influence of Dr. Sydney Farber, the famous pediatric pathologist who had described lipogranulomatosis or Farber’s disease. Dr. Farber was also a pioneer in the chemotherapeutic treatment of leukemia in pediatric patients. She then joined the pathology department at Mount Sinai Hospital in New  York. She also performed collaborative studies with Dr. Jacob Churg. Their collaborative studies led to the description of Churg–Strauss syndrome or eosinophilic granulomatosis with polyangiitis in 1951 [22]. Dr. Strauss was also encouraged by another mentor, Dr. Paul Klemperer, to focus on pediatric pathology. She became the first pediatric pathologist at Mount Sinai and was a pioneer in establishing the specialty of pediatric and perinatal pathology. She was also a cofounder of the Society for Pediatric Pathology in 1965. Dr. Strauss taught at Columbia University in New York and was a consultant in pathology at Elmhurst City Hospital and a special consultant in perinatal pathology at the National Institutes of Health in Bethesda [21]. She published over 100 scientific papers. The Lotte Strauss Prize in pediatric pathology, given to a pathologist under 40 years of age, was established in her honor by the Society of Pediatric Pathology. Dr. Strauss was also a founding faculty at Mount Sinai School of Medicine in 1966. She received the Jacob Medallion as a Mount Sinai Alumnus in 1983. Dr. Strauss died on July 4, 1985.

Enid Gilbert-Barness (1927–2022) Dr. Gilbert-Barness was another outstanding pediatric pathologist. She was born in Sydney, Australia in 1927. She graduated from St. George Girls High School at age 16. She attended medical school at the University of Sydney and then she left Australia in 1951 for further training in medicine. She trained in England and the USA with specialty training in pathology and pediatrics. After completing her residency training in England, she trained at Children’s Hospital of Philadelphia in pediatrics and at Washington Children’s Hospital in 1956. She also had additional residency training in 1963 in Austin, Texas. Dr. Gilbert-Barness worked at the University of Wisconsin in Madison, Wisconsin between 1970 and 1992, rising to the rank of professor of pathology and head of surgical pathology [23]. Dr. Gilbert-Barness was married to Dr. James Bryson Gilbert in 1954 for over 30 years. They had four daughters and one son. She married Dr. Lewis Barness, a pediatrician, in 1987 after the death of her first husband. He was an outstanding pediatrician and they did many collaborative studies together over many years. Dr. Gilbert-Barness was an excellent researcher and diagnostic pathologist. She performed studies on congenital heart diseases, sudden infant death syndrome, and the developmental aspects of chromosome abnormalities. She published over 600 original manuscripts, many book chapters, and eight books [24].

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She was such an outstanding teacher that the residents and fellows at the University of Wisconsin Medical Center voted her as the outstanding teacher of the year for five consecutive years! Dr. Gilbert-Barness retired from the University of Wisconsin in 1992 and joined the University of Florida as professor of pathology and pediatrics. She was on the editorial board of many leading journals in pediatric pathology. She was also the president of the Society for Pediatric Pathology. She received three honorary degrees from several universities including the University of Sydney. She was also honored with the Order of Australia in 2004 which was awarded by Queen Elizabeth II. The Enid Gilbert-Barness Prize was established in 2008 to honor her achievements and contributions to pathology and pediatrics. She had stroke at the age of 85. She died in 2022 at the home of her eldest daughter, Dr. Mary Lawrence.

Dr. Margaret Evelyn Billingham (1939–2009) Dr. Billingham was born in Tanzania in East Africa in 1939. Her father was in the British Diplomatic Services. She attended school in Kenya at the Loreto School. Her medical education was at the Royal Free Hospital in London, the United Kingdom and she graduated in 1954 [25, 26]. In 1956 she married John Billingham and they had two sons. The family moved to the USA in 1963 and lived for a while in Houston Texas, but later moved to San Francisco. Dr. Billingham did a research fellowship at Stanford then she became part of the cardiopulmonary medicine group and finally joined the pathology department and made great academic strides. In the pathology department, she rose through the ranks to become a full professor in 1988. Her work on cardiovascular diseases with Dr. Philip Cave and Dr. Norman Shumway, the famous heart transplant surgeon, in the cardiac transplantation unit in which serial biopsies were done of transplanted hearts provided her with a great deal of insight into the pathologic changes, including rejection, that occurred after cardiac transplantation. Dr. Shumway, who did the first human heart transplant in the USA provided Dr. Billingham with many biopsies of transplanted hearts for pathologic diagnoses. Dr. Billingham went onto develop her own system to grade rejection in cardiac biopsies and this system became widely accepted as the Billingham criteria [27]. Dr. Billingham also analyzed the effects of anthracycline chemotherapy and the complications from these drugs on the heart. She also developed a grading system to evaluate the severity of anthracyclines on the heart. Because of her expertise in cardiac transplant interpretation, she had many visitors from the USA and many other countries who came to study her [28]. Dr. Billingham had a very productive academic life. She wrote more than 500 original papers, abstracts, and chapters in books. She was also a strong advocate for the professional development of women and she received many honors for her

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pioneering work in the interpretation of cardiac biopsies including being selected as a fellow of the Royal College of Pathology. She was the first female president of the International Society of Heart and Lung Transplantation and served as president of the Society of Cardiovascular Society. She received the Distinguished Pathologist Award from the United States and Canadian Academy of Pathology. She retired in 1994 and she and her family moved to Northern California. Dr. Billingham died in 2009.

Julia Margaret Polak (1934–2014) Dr. Julia Polak was born in Buenos Aires, Argentina in 1939. Her grandparents had emigrated from eastern Europe, so her parents were also born in Argentina. She considered becoming an actress when she was very young, but decided to study medicine later on. She met her husband when they were both in medical school in Argentina. They were married and had three children. She completed medical school in 1961 and decided to become a pathologist. She worked as a pathologist for a while in Buenos Aires, but she and her husband decided to go to England to further their medical education. Dr. Polak did postgraduate studies at the Royal Postgraduate Medical School, Hammersmith Hospital under the guidance of Dr. Anthony Pearse, a famous histochemist with interest in endocrine cells and tumors [29, 30]. Dr. Polak became an outstanding histochemist and made many major findings using the emerging sophisticated technique of immunohistochemistry. She and her colleagues elucidated the neuroendocrine system of the gastrointestinal tract and then studied the neuroendocrine system in the lung [31]. Dr. Polak’s work led to the understanding of the intricacies of the neuroendocrine system of the gastrointestinal tract and she described major regulatory roles of neuroendocrine cells and tumors in the digestive system [31, 32]. Dr. Polak’s research led to other major discoveries including studies and localization of nitric oxide in health and disease [33, 34]. Dr. Polak developed pulmonary hypertension and had to undergo a lung and heart transplant in 1995. She lived for 19 years after the transplant, probably representing the longest human survival after these complex transplantations at that time. She enjoyed dramatic presentations and was the presenting pathologist at a medical grand rounds conference at the Hammersmith Hospital in 1995 where she illustrated the pathological and immunohistochemical findings in her native lungs! After her recovery from the transplants, Dr. Polak changed research fields to study regenerative medicine and became a worldwide leader in this field. She established the Imperial College Tissue Engineering and Regenerative Medicine Centre in 1997 and became the first professor of this center. Her research laboratory made significant advances in the field of regenerative medicine and used pluripotent stem cells to try to restore normal lung function. She also did research on creating three-­ dimensional lung structures.

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Dr. Polak was extremely productive during her academic career, in spite of her major illness. She published more than 1000 original papers, many of them with her colleague Dr. Stephen Bloom [32–34]. She did major studies in neuroendocrinology, studies of nitric oxide, and studies in regenerative medicine. Dr. Polak received many honors and awards in the United Kingdom and from many other countries. She was awarded the DBE (Dame Commander of the most Excellent Order of the British Empire) by the Queen in 2003 and the Ellison Ciffe Medal from the Royal Society of Medicine. Dr. Polak died in 2014 at the age of 75. She was one of the world’s longest surviving lung transplant patients.

Elaine Sarkin Jaffe (Born 1943) Dr. Jaffee was born in Brooklyn, Ney York in 1943. She attended college at Cornell University then went on to the University of Pennsylvania for medical school and graduated in 1979. Dr. Jaffee did an internship at Georgetown University Medical Center and then a residency in anatomic pathology at the National Institutes of Health. She has spent her entire professional career at the National Institutes of Health in pathology and hematopathology [35–37]. Dr. Jaffee married Dr. Michael Evan Jaffee, a law professor in1967. They raised two children. Her research work has focused on lymphomas and lymphoproliferative disorders and on the diagnosis and classification of lymphomas. Some of her early work helped to establish the classification of lymphomas into B- and T-cell types. She has published over 700 original papers and authored or edited 7 major textbooks. She has also been an outstanding teacher and mentor at the National Institutes of Health. Dr. Jaffee was one of the leaders in establishing the Revised European-American Classification of Lymphoid Neoplasms (REAL Classification). This classification was adopted by the World Health Organization and retains an important role in the classification of lymphomas. Her outstanding teaching has been recognized by awards from the National Institutes of Health including the Outstanding Mentor Award and the Distinguished Teacher Award. Her outstanding research and diagnostic skills have been recognized with many awards. She was elected as a member of the Institute of Medicine. She has won many distinguished awards including the American Society of Investigative Pathology Gold-Head Cane Award, the American Society of Investigative Pathology Rous-Whipple Award, and the American Society of Investigative Pathology Award for Excellence in Mentoring and Scholarship. She was also the inaugural recipient of the recently established James S. Ewing–Thelma B. Dunn Award for outstanding achievements in pathology and in cancer research. Dr. Jaffee is currently head of the hematopathology section at the National Cancer Institute/National Institutes and a National Institutes of Health Distinguished Investigator.

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Virginia LiVolsi (Born 1943) Dr. Virginia LiVolsi was born on July 29, 1943 in New York City. She graduated from Academy of Mount St. Ursula High School in Bronx, New York in 1961. An ophthalmologist had examined a close relative and diagnosed hypertension during the eye examination. Virginia was very impressed by this finding and decided that she would study medicine [38]. Dr. LiVolsi attended Mount Saint Vincent College and received her BS with honors in 1965. She attended medical school at Columbia University College of Physicians and Surgeons in New York City. During medical school there was a union strike in New York City and the microbiology labs were not constructed in time, so lectures in microbiology were postponed and they doubled up on the pathology lectures giving her a more concentrated exposure to pathology. She and another student in the class became fascinated and enamored with pathology and they both decided to go into pathology [38]. She graduated from medical school in 1969. After completing an internship in 1970, she trained in pathology at Columbia University Medical Center. She served as chief resident in pathology during her final year of residency training. After graduation, she was recruited by Yale University Department of Pathology and worked at this institution until 1983 when she joined the University of Pennsylvania Perelman School of Medicine [39, 40]. She has been an excellent teacher and mentor and has trained many residents in endocrine pathology and head and neck pathology. She has trained over 500 residents and fellows at Yale University and the University of Pennsylvania [38]. She is an excellent diagnostic pathologist. She has published more than 450 original manuscripts, 4 books, and many book chapters and scholarly books. One of the books that she wrote was Surgical Pathology of the Thyroid in the Major Problems in Pathology series in 1990. This classical book on the morphological aspects of thyroid diseases has been used by many pathologist and pathology residents over many decades. It contains excellent photographs and links the biology of the thyroid to the pathological diagnosis of thyroid lesions. Dr. LiVolsi has made many seminal observations in the field of endocrine pathology. She described several original thyroid tumors including the Warthin-like tumor of the thyroid, which can be readily recognized because of its unique morphological appearance [41]. She published a comprehensive study on grading thyroid tumors in 2000. Although this was not readily accepted initially, pathologists have slowly come around to grading papillary thyroid tumors, since this was one of the few areas in diagnostic pathology that was not associated with a grading system. Her proposed grading system was finally accepted during the publication of the fifth edition of the WHO Endocrine Blue Book. Dr. LiVolsi has made multiple original observations in basic biology that have been used in diagnostic pathology. Her team showed for the first time that many thyroid nodules could be analyzed for clonality with molecular approaches and they showed that many thyroid nodules that were morphologically thought to be benign nodules were actually monoclonal proliferations or neoplasm [42]. Her group also showed that tissues stored for up to 10 years in an ultracold freezer could yield high-quality

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nucleic acids for pathological analyses. These studies have influenced many prospective studies on thyroid cancers and other tumors [38]. Dr. LiVolsi has received many awards and honors for her outstanding teaching, research, and other scholarly activities. Some of these have included serving as president of the Endocrine Pathology Society and the United States and Canadian Academy of Pathology. She received the Distinguished Service Award from the American Association of Clinical Pathology, the Mostofi Award from the United States and Canadian Academy of Pathology, and the Harvey Goldman Teaching Award from the United States and Canadian Academy of Pathology among many others. She has been an inspirational diagnostic pathologist, teacher, researcher, and mentor to many young pathologists over many decades.

Sharon Ann Whelan Weiss (Born 1945) Dr. Weiss was born in Lynn, Massachusetts in 1945. She was one of six children. Her father was a surgeon and this undoubtedly helped to stimulate her interest in medicine. She attended Wellesley College in Massachusetts and graduated with honors in 1966. She attended medical school at Johns Hopkins and graduated in 1971. She was always fascinated by the structure and function of cells and this was one of the reasons that she decided to specialize in pathology [43, 44]. She remained at Johns Hopkins for residency training in pathology. During her final year of residency training, she was chief resident—and this was the first time a woman had been chief resident in pathology at this venerable institution. Dr. Weiss worked at the Armed Forces Institute of Pathology from 1976 until 1989. She flourished under the influence of her mentor, Franz Enzinger, the renown soft tissue pathologist. She moved to the University of Michigan in Ann Arbor, MI in 1989. She also served as the director of anatomic pathology at Michigan and was named the A. James French Professor of Pathology and the chief of surgical pathology. In 1998, Dr. Weiss moved to Emory University Hospitals in Atlanta, GA to become professor of pathology and laboratory medicine and director of the Expert Consultation Services at Emory. She was also the dean of faculty development at Emory University Medical Center. Dr. Weiss has published many original descriptions of soft tissue tumors during her career, having described more than nine new entities including epithelial hemangioendothelioma [45] and myxofibrosarcoma [46]. The precise description and diagnosis of these new entities have contributed to progress in the important area of tumor classification. Dr. Weiss has made important contributions to pathology as a teacher and mentor for young pathologists. Her fellowship program in soft tissue pathology has trained many of the outstanding soft tissue pathologists in practice today. She has won many awards and honors including serving as president of the United States and Canadian Academy of Pathology, president of the International Society of Bone and

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Soft Tissue Pathology, and as a trustee of the American Board of Pathology. Some of her honors include the Harvey Goldman Master Teacher and Mentoring Award, the Distinguished Pathologist Award from the United States and Canadian Academy of Pathology, and the Philip Levine Award for Outstanding Research from the American Society of Clinical Pathology. She has published more than 130 original manuscripts. One of her major contributions to pathology is the classical textbook, Enzinger and Weiss Soft Tissue Tumors, which she coauthored with Dr. Enzinger and which has gone through five editions. Dr. Weiss is currently emeritus professor at Emory University. She is greatly admired by the many residents and fellows who trained under her for her vast knowledge of pathology and her great sense of humor.

Fattaneh Tavassoli (Born 1949) Dr. Tavassoli was born in Teheran, Iran in 1949. She migrated to the USA in 1963. Dr. Tavassoli earned her bachelor’s degree with a major in chemistry from South West Missouri State College in 1968. She attended medical school at St. Louis University and received her medical degree in 1972. She did an internship at Barnes Hospital and Washington University School of Medicine in Anatomic and Experimental Pathology. She did her residency in pathology at Barnes Hospital and completed training in 1975 [47]. Because of her growing interest in gynecologic pathology, she received further training in this field at St. Johns Mercy Medical Center in St Louis. She was offered a position at the Armed Forces Institute of Pathology and rose from a staff pathologist to vice chairman of the gynecology and breast pathology between 1976 and 1992. She then served as director of gynecology and breast pathology and director of research laboratory of pathology at Fairfax Hospital in Virginia from 1992 to 1994. Between 1987 and 1992, she was also a consultant at the National Institutes of Health in Bethesda, MD. She then moved to Yale University Medical Center and served as professor of pathology and reproductive sciences and director of the Pathology Women’s Health Program from 2003 to 2016. Dr. Tavassoli has been very productive in her academic life having published more than 220 original manuscripts. One of her major contributions to the field was her textbook on pathology of the breast which was first published in 1992. This comprehensive book on breast pathology was well received and was followed by a second edition in 1999. Dr. Tavassoli described the concept of ductal intraepithelial neoplasia (DIN) to cover the range of intraductal proliferative lesions from intraductal hyperplasia to atypical ductal hyperplasia and ductal carcinoma in situ. [48]. Dr. Tavassoli’s team also showed the expression of androgen receptor protein in invasive and noninvasive breast carcinomas and suggested that the presence of this receptor may be of potential therapeutic value [49].

References

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She was also coeditor of the Armed Forces Institute of Pathology fourth series on Tumors of the Breast with the renown breast pathologist, Dr. Vincenzo Eusebi, from Italy in 2009. Dr. Tavassoli was also one of the editors of World Health Organization Classification of Tumours of the Breast and Female Genital Organs Pathology and Genetics in 2003. She is an enthusiastic teacher and has given hundreds of lectures in the USA and in many other countries. She has received many professional honors and awards. Some of these include the Cylinder of Cyprus Award from Encyclopedia Iranica as a Distinguished woman in medicine, the Lifetime Achievement Award at the third Vienna Breast Surgery Day, and she was the recipient of the Fourth Annual WAD Anderson Award from the department of pathology at the University of Miami Miller School of Medicine [47]. Dr. Tavassoli became an emeritus professor of pathology at Yale University School of Medicine in 2016. She has remained active in academic pathology presenting lectures and doing research.

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17. Alessandrini L, Canzonieri V.  Sophie Spitz (1910–1956). In: van den Tweel JG, editor. Pioneers in pathology. In: van Krieken JHJM, series editor. Encyclopedia of pathology. Cham: Springer International Publishing; 2017. p. 490–493. 18. Spitz S. Melanomas of childhood. Am J Pathol. 1948;24:591–609. 19. Agnantis NJ, Batistatou A. Piringer-Kuchinka, Alexandra (1912–2004). In: van den Tweel JG, editor. Pioneers in pathology. In: van Krieken JHJM, series editor. Encyclopedia of pathology. Cham: Springer International Publishing; 2017. p. 436–439. 20. Piringer-Kuchinka A, Martini I, Thalhammer O.  Superior cervical-nuchal lymphoid nidus with small groups of epithelial cell proliferation. Virchows Arch. 1958;331(5):522–35. (In German). 21. Ramieri MT, Marino M. Strauss, Lotte (1913–1985). In: van den Tweel JG, editor. Pioneers in pathology. In: van Krieken JHJM, series editor. Encyclopedia of pathology. Cham: Springer International Publishing; 2017. p. 499–501. 22. Churg J, Strauss L. Allergic granulomatosis, allergic angiitis and polyarteritis nodosa. Am J Pathol. 1951;27(2):277–301. 23. Obituary. Enid May (Gilbert) Barness, MD. Minneapolis: Star Tribune; May 11, 2022 https:// www.startribune.com/obituaries/detail/0000425106/. Accessed 20 May 2022. 24. Haust MR, Lonkey K. Festschrift: Dr. Enid Gilbert Barness. Enconmium. Fetal Pediatr Pathol. 2014;33:262–7. 25. Berry GJ, Tazelaar HD.  Billingham, Margaret Evelyn (1939–2009). In: van den Tweel JG, editor. Pioneers in pathology. In: van Krieken JHJM, series editor. Encyclopedia of pathology. Cham: Springer International Publishing; 2017. p. 74–76. 26. Billingham ME, Cary NR, Hammond ME, et al. A working formulation for the standardization of nomenclature in the diagnosis of heart and lung rejection: Heart Rejection Study Group. J Heart Transplant. 1990;9:587–93. 27. Billingham ME. Some recent advances in cardiac pathology. Hum Pathol. 1979;10:367–86. 28. Billingham ME, Mason J, Bristow M, et al. Anthracycline cardiomyopathy monitored by morphological changes. Cancer Treat Rep. 1978;62:865–72. 29. Haworth SG. Professor Dame Julia Polak, DBE Pulmonary. Circulation. 2014;4(4):736. 30. Laurance J.  Julia Margaret Polak. Lancet. 2014;384(9951):1342. https://doi.org/10.1016/ s0140-­6736(14)61809-­2. 31. Conversations with Pathologists. Interview with Julia Polak: full Transcript. July 25, 2007; Available from: https://conversations.pathsoc.org/index.php?option=com_ content&view=article&id=80:julia-­polak-­full-­transcript&catid3&Itemid=194. Accessed 20 Apr 2022. 32. Polak JM, Bloom SR.  Some aspects of neuroendocrine pathology. J Clin Pathol. 1987;40(9):1024–41. 33. Polak JM, Bloom SR. The diffuse neuroendocrine system. Studies of this newly discovered controlling system in health and disease. J Histochem Cytochem. 1979;27(10):1398–400. 34. Robbins RA, Barnes PJ, Springall DR, et al. Expression of inducible nitric oxide in human lung epithelial cells. Biochem Biophys Res Commun. 1994;203(1):209–18. 35. Elaine S.  Jaffee, MD.  Research summary. Center for Cancer Research. National Cancer Institute. https://ccr.cancer.gov/staff-­directory/elaine-­s-­jaffe. Accessed 20 May 2022. 36. Cossman J, Neckers LM, Hsu S, et al. Low grade lymphomas. Expression of developmentally regulated B-cell antigens. Am J Pathol. 1984;115(1):117–24. 37. Hsu SM, Yang K, Jaffe ES. Phenotypic expression of Hodgkin’s and Reed Sternberg cell in Hodgkin’s disease. Am J Pathol. 1985;118:209–17. 38. Livolsi VA. Pathologist. Personal communication by email. 26 May 2022. 39. Virginia A. LiVolsi, MD. Meet Dr. LiVolsi. Penn Medicine: University of Pennsylvania. https:// www.pennmedicine.org/providers/profile/virginia-­livolsi. Accessed 26 May 2022. 40. Virginia A.  LiVolsi, MD Curriculum Vitae. Perelman School of Medicine University of Pennsylvania: Pathology and Laboratory Medicine. https://pathology.med.upenn.edu/department/people/458/virginia-­livolsi. Accessed 26 May 2022.

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41. Apel RL, Asa SL, LiVolsi VA.  Papillary Hurthle cell carcinoma with lymphocytic stroma “Warthin-like tumor” of the thyroid. Am J Surg Pathol. 1995;19(7):810–4. 42. Hicks DG, LiVolsi VA, Neidich JA, et al. Clonal analysis of solitary follicular nodules of the thyroid. Am J Pathol. 1990;137(3):553–62. 43. Johns Hopkins Medicine: Pathology. Our Alumni: Director of the Expert Consultation Service, Emory. https://pathology.jhu.edu/education/residency/our-­alumni/sharon-­weiss. Accessed 4 June 2022. 44. National Library of Medicine. Meet Local Legend: Sharon Weiss, MD. May 30, 2022. https:// www.nlm.nih.gov/exhibition/locallegends/Biographies/Weiss_Sharon.html. Accessed 10 June 2022. 45. Weiss SW, Enzinger FM.  Epithelioid hemangioendothelioma. A vascular tumor often mistaken for a carcinoma. Cancer. 1982;50(5):970–8. 46. Weiss SW, Enzinger FM.  Myxoid variant of malignant fibrous histiocytoma. Cancer. 1977;39(4):1672–85. 47. Prabook, Fattaneh Abbas-zadeh Tavassoli, MD. New York: World Biographical Encyclopedia. https://prabook.com/web/fattaneh_abbas-­zadeh.tavassoli/3346609. Accessed 30 May 2022. 48. Tavassoli FA. Ductal carcinoma in situ: introduction of the concept of ductal intraepithelial neoplasia. Mod Pathol. 1998;11(2):140–54. 49. Moinfar F, Okcu M, Tsybrovskyy O, et  al. Androgen receptors frequently are expressed in breast carcinomas: potential relevance to new therapeutic options. Cancer. 2003;98(4):703–11.

Chapter 22

Nobel Laureates in Pathology

The Nobel Prize is indisputably the most prestigious international prize that exists today. The Nobel Prize, as originally designed by Alfred Nobel, was started in 1901, 5 years after his death, is awarded for discoveries in physics, chemistry, physiology or medicine, literature, and peace among nations. The prize in economics was established in 1968 and first awarded in 1969. Alfred Nobel was a most unusual person. Although he is well known as the inventor of dynamite, he was also a chemist, a prodigious inventor with hundreds of patents to his name, an entrepreneur, and a businessman [1]. He and his brothers were homeschooled and at age 17. His father had him visit several countries including France, Germany, Italy, and the USA. He was a polyglot who spoke at least six languages (Swedish, French, English, Italian, Russian, and German). Although Ascono Sobrero from Italy had invented nitroglycerine by mixing glycerine with sulphuric and nitric acid, the inventor had considered nitroglycerine too dangerous to be of practical use. However, Nobel wanted to develop a more stable and commercially useful product that could be used in the construction industry. He discovered that by mixing nitroglycerine with the fine sand kieselguhr he was able to make the liquid into a paste that could be shaped into rods and inserted into drilling holes. His work to develop dynamite was not without many dangers. During the development of dynamite, many people were killed or injured including his younger brother. He took out a patent for dynamite in 1864, just when the Civil War was ending in the USA [2]. Alfred Nobel reportedly had an aversion to publicity. When he was invited to participate in a project listing famous and outstanding Swedes, he agreed to participate in the work, but did not want his portrait included in the collection [2]. He was reported to be a hard worker who worked 15–20 h a day and seldom went to bed before midnight. He was also known as someone who would write 15–20 letters a day [2, 3]. By donating most of his wealth to establish the Nobel Prizes, one can imagine that there were some conflicts with the surviving members of Nobel’s family about © The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 R. V. Lloyd, Pathology: Historical and Contemporary Aspects, https://doi.org/10.1007/978-3-031-39554-3_22

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his estate. He had never been married and had no children, so there were no potential conflicts with direct descendants or spouses. As one story goes, after the death of his brother Ludwig in 1883 some newspapers mistakenly published obituaries about Alfred. A French newspaper was reported to proclaim “The merchant of death is dead’ in reference to his “invention of dynamite.” As the speculation goes, this may have been a motivating factor for his establishing the Nobel Prize in his will [3]. There have been six pathologists who have been trained in pathology and have participated in the practice of pathology that have won the Nobel Prize [4]. These have included experimental pathologists, basic researchers, a surgical pathologist, and a clinical pathologist. There are other Nobel laureates who have worked in pathology departments at academic institutions with expertise in some are of pathology who have won the Nobel Prize, but have not undergone training in traditional diagnostic pathology training programs. All of these six Nobel laureates that are discussed below have been in the physiology or medicine category.

Karl Landsteiner (1868–1943) Karl Landsteiner was a pathologist and an early pioneer in immunology. He was the son of Leopold and Fanny Landsteiner from Vienna, Austria. His parents were well off financially. They spent their summers in Baden which was just outside of Vienna. Karl’s father died of a myocardial infarction when he was only 7 years old, so he was raised mainly by his mother. During this period in history, Jewish families who wanted to advance in their careers often converted to the catholic faith, so Karl and his mother did this in 1890 [5]. After medical school, he joined the department of internal medicine in Vienna in 1891. However, Karl was very interested in science, so he studied chemistry in Germany and Switzerland for 2 years with an emphasis on organic chemistry. A few years later, he joined the pathology department in Vienna and studied pathological anatomy and histology. His research on serum agglutinins was very productive and he was able to show agglutination between various sera of blood cells between different individuals. Based on the antigens present on the cell membrane, he was able to demonstrate at least three types of blood groups including A, B, and C (C was later changed to O). Several of his coworkers were later able to show a fourth group, type AB. These original discoveries had major impacts in surgery by understanding and avoiding transfusion reactions [6, 7]. As a result, applications in other areas of medicine including forensic medicine became evident. In other studies of blood cells Landsteiner and his colleagues were able to develop tests for cold agglutinins that could be used in the diagnosis of paroxysmal cold hemoglobinuria. Landsteiner’s interest in pathology research encompassed several fields. For example, he was able to develop models for syphilis using a monkey model. He and his colleagues showed that infecting animals with Treponema resulted in syphilitic

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lesions in the brain and other organs. It was around this time in 1905 that he and his collaborators developed darkfield microscopy to identify syphilitic bacteria. In 1908, he became the head of pathology at a hospital in Vienna. His research during this time continued to be very broad and included the study of poliomyelitis. He was able to show that autopsy tissues from polio victims could infect monkeys and lead to paralysis as was seen in humans. The histopathologic changes in the central nervous system of his experimental models were surprisingly similar to those in humans. Since they could not demonstrate bacterial organisms in the lesions, they speculated about a viral etiology, which proved to be correct from future studies by others. In 1922, he emigrated to the USA with his wife and son and joined the Rockefeller Institute in New York City. Additional studies in immunology in the 1920s showed that all antigens were not simply proteins, but that smaller molecules such as haptens could be part of the antigen-antibody reaction. He continued his work on blood groups at the Rockefeller Institute and his group described other blood antigens including the M, N, O, and P groups in 1927. He formed a close collaboration with Philip Levine at Rockefeller [8]. They described individual serologic specificity in humans. Working with several collaborators they extended their understanding of transfusion reactions and described the Rh factor which had a major impact on maternal fetal rejections in multiple pregnancies. He was awarded the Nobel Prize in physiology or medicine in 1930 for his elegant work on human blood groups. In his later years he was very concerned when his wife developed thyroid cancer. He died of a myocardial infarction in 1943 and his wife died a few months later. Landsteiner was recognized not only a brilliant pathologist-scientist, but he was also seen as a shy, modest person with a generous heart.

Johannes Fibiger (1867–1928) Johannes Fibiger was a professor of anatomic pathology at the University of Copenhagen in Denmark. He studied medicine at the University of Copenhagen [4]. After graduation in 1890, he continued his studies in Berlin and was influenced by outstanding scientists including Robert Koch and Paul Ehrlich in Frankfort. His doctoral research involved the study of diphtheria [9]. He made several unique contributions to science in his doctoral research. He had developed a more efficient way of growing bacteria. Some of his studies involved testing blood serum for diphtheria and he was reportedly the first scientist to use studies with controlled clinical trials [9]. In his examination of diphtheria serum, he had separated the sera into those from treated and untreated patients and found that more of the untreated patients died compared to the treated ones. Fibiger won the Nobel Prize in 1934 for studies implicating a nematode worm in the etiology of stomach cancer in rodents. Unfortunately, this work became very controversial after his death and has remained quite controversial more than 100 years after the studies were published [10, 11]. Fibiger had fed rats cockroaches

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infected with worms and their eggs. These included healthy rats some of which were wild rats. The infections were associated with epithelial hyperplasia or proliferation of the lining cells of the stomach, inflammation, and tumor formation. Fibiger also concluded that there were metastases of these tumors to the lungs, defining the growths as malignant tumors or carcinomas capable of metastasizing, which is part of the basic definition of cancers. He published his first studies in 1913 and received the Nobel Prize in 1934, the sole recipient in physiology or medicine. But it was not until some years that his discoveries would be proven erroneous. There was a great deal of enthusiasm for research related to irritation leading to carcinogenesis in the 1920s. For example, Dr. Katsusabure Yamagiwa in Japan had shown with his collaborators that coal tar painted on the ears of rabbits could induce cancer (tar cancer) [12]. Dr. Yamagiwa had also been nominated for a Nobel Prize in physiology or medicine around this time for his cancer initiation model. Fibiger was a careful scientist and several other prominent histopathologists had examined his specimens and concluded that they were indeed malignant tumors. However, histologic techniques were not very sophisticated at this time. In 1952 two American scientists, Hitchcock and Bell, tried to repeat Fibiger’s experiments using animals with and without vitamin A deficiency. They concluded that the lack of vitamin A produced the large papillomas (benign tumors) that Fibiger had interpreted as cancers. The lesions in the lungs that were interpreted by Fibiger and his colleagues as metastases were interpreted as [12] metaplastic lesions secondary to vitamin A deficiency. Metaplasia refers to one adult tissue becoming another adult tissue, but this is usually not involved in cancer development. They concluded that Spiroptera carcinoma (later correctly identified as Gongylonema), which originated in South America and the West Indies and came to Europe through the transportation of sugar, did not lead to malignant tumors or carcinomas and that the histology of the growths had been misinterpreted. Some might argue that the studies of Fibiger, although misleading due to misinterpretation of the histological findings, were ahead of its time, since parasites and other organisms were subsequently shown to be associated with cancers such as Schistosoma haematobium infection with bladder cancer in humans [13] and liver fluke infection associated with cholangiocarcinoma or cancer of the bile duct [14].

George Hoyt Whipple (1878–1976) George Whipple was born in New Hampshire in 1878. He attended Yale University and then continued the tradition of his father and grandfather and decided to study medicine [15]. One of his most influential mentors was William H. Welch at Johns Hopkins who was the head of pathology. This experience probably influenced George to pursue this specialty. Dr. Whipple trained as an anatomic pathologist and an experimental pathologist. While at Hopkins he was involved with the autopsy of a young physician who had been working as a missionary in Turkey. The patient had died after being symptomatic for 5 years. His symptoms were multisystemic and

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included arthritis, diarrhea, and progressive wasting. Whipple concluded that this was related to abnormal fat metabolism and coined the term intestinal lipodystrophy. The case report was published in the Johns Hopkin Hospital Bulletin in 1907 and Whipple had the foresight to suggest that it was possibly caused by an infectious agent. This latter hypothesis was proven to be correct after a bacterium was shown to be the etiological agent and the first successful treatment with chloramphenicol was performed in 1952. In 1992, it was shown that the bacterium Tropheryma whipplei was the etiological agent. Today, the polymerase chain reaction is commonly used to assist in the diagnosis of this previously fatal disease. It is of interest that Whipple’s description was not the first one of this disorder, since the first description was by Allchin and Gebb in the United Kingdom in 1895 [16]. A great deal of the work on the characterization of the etiological agent for Whipple’s disease occurred after the death of Dr. Whipple [17]. Ultrastructural and other studies have shown lipid deposition in the intestinal mucosa, mesenteric and retroperitoneal lymph nodes. The glycoproteins within macrophages stain positive with periodic acid–Schiff (PAS) stains. Electron microscopic studies revealed a Gram-­ positive bacterium as the etiological agent. In 1992, molecular sequencing revealed a 16s ribosomal RNA with homology to actinomycetes. Treatment includes an induction therapy followed by several months of suppressive maintenance therapy to prevent relapse. Relapse is often characterized by neurologic symptoms. Neurologic symptoms may rarely indicate primary central nervous system Whipple’s disease. Cardiac involvement occurs in some patients and ocular involvement may also occur. Whipple was very productive over many decades and published over 300 papers on blood formation, hemoglobin, plasma protein synthesis, and metabolism. He gave the name of thalassemia to the Mediterranean anemia of Cooley which was among his many contributions to hematology and general pathology. The major studies that led to the Nobel Prize in 1934 involved his studies of biliary pigments in the liver as related to hemoglobin synthesis in a canine model. His studies of anemia as a deficiency of hemoglobin used a dog model which was made anemic by blood-letting. His search for nutrients to reverse the anemia led to the use of raw liver as an efficient way to stimulate blood regeneration. The two other investigators that shared the Nobel Prize with Whipple applied his experimental approach to the study of pernicious anemia in human patients. Whipple had encouraged physicians to consider dietary factors in managing anemic patients based on his work in dogs [18]. There has been some controversy by scholars as to whether Whipple should have shared the Nobel Prize with Murphy and Minot or if another investigator, William Castle from Harvard, should have been the third recipient for his discovery of intrinsic factor in pernicious anemia [15]. William Murphy who had started medical practice 1  year earlier after finishing his training was working with George Minot at Peter Bent Brigham and they had used beef liver for several months to cure patients with pernicious anemia. Minot had credited Whipple for his role of nutrition in treating pernicious anemia [18]. However, detractors had pointed out that Whipple was studying iron-deficiency anemia in his dog model which was helped by ingesting liver while Murphy and Minot were studying vitamin B12 deficiency in their

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patients because of the absence of intrinsic factor in the gastric juice needed for absorption of vitamin B12. So, it was fortuitous that the large amounts of liver fed to the patient had a mass effect on B12 absorption, since only minute amounts of the vitamin were needed to cure the pernicious anemia. Whipple lived a long and productive life. After retiring as dean of the University of Rochester Medical School in 1953, he became an emeritus professor in 1955 and lived in Rochester, New  York, for the rest of his life. He continued doing some research for a while and enjoyed many outdoor activities. He died in 1976 at the advanced age of 98.

Renato Dulbecco (1914–2012) Although he was trained as an anatomic pathologist and worked as a pathologist briefly earlier in his career, Renato Dulbecco was a basic scientist whose research in virology and cancer biology contributed greatly to advances in these areas [19–21]. He was born in Catanzaro, Italy, at the start of World War I. He was an outstanding student and entered the University of Turin at age 16 to study medicine. He received his degree in morbid anatomy and pathology in 1936. He started military service as a medical officer and physician and was later discharged after which he returned to work in pathology. However, in 1939 he had a second round of military services at the inception of World War II and he served in France and Russia. In 1942 while on duty in the Russian offense he had a narrow escape on the front lines. He was hospitalized for several months and then sent home [19, 20]. He worked in a small village near Turin, Italy, and served as a physician to the local people resisting the German occupation. After the war, he returned to school to study physics for 2 years. He had been interested in physics and mathematics from his earlier days at the University. It was at the University of Turin that he became friends with two fellow students who also became Nobel laureates, Salvador Luria and Rita Levi-Montalcini. They both had a great deal of influence on his career as a basic research scientist. In 1946, he was invited to the USA by Luria. Dulbecco joined Luria at the University of Indiana in Bloomington [19–21]. He accepted and started working on bacteriophages in Luria’s lab. During this time at Indiana, he shared a lab with another future Nobel laureate, James Watson. In his early research, Dulbecco discovered that visible light could reactivate bacteriophages that had been inactivated by ultraviolet light. These observations contributed to his growing fame and he was invited by Max Delbruck at California Institute of Technology (Caltech) to be a research fellow. He accepted the position in 1949. At Caltech, he worked on animal viruses and was the first person to develop a plaque assay for various viruses. This was a powerful technique that could count viruses to determine the number of biologically active virus particles. He also worked on developing quantitative assays for poliomyelitis, which was a very common infection in the 1940s and 1950s, as well as for other polyoma viruses. His work was in the animal virus field. One of the reasons that he became involved in this area was that a wealthy citizen had given

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Caltech funding in this area. His medical background probably had some influence in the decision to pursue this line of research. His work at Caltech was so outstanding that he was promoted to associate professor and then full professor. In 1963, he left Caltech for Salk Institute which was founded by Jonas Salk, who had developed the famous Salk polio vaccine. He continued working on viruses and their role in cell transformation to cancer at the Salk Institute [22]. In 1972, Dulbecco moved to the United Kingdom to work at the Imperial Cancer Research Fund and started working on breast cancer including the role of stem cells in these cancers. He identified tumor-initiating cells (stem cells) in rat mammary cancer [23]. He subsequently returned to the USA and became president of the Salk Institute in 1988. He won the Nobel Prize in physiology or medicine in 1975 for his work on viruses, having shown that genetic material from viruses could be incorporated into human cells as one host organism and that this could lead to abnormal growth in the host. The two other laureates in that year included Howard Temin who had trained in his laboratory at Caltech and David Baltimore with whom he had also worked earlier in his career. Temin and Baltimore had both discovered reverse transcriptase in viruses independently. Reverse transcriptase proved to be indispensable for the development of the polymerase chain reaction about a decade and a half later [24]. Dulbecco lived a long and very productive life, dying 3  days before his 98th birthday in 2012. Verma considered him to be a giant in the field of cancer biology who was distant yet approachable [19]. While David Baltimore considered him to be “a gentle superman—both gentle and remarkable” [20].

Baruj Benacerraf (1920–2011) Although he received some training in pathology after medical school, Baruj Benacerraf was primarily a basic scientist who rose to great heights in his chosen field by hard work and dedication to his students. He was born in Caracas, Venezuela, in 1920, 2 years after the end of World War I. His father was originally from Spanish Morocco and his mother was from French Algeria. The family moved to Paris when Baruj was 5 years old, so all of his primary and secondary education was in Paris. The family returned to Venezuela in 1939 just before the inception of World War II. His father was a successful businessman. The family moved to New York in 1940 and Baruj enrolled at Columbia University where he earned his BS degree in 1942 as a pre-med student. Although he was an outstanding student, he was admitted to only one medical school at the last minute because of the discrimination suffered by Jewish students in that era. He indicated that he was only granted an interview at the Medical College of Virginia because of the father of a close friend [25, 26]. Since his medical training occurred during the war years, he was drafted and was able to complete medical school in 3 years. He did an internship at Queens General Hospital in New York. He trained in pathology at Columbia University School of Physicians and Surgeons, and then became a first lieutenant in the US Army Medical Core in 1946. Dr. Benacerraf decided to become a medical researcher rather than to practice

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medicine. He chose immunology as his field of interest partly because of his history of bronchial asthma from childhood and his curiosity about the biology of allergies. An important person in his scientific training was Elvin Kadat, an immunochemist at Columbia University School of Physicians and Surgeons. Dr. Kadat practiced a very rigorous approach to scientific problems and stressed a quantitative approach to important scientific problems. In 1949, Benacerraf moved to Paris and worked in Bernard Halpers’ laboratory studying reticuloendothelial function and its role in immunity. However, after a few years he realized that there was a dearth of laboratory openings in France and he decided to return to the USA. With the help of Lewis Thomas, he was able to get a position at New York University where he studied hypersensitivity mechanisms at a cellular and humoral level. Although he had continued assisting in his father’s business it was around this time that he decided to become more focused on his basic research in immunogenetics. Dr. Benacerraf had extensive experiences at other institutions. He spent some years at the National Institute of Allergy and Infectious Disease at the National Institutes of Health. In 1970, he accepted the position as chair of pathology at Harvard Medical School. He indicated that one of the reasons for going to Harvard was that he had missed the university environment and interacting with students and fellows. At Harvard he started an interdepartmental immunology graduate program and this influenced the increased attention to immunology, which was rapidly growing field. He also continued his research on immune response genes at Harvard [27]. This work had many practical applications including the regulation of transplant rejection. Organ transplantation was a rapidly growing field at this time. His work on immunoglobulins and antigens controlled by dominant autosomal genes or immune response genes that were located in the major histocompatibility complex of mammals led to his receiving the Nobel Prize in 1980 along with two other colleagues [26–28]. After winning the Nobel Prize in 1980 he continued to receive many other awards and important positions including president of the Sidney Farber Cancer Institute in 1980, election to the Institute of Medicine in 1985 and president of the Dana-Farber Cancer Center from 1980 to 1992. He died on August 2, 2011 [28].

John Robin Warren (Born 1937) John Robin Warren is a diagnostic pathologist from Australia. He did not have a research laboratory to perform experimental work like most Nobel laureates in the sciences. He was a practicing pathologist who used his powers of observation and his determination to solve a common problem to track down the etiology of peptic ulcers and gastritis. Together with his friend and colleague Barry James Marshall, they solved one of the medical problems that had challenged medical science for many years. Robin Warren was born in 1937 in Adelaide, Australia. Many of his immediate ancestors were in the medical profession and that undoubtedly influenced his decision to pursue a career in medicine [29, 30]. He was an avid reader. At the age of 10,

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he received a Kodak box camera for his birthday. That gift started his lifelong hobby and attraction to photography. He was usually in the top level of his class at school, although for one period he was one of the students in the bottom of his class. That experience motivated him to always try to do his best academically. After high school, he received a commonwealth scholarship to attend the university. While growing up, he had suffered a grand mal epileptic seizure, but fortunately this was not recurrent after he was placed on seizure medications. He entered medical school at Adelaide University in 1955. After 6 years of medical school, he became a junior medical officer. As a resident he worked between 100 and 120 h each week with minimal pay, but the young doctor enjoyed the hard work. He married his wife while he was a resident and they had five children. Since his wife was training to be a psychiatrist, that made it challenging for both of them to manage with so many young ones. He received pathology training as a registrar in clinical pathology at the Institute of Medical and Veterinary Science which included the Royal Adelaide Hospital. He and his family moved to Melbourne as a clinical pathology registrant at the Royal Melbourne Hospital for further training in morbid anatomy and histopathology. After 4 years of training, he became a fully-fledged pathologist. In 1968, he and his family moved to Royal Perth Hospital where he started his practice of pathology. During the 1970s, he became interested in the new technology of endoscopic gastric biopsies. In 1979, he noticed bacteria growing on the surface of gastric biopsies and started to make a systematic study of all the gastric biopsies submitted for diagnosis. He met Barry Marshall, a gastroenterologist, in 1981 and the two men started collaborative studies of gastric biopsies [29, 30]. The collaboration was slow initially, but became more intense with time. Together they studied the clinicopathological aspects of duodenal ulcers in gastric biopsies with good follow-up data of the patients. Their interest and areas of expertise were complementary. They correlated the histopathological, ultrastructural, microbiological findings with the endoscopic findings, clinical histories and follow-up of patients with gastric ulcers and were able to link the infections to duodenal ulcer and successfully cultured the organisms (Helicobacter pylori). Marshall even infected himself with Helicobacter and became symptomatic. In the early 1980s, they published two papers in the Lancet [31]. Their findings were only recognized slowly at first, but then their observations became highly cited over the following decade. During this time, he and Marshall studied the effects of eradicating the bacteria and the relapse rate of duodenal ulcers over a 7-year period. They showed that with successful treatment of the infection, recurrence of peptic ulcer was uncommon. They even developed a laboratory test with other colleagues to detect the Helicobacter organism in patients. By 1990, the medical community had corroborated their findings multiple times with many studies. They traveled extensively giving lectures about their findings in many countries. Unfortunately for Warren and his family, after a trip to Japan in 1996, his wife was diagnosed with pancreatic cancer and died a few months later. Warren retired from his practice of pathology shortly after his wife’s demise. Warren and Marshall were awarded the Nobel Prize in physiology or medicine in 2005 for their discovery of Helicobacter as the etiological agent of peptic ulcers.

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Subsequent work by other investigators showed the relationship between Helicobacter-associated peptic ulcers and cancers such as gastric lymphomas and adenocarcinomas [32, 33]. These later findings reemphasized the early reports of Fibiger (which were unfortunate misinterpretations of pathological findings in the stomach) indicating that organisms could be linked to gastric cancers [10–12].

References 1. Lichtman MA. Alfred Nobel and Prizes: from dynamite to DNA. Rambam Maimonides Med J. 2017;8(3):e0035. https://doi.org/10.5041/RMMJ.10311. 2. Fant K. Alfred Nobel. A biography. New York: Arcade; 1993. 3. Nicholls M. Alfred Nobel founder of Nobel Prize. Mark Nichols profile the life and work of Alfred Nobel and the background to the establishment of the coveted Nobel Prize. Eur Heart J. 2019;40(17):1315–7. 4. Hajdu SI. Nobel laureate pathologist. Ann Clin Lab Sci. 2009;39(2):196–8. 5. Sedivy R. Landsteiner Karl (1868–1943). In: van den Tweel JG, editor. Pioneers in pathology. In: van Krieken JHJM, series editor. Encyclopedia of pathology. Cham: Springer International Publishing; 2017. p. 322–27. 6. Gottlieb AM.  Landsteiner, the melancholy genius: his time and his colleagues, 1868-1943. Trans Med Rev. 1998;12(1):18–27. 7. Zetterstrom R. Alfred Nobel’s will and the Nobel Prize to Karl Landsteiner for the discovery of human blood groups. Acta Paediatr. 2006;97(4):396–7. 8. Bayne-Jones S.  Dr. Karl Landsteiner- Nobel Prize laureate in medicine, 1930. Science. 1931;73(1901):599–604. PMID 17839684. 9. Hrobjartsson A, Gotzsche PC, Gluud C. The controlled clinical trial turns 100 years: Fibiger’s trial of serum treatment of diphtheria. BMJ. 1998;317(7167):1243–5. https://doi.org/10.1136/ bmj.317.7167.1243. 10. Fibiger J. On Spiroptera carcinomata and their relation to true malignant tumors, with some remarks on cancer age. J Cancer Res. 1919;4(4):367–87. 11. Stolt C-M, Klein G, Janssen ATR. An analysis of a wrong Nobel Prize—Jonannes Fibiger, 1926: A study in the Nobel archives. Adv Cancer Res. 2004;92:1–12. 12. Hitchcock CR, Bell ET.  Studies on the nematode parasite Gongylonema Neoplasticum (Spodoptera Neoplasticum) and avitaminosis A in the forestomach of rats: comparison with Fibiger’s results. J Natl Cancer Inst. 1952;12(6):1345–87. 13. Malat SN, Real FX.  Epidemiology of bladder cancer. Hematol Oncol Clin North Am. 2015;29(2):177–89. 14. Sripa B, Brindley PJ, Mulvenna J, et al. The tumorigenic liver fluke Opisthorchis viverrini-­ multiple pathways to cancer. Trends Parasitol. 2012;28:395–407. 15. Damjanov, I. Whipple George Hoyt (1878–1976). In: van den Tweel JG, editor. Pioneers in pathology. In: van Krieken JHJM, series editor. Encyclopedia of pathology. Cham: Springer International Publishing; 2017. p. 553–5. 16. Morgan AD. The first recorded case of Whipple’s disease? Gut. 1961;2:370–2. 17. Afshar P, Redfield DC, Higginbottom PA.  Whipple’s disease: a rare disease revisited. Curr Gastroenterol Rep. 2010;12(4):263–9. 18. Minot GR, Murphy WP.  Treatment of pernicious anemia by a special diet. JAMA. 1926;87:470–6. 19. Nobel Foundation. Dulbecco, Renato Biographical. The Nobel Prize in physiology or medicine, 1975, Addendum August 2005. Stockholm: Nobel Foundation. Renato Dulbecco  – Biographical -­ NobelPrize.org.

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20. Verma IM. Renato Dulbecco (1914–2012) Molecular biologist who proved that virus-derived genes can trigger cancer. Nature. 2012;483(7390):408. 21. Baltimore D. Retrospective: Renato Dulbecco (1914–2012). Science. 2012;335:1587. 22. Kevles DJ. Renato Dulbecco and the new animal virology: medicine, methods, and molecules. J Hist Biol. 1993;26(3):409–42. https://doi.org/10.1007/BF01062056. PMID: 11613167. 23. Zucchi I, Sanzone S, Astigiano S, et al. The properties of a mammary gland cancer stem cell. Proc Natl Acad Sci U S A. 2007;104(25):10476–81. 24. Mullis KB. Target amplification for DNA analysis by the polymerase chain reaction. Ann Biol Clin (Paris). 1990;48(8):579–82. 25. Odelberg W, editor. Les Prix Nobel. The Nobel prizes 1980. Stockholm: Nobel Foundation; 1981. 26. The Nobel Lectures in Immunology. The Nobel Prize for physiology or medicine, 1980 awarded to Baruj Benacerraf, Jean Dausset & George D. Snell. Scand J Immunol. 1992;35(4):373–98. 27. Germain RN, Paul WE.  Baruf Benacerraf (1920–2011) immunologist who won the Nobel prize for genetics of T-cell antigen recognition. Nature. 2011;477:34. 28. Benacerraf B. When all’s said and done…. Ann Rev Immunol. 1991;9:1–26. 29. Hellstrom PM. Spotlight on gastroenterology – the Nobel laureates in physiology or medicine 2005: John Robin Warren and Barry James Marshall. Scand J Gastroenterol. 2005;40:1383–5. 30. Van Der Weyden MB, Armstrong RM, Gregory AT. The 2005 Nobel prize in physiology or medicine. Med J Austr. 2005;183:612–4. 31. Warren JR, Marshall B. Unidentified curved bacilli gastric epithelium in active chronic gastritis. Lancet. 1983;321(8336):1273–5. 32. Yakirevich E, Resnick MB. Pathology of gastric cancer and its precursor lesions. Gastroenterol Clin N Am. 2013;42(2):261–84. 33. Isaacson PG, Spencer J.  Gastric lymphoma and Helicobacter pylori. Important Adv Oncol 1996;111–21.

Chapter 23

Future Directions in Pathology

Histochemical stains have been very useful in the development and for advances in pathology, especially in anatomic diagnostic pathology. However, they no longer represent the state-of-the-art techniques in diagnostic pathology. Immunohistochemical stains remain an important diagnostic tool in anatomic pathology that is used daily by diagnostic pathologists [1, 2]. A few histochemical stains are also very helpful for diagnostic pathology especially in medical renal pathology, neuropathology, and in a few other areas of anatomic pathology. Newer developments in diagnostic pathology that are leading to major advances in the field include molecular immunohistochemistry, molecular diagnostics, and digital pathology. These new areas will continue to lead to the development of new diagnostic techniques to make advances in the classification of disease processes, especially in anatomic pathology for the foreseeable future. These fields will also help to advance newer disease classifications in many areas of pathology.

Molecular Immunohistochemistry Molecular immunohistochemistry is an emerging field that combines traditional immunohistochemistry with molecular diagnostics. This combination of techniques represent a fusion of traditional classical immunohistochemistry with molecular diagnostics and should remain in the forefront as advances in pathology are made over the next few decades. Molecular immunohistochemistry provides genetic information about disease processes. Many of these procedures are used to analyze neoplastic tissues. One common application that has been developing for a few decades is to use tissue sections or cytological preparations to demonstrate pathologic changes in tissues such as gene translocations, chromosomal translocations, and genetic mutations. An example of the immunohistochemical detection of translocations in tissue sections is the detection of BCL2 chromosomal translocation in © The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 R. V. Lloyd, Pathology: Historical and Contemporary Aspects, https://doi.org/10.1007/978-3-031-39554-3_23

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follicular lymphomas [3]. The translocation of chromosomes such as t(14:18) (q32q21) occurs in around 70–90% of follicular lymphomas and is relatively uncommon in most other lymphomas. In this translocation, the BCL2 gene on chromosome18 goes to the IGH gene on chromosome 14. This leads to the overexpression of BCL2 protein in the tumor cells of follicular lymphomas. The diagnosis of follicular lymphoma is assisted by BCL2 immunostaining in the follicular center cells. The detection of this translocation by molecular immunohistochemistry is thought to be more sensitive than the polymerase chain reaction, since the morphological findings can be combined with the immunohistochemical visualization of the positively stained cells [3]. Antibodies have been used to detect epidermal growth factor receptor mutations in some lung cancers and in other malignancies and these analyses have significant therapeutic implications. Mutations in exons 19 and 21 of EGFR are some of the most common ones and are useful in the treatment of lung cancers and other cancers with these mutations [4]. Other immunohistochemical studies that correlate very highly with molecular analysis include the BRAFV600E mutation in many human cancers including papillary thyroid carcinoma of the thyroid, ameloblastomas, colonic and ovarian adenocarcinomas, and malignant melanomas [4, 5]. The correlation between immunohistochemical analysis and molecular analysis is usually greater than 95% [5]. The use of molecular immunohistochemistry to detect gene rearrangements and mutations is usually less expensive and easier to perform in the pathology laboratory, so these methods will continue to increase in diagnostic pathology. It is important to note that initial experiments that are not successful in molecular immunohistochemistry may not be the final answer for advances in this field. For example, antibodies to detect chromosomal translocations in synovial sarcomas reported a few years ago were not very successful because of a relative lack of specificity [6]. However, recent studies have generated highly specific monoclonal antibodies to detect t(x;18) (p11q11) translocations that are highly sensitive (95%) and highly specific (100%). This has made molecular immunohistochemical approaches to the diagnosis of synovial sarcomas, simpler and easier to perform in the pathology laboratory in the future [7]. Future applications of this approach in the pathology laboratory will include generation of many more highly specific monoclonal antibodies that can be readily applied to formalin-fixed paraffin embedded tissue sections that are routinely available in pathology laboratories. When coupled with automated immunohistochemical staining machines, this becomes a powerful approach for diagnosis in the pathology laboratory. In addition, the tumor cells that show the molecular alterations can be easily visualized under the microscope. With these tools, studies of tumor heterogeneity become a lot easier to perform.

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Multiplex Immunohistochemistry and in Situ Hybridization Earlier studies with immunohistochemistry and/or in situ hybridization to detect protein and RNA or DNA in tissue sections usually focused on one target in the tissue section. There were a few exceptions such as using three antibodies to assist in the diagnosis of prostate cancer by immunohistochemistry, but this approach was rather limited. In addition, these techniques using antibodies or nucleic acid probes were all directed at the same target rather than multiple structures within the same cell. Multiplex immunohistochemistry with or without immunofluorescence labeling can be used to detect multiple antigens in the same tissue sections and usually in different cells within the tissues [8, 9]. Antibody or nucleic acid detection is usually combined with sophisticated imaging techniques that allows for a more detailed and sophisticated analysis about the interaction of different molecules within the tissues being studied. Some of the early work with multiplex immunohistochemistry have focused on analysis of various tumor infiltrating inflammatory cells such as B and T cells as well as other inflammatory cells. Targets include program cell death protein-1 (PD-1) and program cell death ligand-1 (PDL-1). PD-1 and PD-L1 have also been analyzed to study the interaction of tumor cells with inflammatory cells [10, 11]. These studies can be used as predictive as well as prognostic markers for specific cancer types [11]. The analyses can be done with digital spatial profiling for proteins and/or RNA/DNA targets and with other methods [10]. It is possible to analyze multiple tissue sections with automated methods over a 24-h period [10]. These multiplexing techniques should soon be more widely applicable for clinical practice as well as for research. In a recent study examining tumor mutational burden, gene expression profiling for interferon gamma gene signatures, PD-L1 immunohistochemistry and multiplex immunofluorescence/multiplex immunohistochemistry analyses showed that multiplex immunofluorescence and multiplex immunohistochemistry were more effective than other approaches in predicting objective response to anti-PD-L1 therapy [12]. This approach supports the importance of both biomarker expression and spatial distribution interactions in the tumor microenvironment [13]. The tumor microenvironment is involved in many interactions between tumor cells and the host cells. It includes B and T lymphocytes, natural killer cells, dendritic and other inflammatory cells such as macrophages and white blood cells as well as PD-1 and PDL-1, stromal cells and cytokines. Studies of tissue specimens after treatment have provided insights into the way immunotherapy can remodel the tumor microenvironment as well as the mechanisms of action of thee targets on the effectiveness of specific therapies [13]. The use of multiplex immunofluorescence or immunohistochemistry with in situ hybridization has been reported in recent studies to localize specific RNAs and various proteins. In situ hybridization has been used to localize specific RNAs and various proteins within tissue sections such as kidney biopsies [14]. These combined procedures will become more useful in the future to analyze the spatial distribution and interactions of various target molecules within cells in both diagnostic and

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research pathology [14, 15]. An example of one of these approaches used GeoMx digital profiling from nanotechnology which allowed detection of nearly 100 proteins or 1000 cancer transcriptome-related RNA targets. Protocols such as GeoMx have already been used in clinical studies to show the prognostic value of tumor-­ infiltrating lymphocytes in solid tumors and in hematologic malignancies [16]. We can anticipate many more studies like these in the near future.

Molecular Diagnostics The sequencing of the human genome in 2001 was a great stimulus for the continuing development of molecular biology. This achievement accelerated the adaptation of molecular techniques in molecular medicine and molecular pathology [17–19]. Molecular techniques are now becoming a standard method of analysis in pathology, especially with the continued decrease in the cost of sequencing by whole-­ exome or whole-genome methods [20]. Because pathologists are in charge of the diagnoses of tissue biopsies and tissue resections, they have become intimately involved in molecular diagnostics which is used for the diagnosis and treatment of an ever-increasing number of diseases [21]. One unique contribution of molecular diagnosis in pathology is to provide a greater level of certainty about specific diagnoses that cannot be obtained with other approaches. Molecular diagnoses have also contributed greatly to the diagnosis of new entities in anatomic pathology. This has been noted in the more precise classification of lesions in hematopathology and in the central nervous system and soft tissue tumors as well as in most other subspecialties in diagnostic surgical pathology. Tumor classification in neuropathology has been revolutionized with the inclusion of molecular diagnostics for specific tumor types [22]. For example, the use of isocitrate dehydrogenase mutations in the classification of gliomas and the use of both co-deletion of chromosomes 18/19 in combination with isocitrate dehydrogenase mutations are routinely used in the classification of oligodendrogliomas [22]. Similarly, low-grade pediatric gliomas have unique features compared to tumors in adults with driver events involving genes such as FGFR1, BRAF, or MYB/MYBL1 [22]. Thus, a wide variety of biomarkers are currently used for more reliable and reproducible classification of gliomas which extends beyond the historical morphological classification [22]. With rapid advances in molecular diagnostics and tumor analyses, we can anticipate further advances in molecular tumor analysis and classification in most areas of pathology, especially oncologic pathology. The practicing pathologist will continue to have a major role in deciding the portion of tumors to analyze, in assessing tumor heterogeneity and in the correct morphological classification of tumors as well as tissue quality with respect to preservation of nucleic acids for the optimal analysis for therapeutic purposes. Some molecular techniques have been used for more than a decade. For example, the detection of microsatellite instability (MSI) with abnormal mismatch repair of DNA has been associated with Lynch syndrome for many years [23]. Microsatellites

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consist of 10–60 base pair regions of DNA that contain multiple repeats of 1–5 base pair motifs. Microsatellites occur at microsatellite loci which are widely dispersed in the genome. Impairment of MSI impairment can make cells unable to regulate the length of microsatellites during cell division [24]. Patients with MSI-high have a better prognosis compared with MSI stable tumors. Patients with MSI-high colon cancers are more susceptible to immune enhancer therapy such as the PDL-1 inhibitor pembrolizumab which has been approved for unresectable or metastatic solid tumors [25]. Cells with abnormal microsatellite repair have proteins that cannot bind to mismatch repair errors in DNA to correct the mismatch by excision of the error and repairing the mismatch, so the cells accumulate errors instead of repairing the mismatch. Although first described in Lynch syndrome involving colonic adenocarcinoma, many other tissues and tumors have been shown to be affected by lack of mismatch repair including ovarian, endometrial, adrenal cortical, and many other types of cancers [23]. Although immunohistochemistry can be used to detect MSI, molecular analysis with the polymerase chain reaction is a more reliable method to analyze for MSI using immunohistochemistry as an initial screening method. We can anticipate more tumor types will be discovered with MSI in the future and that unresectable tumors may be treated with immunotherapy to improve patient prognosis. Currently, computerized methods are being developed that analyze next-generation sequencing data to detect MSI in tumors [24]. Another area with great advances in pathology which has a promising future is next-generation sequencing (NGS), also known as massive parallel sequencing. In this procedure, NGS captures the individual sequences of many millions of DNA molecules from multiple targets. With this approach, detection of low-frequency allelic variants is much easier. NGS is relatively flexible, since it can be done on whole exomes, on whole genomes or on a targeted gene panel. The use of NGS is also flexible, since it can be used to detect mutations in cancers as well as analyze many other diseases [26]. Pathologists play a major role in NGS sequencing, since they can examine the tissues to be analyzed and avoid using necrotic or partially degraded tissues as well as selecting tissues with adequate amounts of tumor or related samples for the analysis. If unaffected tissues that do not involve the disease process are used for analysis, the results will produce false-negative results in spite of the sensitivity and sophistication of the NGS protocol. Although a growing number of applications are being reported, we can anticipate that NGS will be applied to tissues with limited amounts of specimens such as cytological specimens and small tissue biopsies [27]. Earlier studies of NGS for advanced non-small-cell lung cancer therapeutic treatment with predictive biomarkers illustrate the power and utility of this approach [28]. As the field advances, NGS may be useful not only to predict tumor response as targeted treatment in patients with advance cancers, but also in borderline cases with risk factors that may be of undetermined significance [27]. As NGS becomes even less expensive and more sensitive, it will be used also for predictive treatment response and assist pathologists in defining malignancy risk with borderline and in other indeterminate cases.

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Digital Pathology Digital pathology is the branch of pathology that uses digitized glass slides usually made with a whole slide image scanner and then analyzes the image with a computer. The digital slide can be analyzed by artificial intelligence (AI) using a machine to perform cognitive tasks to reach a specific goal with the data provided. One of the major goals in pathology is to use AI to assist in the diagnosis and grading of cancers or to detect metastatic malignancies in tissues such as lymph nodes with only a small focus of metastatic malignancy that is usually difficult for the pathologist to detect without exhaustive examination of the tissue. The use of AI is stimulating many advances in health care and in many areas of medicine and pathology [29]. The development of ever-increasing computer power, sophisticated pattern recognition algorithms, and advanced imaging processing software have all contributed to computer-based systems that can perform complex analyses in medical imaging and bioinformatics [29]. The computer can scan billions of unstructured information, extract the relevant information, and recognize complex patterns generated by “big data.” These systems use machine learning (ML) to recognize complex patterns such as seen in histopathological slides. These approaches have begun to revolutionize diagnostic pathology [28, 29]. The use of digital pathology analysis includes digitizing histopathologic slides with whole-slide scanners and subsequent analysis of the slides with whole-slide images assisted by computer analysis [30–32]. Digital pathology started a few decades ago when investigators developed techniques to analyze images from a microscopic field of blood smears and then converting the optical data from the images into a matrix of optical density values and examined different cell types based on information in the scanned images [31]. With the use of AI, this branch of computer science defines a machine-based approach that is used to make specific predictions that can analyze data just as an intelligent human might do under similar circumstances [31]. The data is fed into a machine to enable machine learning. Deep learning is a specific machine-learning approach using artificial neural networks or artificial neural architecture that simulates those in humans [31]. Digital pathology has been aided by these advances, especially from deep learning such as training machines to make diagnoses from slide images in the same way that pathologists make diagnoses from glass slides. A major advantage to using AI in digital pathology is that it decreases a large number of time-consuming tasks that pathologists perform daily. With this extra assistance, the pathologist can focus on high-level decision-making tasks [31]. For example, instead of spending a great deal of time scanning a glass slide with a lymph node under the microscope from a patient with prostate cancer to evaluate for metastatic disease, this step can be done by AI [31]. The detection of these small foci of metastatic carcinoma is extremely important, since it will determine the staging and treatment of the patient with metastatic disease. The pathologist can then check the findings of the machine for accuracy to validate them and can then concentrate on integrating all of the information from the case (synoptic coding) used in staging and treatment of the patient with known metastatic disease.

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One of the areas in which AI has been used in digital pathology is in the diagnosis and Gleason grading of prostate cancer biopsies and radical resections [32–35]. Historically, there has been a great deal of variability among pathologists in the Gleason grading of prostate cancer and this can lead to undertreatment or overtreatment of patients with the incorrect diagnosis. Gleason grading studies with AI using specific algorithms for prostate cancer diagnosis and grading have shown that a sensitivity of 97.7% and a specificity of 99.3% can be readily achieved [34]. Another study using a large series of cases (4712 cases) of prostate biopsies with a deep learning system compared to three urologic pathologists achieved a kappa statistic of 0.854 in diagnosing and grading prostate cancer biopsies which is an excellent result and helps to validate this approach [35]. In areas where the diagnosis of a lesion can be very subtle and present difficulties, even for experienced pathologists, deep learning can also assist in these analyses. For example, a newly described thyroid neoplasms designated as follicular neoplasm with papillary-like nuclear features (NIFTP) has been analyzed by artificial intelligence. The nuclear features assist in making the diagnosis, and these features are sometimes difficult to detect even by experienced pathologists. The reason to make this distinction is that patients with NIFTP are treated more conservatively than patients with conventional papillary thyroid carcinoma. They receive only a thyroid lobectomy and no radioactive iodine follow-up treatment unlike some patients with papillary thyroid carcinoma who sometimes receive a total thyroidectomy and may sometimes receive follow-up radioactive iodine treatment [36]. A recent study with digital pathology showed that using AI, histological features associated with NIFTP could be evaluated by machine learning to distinguish NIFTP from papillary thyroid carcinoma [37]. This is a significant achievement, since cytological examination of thyroid lesions cannot distinguish between these two lesions and whole tissue sections with examination of the entire capsule of the tumor is needed to make that distinction in some cases. Even with whole section analysis some pathologists cannot make this distinction which is important for the clinical and surgical treatment of these patients. Analogous findings in many subspecialty areas of surgical pathology can be anticipated in the future with the assistance of digital pathology and AI. Another area where the application of AI will have a great impact in the future will be in combining genomics with reliable image registration methods [30]. There are many variables in histological analysis such as tissue heterogeneity, staining quality, thickness of the sections, and tissue processing. Future studies will have to address all of these variables to make the final diagnosis with digital pathology when using deep learning. These issues have started to be addressed [32]. In one study, these variables were analyzed with independent patient cohorts and the original training cohort for control purposes. It was discovered that many histological artifacts can lead to losses in model performance depending on the severity of the artifacts [32]. The authors recommended stress-testing diagnostic models using synthetically generated artifacts to improve diagnostic accuracy with digital pathology [32]. Future studies in digital pathology and artificial intelligence may incorporate this model with synthetic

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artifacts to improve diagnostic accuracy and reliability with artificial intelligence analyses. The future of digital pathology with AI and machine learning in diagnostic pathology is very promising. Numerous challenges remain before this approach becomes a standard procedure in diagnostic pathology. However, the potential utility of AI and machine learning in diagnostic pathology holds much promise and should help to revolutionize medicine and pathology by improving diagnostic accuracy and lead to increased efficacy in making histological diagnoses with improved patient diagnosis and treatment [38, 39].

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Correction to: Breast Pathology Ricardo V. Lloyd

Correction to: Chapter 7 in: R. V. Lloyd, Pathology: Historical and Contemporary Aspects, https://doi.org/ 10.1007/978-­3-­031-­39554-­3_7 In Chapter 7, one of the authors noticed that there is an error with their figure numbering and figure legend and has requested for corrigendum. The correct information has been given below: In Section “Breast Carcinomas” and Subsection “Ductal Carcinoma In Situ” on page 58, the figure numbers listed as (Figs. 7.2 and 7.3) have been replaced with (Fig. 7.2). The legend of Fig. 7.3 has been updated as “Invasive ductal carcinoma with infiltration of stroma by carcinoma”. Previously, it was “Ductal carcinoma in situ of breast showing nests of proliferating tumor cells without stromal invasion”. In the sentence on page 59, line 05, Ductal carcinoma in situ is more frequently associated with calcifications seen mammographically compared to invasive ductal carcinoma, figure citation (Fig. 7.3) has been added.

The updated original version of this chapter can be found at https://doi.org/10.1007/978-­3-­031-­39554-­3_7 © The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 R. V. Lloyd, Pathology: Historical and Contemporary Aspects, https://doi.org/10.1007/978-3-031-39554-3_24

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Appendices

Appendix 1: Pathology Organizations There are several major pathology organizations. Most of these were formed early in the twentieth century, so they have been in existence for a relatively long period of time. All of these major organizations have thousands of pathologists or researchers in pathology as members. The organizations also publish scholarly journals and/ or other publications that are subscribed to by members of their society and other interested parties. American Society of Investigative Pathology The American Society of Investigative Pathology (ASIP) is a society of biomedical scientists who perform research focused on elucidating the mechanisms involved in the development of human diseases. Many researchers currently study the cellular and molecular pathobiology of these diseases. One of the ancestral societies of the ASIP included the Boston Society of Medical Sciences which began in 1869 by a group of Harvard Medical School Faculty. Predecessors of the ASIP, the American Association of Pathologists and Bacteriologists (AAPB) began in 1900 and the American Society of Experimental Biology (FASEB) was founded in 1912 by the American Physiological Society, The American Society of Biological Chemistry (Currently the American Society of Biochemistry and Molecular Biology) and the American Society of Experimental Therapeutics all had some influence on the development of the ASIP [1, 2]. A leading group of experimental pathologists met to create the American Society for Experimental Pathology in 1913. The founders included the Nobel laureate George H. Whipple and several other outstanding experimental pathologists. The American Society for Experimental Pathology held its annual meeting with the Federation of American Society for Experimental © The Editor(s) (if applicable) and The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 R. V. Lloyd, Pathology: Historical and Contemporary Aspects, https://doi.org/10.1007/978-3-031-39554-3

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Biology (FASEB) for many years. The ASIP was one of the members of FASEB, which included 27 societies with over 125,000 biomedical scientists from the USA and many other countries. The two branches of the ASIP’s genealogical tree, the American Association of Pathologist and Bacteriologists and the American Association for Experimental Pathology, merged in 1976 to become the American Association of Pathologists and this organization was incorporated in 1992 as the American Society of Investigative Pathology [1, 2]. The ASIP publishes two outstanding journals, The American Journal of Pathology and the Journal of Molecular Diagnostics. Membership in the ASIP has grown over the years and have included many outstanding scientists including eight Nobel laureates as well many other scientists who have won many prestigious national and international awards. United States and Canadian Academy of Pathology (USCAP) The International Association of Medical Museums (IAMM) was formed in 1906 by Maude Abbott, William MacCallum, and James Carroll in Washington DC. The goal was to form an international organization that allowed the exchange of museum materials for teaching and research [3]. Multiple IAMM divisions were established subsequently in many countries including Austria, Denmark, France, Holland, Italy, Japan, and Scotland [3]. The first meeting in 1907 attracted famous scientists such as Dr. William Osler and others. Dr. Osler had also played a major role in founding the IAMM while he was in Montreal, Canada because of his interest in pathology museums. The first president of the organization was Dr. James Carrell, a US Army physician who presided over the first meeting in 1907. The name of the IAMM was changed to the International Academy of Pathology (IAP) in 1955 and new constitution bylaws were formalized in 1969. The IAMM has played a major role in the continuing medical education of pathologists over many decades. The efforts of Dr. Mostofi from the Armed Forces Institute of Pathology helped to restructure the USCAP and IAP in 1986. The IAP was incorporated in 1955. But the USCAP Division was not incorporated until 1986. The USCAP Division had been previously known as the US/ Canadian Division of the International Academy. Upon incorporation in 1986 the US-Canadian branch of the IAP became the USCAP [3, 4]. The IAP has about 50 divisions throughout the world with over 18,000 members. The USCAP is the largest division with over 10,000 members. The USCAP emphasizes diagnostic pathology and clinical research as well as a large variety of courses at its annual meeting in the USA and Canada. The two major pathology journals sponsored by the USCAP include Modern Pathology and Laboratory Investigation. Modern Pathology emphasizes diagnostic pathology with current emphasis on molecular and digital pathology, while Laboratory Investigation emphasizes animal and human models of diseases.

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American Society for Clinical Pathology (ASCP) The American Society for Clinical Pathology (ASCP) was started in St. Louis Missouri in 1922. During this period, there were only 35–40 physicians listed as clinical pathologists [5]. Some of the initial goals of the ASCP included promoting the practice of scientific medicine by a wider application of clinical laboratory medicine to diagnose diseases, to stimulate and to encourage cooperation between original research in all branches of clinical pathology, and to encourage cooperation between the practitioner and the clinical pathologist [5]. One of the practical goals of the ASCP was to provide continuing education for pathologists and for laboratory professionals with workshops, symposia, and other means as well as with modern techniques such as teleconferences and e-courses. The first president of the ASCP was Dr. Philip Hillkowitz. In 1936, the American Board of Pathology was established by the ASCP so this added credibility and professionalism to diagnostic pathology. In 1941, Dr. Emma S. Moss from Charity Hospital in the Louisiana School of Medicine became the first female president of the ASCP and also of any major pathology organization. The ASCP helped to create the College of Pathologists (CAP) in 1946 which has become a major organization and leader in the field. The ASCP has over 130,000 members of physicians and laboratory professionals. The ASCP has also provided continuing medical education for pathologists and laboratory workers in the daily practice of diagnostic pathology. The organization publishes an important academic journal, The American Journal of Clinical Pathology. The ASCP is also a major publisher of medical textbooks and multiple other medical publications [5]. College of American Pathologists (CAP) The College of American Pathologists (CAP) was founded in 1946 and its inception was stimulated by the ASCP. This organization is certified by the American Board of Pathology and it is a leader in laboratory quality assurance [6]. The CAP inspects and accredits medical laboratories. The organization is a major advocate or high quality and cost-effective medical care. The CAP also provides proficiency testing to pathology laboratories and provides CAP laboratory accreditation. Starting in 1964, the CAP has published checklists with requirements for the performance of laboratory tests. Another major contribution of the CAP in anatomic pathology is the synoptic coding of pathology oncologic reports, which classifies tumors by topography, lymph node involvement, and metastatic disease. These synoptic codes are greatly appreciated by pathologist and clinicians who deal with oncologic diseases. In addition to regular publications for its members and other interested parties, the CAP also publishes a prestigious pathology journal, Archives of Pathology & Laboratory Medicine [6].

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References 1. Madara JL. A new editor on the occasion of the centennial celebration of the Journal (Maybe). Am J Pathol. 2001;159(4):1183–5. 2. American Society for Investigative Pathology: Investigating the pathogenesis of disease [Internet]. Bethesda: ASIP; 2014. An executive summary of the history of ASEP and its Successors. Available from https://web.archive.org//web//20180630173653/http://archive.asip. org/mission/. Accessed 20 Aug 2022. 3. Earle KM [Internet]. International Academy of Pathology; 1981. Our history. The International Academy of Pathology and the United States and Canadian Division. The first seventy-five years, 1906–1981. https://www.iapcentral.org/home/history. Accessed 20 Aug 2022. 4. Allen PW. How USCAP (the United States/Canadian Academy of Pathology) got its name. A real just told you so story. Ann Diagn Pathol. 2022;57:1–5, 151905. https://doi.org/10.1016/j. anndiagpath.2022.151905. Accessed 20 Aug 2022. 5. American Society for Clinical Pathology [Internet]. Chicago: American Society for Clinical Pathology; 2022. Marks the 100th anniversary of the American Society for Clinical Pathology; 2022. https://lp.ascp.org/ascp100anniversary/. Accessed 21 May 2023. 6. College of American Pathologists [Internet]. Northfield: College of American Pathologists. 2023. http://www.cap.org/. Accessed 12 Feb 2023.

Appendix 2: The American Board of Pathology The American Board of Pathology (ABPath) is a member of the 24-member board of the American Society of Medical Specialties. The ABPath was founded in 1946 and it provides official certification in many areas of pathology including Anatomic and Clinical Pathology, Neuropathology, and many subspecialty board certifications to qualified doctors including Doctors of Medicine and Doctors of Osteopathic Medicine [1]. The subspecialty areas of certification include the following: –– –– –– –– –– –– –– –– –– –– ––

Blood Banking/Transfusion Medicine Chemical Pathology Clinical Informatics (computer technology) Cytopathology Dermatopathology Forensic Pathology Hematopathology Medical Microbiology Molecular Genetic Pathology Neuropathology Pediatric Pathology

Many of these subspecialty areas are in Anatomic and Clinical Pathology. However, other areas of Anatomic Pathology such as Soft Tissue Pathology, Gastrointestinal and Liver Pathology, Breast Pathology, Gynecologic Pathology, and Cardiovascular Pathology do not have board certification to date.

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The American Board of Pathology met for the first time in Chicago, IL, in 1936. Individuals who receive a certificate are designated as Diplomates of the American Board of Pathology.

Reference 1. The American Board of Pathology. A Member Board of the American Board of Medical Specialties [Internet]. Tampa: American Board of Pathology; 2015. https://www.abpath.org/ index.php/abpath-­certlink. Accessed 22 Aug 2022.

Index

A Abbott, M., 75, 206, 207, 246 Adenocarcinoma clear cells, 45–46, 54 colonic, 89–90 endometrial, 47, 54 pancreatic, 93, 94 prostate, 67–69 Adenoma adrenal cortical, 136 follicular, 129 tubular, 87, 88 villous, 87, 88 Alanine aminotransferase (ALT), 192–193 Alzheimer’s disease, 115–117 American Board of Pathology (ABPath), 1, 16, 39, 218, 247–249 Ancillary diagnostic techniques, 28 Apharesis, 198 Artificial intelligence (AI), 3, 77, 149, 150, 240–242 Aspergillus, 183 Aspiration cytology, 27 Autopsy, 2, 6–8, 10, 18, 19, 21, 35–41, 53, 69, 70, 104, 116, 131, 135, 146, 152, 166, 167, 185, 205, 207, 209, 225, 226 Avicenna, 9 Azzopardi, J.G., 64–65 B Babes, A., 30 Bacteria, 3, 8, 79, 85, 86, 101, 103, 155–158, 161, 167, 173–175, 177, 178, 225, 231 numbers of, 173

Baltimore, D., 229 B-cell light chain restriction, 147 BCL2 immunostaining, 236 Benacerraf, B., 229–230 Benivieni, Antonio di Paolo, 35 Bethesda system, 28 Billingham, M.E., 213, 214 Biopsy fine needle aspiration, 149 BI-RADS, 57 Blackwell, E., 205 Bladder tumor, 70–71, 226 Blastomyces, 184 Blood components, 197, 198 Blood transfusions, 92, 122, 176, 192, 197 BRAFV600E, 131, 236 Brain tumors, 117–120, 123, 124, 208 Bright, R., 190 C Canavan, M.M., 205 Candida, 182 Carcinoma adenocarcinomas, 28, 47, 50, 67, 68, 93, 105, 106, 236 adrenal cortical, 136, 137 anaplastic, 28, 70, 93, 132–133 clear cell papillary renal cell, 72 follicular, 128, 131, 132, 241 hepatocellular, 90–93, 193 invasive ductal, 58, 59, 61–63 invasive lobular, 60–63 medullary thyroid, 2, 133, 134, 141 papillary thyroid, 129, 131, 141, 236, 241

© The Editor(s) (if applicable) and The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 R. V. Lloyd, Pathology: Historical and Contemporary Aspects, https://doi.org/10.1007/978-3-031-39554-3

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252 Carcinoma (cont.) renal cell, 72 serous ovarian, 48–51 squamous cell, 46, 71, 105, 106, 169, 184 Carcinoma of cervix, 46, 47, 53 Cardiac troponin, 191 Carney, J.A., 140–142 Celsius, C., 6 Chinese medicine traditional, 10 Chronic traumatic encephalopathy (CTE), 41, 116–117 Cirrhosis alcoholic, 90–92, 193 Clinical chemistry, 3, 189–194 Congo Red stain, 77 Coronavirus, 102, 165, 166 Cytopathology, 1, 3, 27–32 thyroid, 27, 28 D Diabetic nephropathy, 81–82 Diagnostic models, 241 DiMaio, V.J.M., 40, 41 DNA viruses, 161 Dorfman, R.F., 23, 146 Ductal carcinoma in situ, 58–60, 218 E E-cadherin, 60 Electron dense immune complex, 80 Endometriosis, 51–52 End stage renal disease, 82 Epidermal growth factor receptor (EGFR) antibody, 118, 236 Ewing, J., 19–21, 23 F Fibiger, J., 225, 226 Fibroadenoma, 57, 58 Fibrolamellar hepatocellular carcinoma, 91, 93 Flexner report, 36 Frozen section, 15, 16, 18, 20, 21, 139, 141, 194 Fungal infection, 103, 181–186 Fungi, 3, 155, 156, 161, 181–184 G Galen, 5, 6, 9, 35 Gastritis chronic, 85, 86

Index Gastroenteritis virus, 163–164 Gene rearrangement, 28, 147–148, 236 Gene therapy, 200 Gilbert-Barness, E., 212, 213 Gleason, D.F., 73–74, 241 Gleason grade prostate carcinoma, 68 Gliomas low grade, 117, 118, 238 high grade, 117 Glomerular disease, 78–83 Glomerulonephritis acute proliferative, 79–80 membranoproliferative, 80 Glycosylated hemoglobin, 192 Grade group prostate carcinoma, 68, 74 H Harvey, W., 6, 10, 37 Hashimoto, H., 11, 128, 129 Helicobacter infection, 85–86 Hemophilia A, 199–200 Hemophilia B, 199 Hepatitis viral, 92 HER2 gene, 60, 63, 64 Herophilus, 5, 35 Hippocrates, 5, 35 Histoplasma, 182–184 Hodgkin disease, 145–147, 150–152, 207 Hodgkin, T., 7, 145, 146 Hu, Z., 11 Hyperplasia adrenal cortical, 134, 135 I Ibn Zuhr (Avenzoar), 9 Immunization bacterial diseases, 174 Immunohistochemistry (IHC), 9, 11, 16, 27, 28, 41, 62, 77, 115, 123, 146–148, 162, 214, 235–237, 239 molecular, 235–236 PD-1, 237 PD-L1, 237 Influenza pandemic, 166–168 Influenza virus, 102, 162, 165–167 In situ hybridization, 9, 28, 60, 156, 162, 174, 237–238 Interstitial pulmonary fibrosis (IPF), 103–105 Intestinal polyp, 86–88

Index J Jaffe, E.S., 215 K Kawasaki, T., 11 Koch, R., 8, 11, 225 Koss, L.G., 30–32 Kurman, R., 54–55 L Laboratory medicine, 3, 41, 74, 247 Landsteiner, K., 197, 224 Lennert, K., 150–152 Liebow, A.A., 112–113 Liver disease alcoholic, 91, 193 Liver function, 189, 192–193 LiVolsi, V., 216, 217 Lobular carcinoma in situ, 60–61, 63 Lukes, R.J., 151–153 Lung neuroendocrine tumors, 107–109 Lyme disease, 176–178 Lymphoma large cell, 149 M Malaria, 157, 184–186, 198, 201, 210 Marshall, B.J., 85, 230, 231 Mass spectrometry, 156, 182 Mayo Clinic, 15, 16, 124, 141, 142 Measles virus, 163 Medullary thyroid carcinoma, 2, 133–134, 141 Meningioma, 119–120, 125 Microsatellite instability (MSI), 89, 90, 238, 239 Molecular diagnostic, 9, 28, 38–40, 54, 163, 190, 235, 238–239, 246 Molecular techniques, 18, 156, 162, 177, 182, 238 Morgagni, G.B., 6, 7, 35, 37 Morison, B.C., 96 Moss, E.S., 209–210, 247 Mostofi, F.K., 74–75, 246 Multiplex immunohistochemistry, 237–238 Mutation BRCA, 50 N Needle biopsy, 17, 83, 101 Neuroendocrine tumor pancreatic, 95

253 Next generation sequencing (NGS), 239 NIFTP, 241 Nobel, A., 223 O Omalu, B.I., 41 Osler, W., 146, 190, 206, 207, 246 P Pandemic 1918, 102, 166–169 Papanicolaou, G.N., 27, 29–31 Paraganglioma, 138–139, 142 Paramyxovirus, 163 Parasites, 3, 70, 155, 184–186, 226 Parasitic infection, 71, 185 Parathyroid hormone, 193–194 Pasteur, L., 157–159 Pathology anatomic, 1–4, 7, 8, 10, 16, 23, 39, 53, 77, 96, 124, 215, 218, 225, 235, 238, 247, 248 autopsy, vi, 2, 38, 40 clinical, 1–4, 16, 19, 36, 38, 39, 54, 77, 96, 97, 116, 145, 147, 150–152, 181, 189, 197, 206, 209, 224, 225, 231, 238, 241, 246, 248 experimental, 1, 3, 4, 7–9, 11, 18, 36, 83, 218, 224, 225, 245, 246 forensic, 2, 38–40 genitourinary, 1, 67–75 gynecologic, 45, 53–55, 218, 248 molecular, 1, 3, 11, 15, 24, 27, 36, 38, 130, 141, 147, 150, 151, 167, 189, 216, 235, 236, 238, 245, 246 pediatric, 2, 146, 212, 213, 238 surgical, 1, 3, 9, 10, 15–18, 20–24, 38, 45, 53, 54, 57, 64, 67, 85, 101, 124, 127, 169, 181, 206, 211, 216, 224, 238, 241 Pearse, A.G.E., 139–140, 214 Pheochromocytoma, 137–139 Phyllodes tumor, 58 Picornavirus, 163 Pituitary tumors, 120–122, 125, 127 Plasmodium falciparum, 186 Plasmodium malariae, 186 Pneumonia acute, 101, 167 chronic, 103 influenza, 167 Sars-Cov-2, 103 viral, 101–103 Polak, J.M., 140, 214, 215 Popper, H., 96–97

254 Pringer-Kuchinka, A., 211 Prion disease, 122–125, 198 Prostate cancer incidental, 69–70 R Reed, D., 145, 207 Renato, D., 228–229 Respiratory syncytial virus, 102, 163 Rhabdovirus, 164 RNA viruses, 161–164, 169 Rokitansky, C., 7, 36, 40 Rosai, J., 15, 22–24 Rubinstein, L.J., 123–124, 208, 209 Russell, D., 123, 124, 208–209 S SARS-CoV-2 virus, 103, 165–166 Scheithauer, B.W., 124–125 Scully, R.E., 45, 52–54 Sequence multiple analyzer with components (SMAC), 190 Short tandem repeats, 18, 40 Sickle-cell disease, 200–201 Smith Egyptian Papyrus, 5 Spencer, H., 110–112 Spirochetes, 175–177 Spitz nevus, 211 Spitz, S., 210–211 Stout, A.P., 21, 22 Strauss, L., 211–212 Stress testing, 241 Syphilis, 20, 145, 175, 176, 224 T Tapeworm, 184, 185 Tavassoli, F., 218, 219

Index Telecytology, 27, 28 Telepathology, 18 Temin, H., 229 Therapeutic erythrocytapheresis, 199 Thyroiditis chronic lymphocytic, 128 Toussaint, H., 159 Transfusion medicine, 3, 9, 197–202, 248 Transfusion reactions, 198, 224, 225 Tuberculosis (TB), 2, 5, 7, 8, 53, 103, 145, 157, 174–175, 206, 207, 209 V Vaccination, 156–157, 169, 174 Vaccines, 47, 156–159, 162, 163, 165, 168, 169, 186, 229 cancer, 47, 229 rabies, 157, 158 Vacuum assisted biopsies, 17 Valsalva, A., 7, 35 van Leeuwenhoek, A., 6, 7 Vibrio cholera, 174, 177–178 Virchow, R., 3, 7, 8, 11, 36, 40, 145, 152, 190 Virtopsy, 38, 39 Viruses, 3, 9, 45–47, 80, 92, 102, 103, 155, 156, 158, 161–164, 168, 169, 200, 228, 229 human papilloma, 32, 46, 54, 162, 169 von Willebrand disease, 201–202 W Warren, J.R., 85, 230–232 Weiss, S., 217, 218 Whipple, G.H., 226–228, 245 Y Yersinia pestis, 174, 177