Comprehensive Cytopathology E-Book

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Description

This best-selling book provides you with a comprehensive guide to the diagnostic applications of exfoliative and aspiration cytology. The book takes a systemic approach and covers the recognized normal and abnormal cytological findings encountered in a particular organ. Appropriate histopathological correlations and a consideration of the possible differential diagnosis accompany the cytological findings. The book is lavishly illustrated, making it the perfect practical resource for daily reference in the laboratory.
  • Provides an accessible guide to diagnostic investigation and screening.
  • Includes a summary of major diagnostic criteria and discusses the pitfalls and limitations of cytology.
  • Utilizes a consistent chapter structure to make finding the answers you need quick and easy.
  • Provides updates to crucial chapters to keep you on top of the latest diagnosis and techniques.
  • Incorporates differential diagnosis tables for easy comparison/contrast of diagnoses.
  • Offers more than 1800 full-color images depicting a full range of normal and abnormal findings.
  • Discusses new concepts on molecular basis of neoplasia.
  • Explores the role of cytogenetics in cancer development.

Sujets

Livres
Savoirs
Medecine
Médecine
United States of America
Oncogén
Intraepithelial neoplasia
Hodgkin's lymphoma
Squamous intraepithelial lesion
Oncology
Cirrhosis
Meningitis
Bethesda System
Robotics
Kaposi's sarcoma
Photocopier
Liver
Viral disease
Surgical suture
Thyroid nodule
Endometrial biopsy
Vaginal intraepithelial neoplasia
Phase contrast microscopy
Endophthalmitis
Chromosome abnormality
Non-small cell lung carcinoma
Viral
Immunocytochemistry
HPV vaccine
Pericardial effusion
Cervical intraepithelial neoplasia
Dysplasia
Opportunistic infection
Fluorescent in situ hybridization
Mediastinum
Neoplasm
Carcinoma in situ
Uveitis
Cholangiocarcinoma
Retinal detachment
Thymoma
Hashimoto's thyroiditis
Urinalysis
Chromosomal translocation
Review
Retroperitoneal space
Flow cytometry
Physician assistant
Retinoblastoma
B-cell chronic lymphocytic leukemia
Terminology
Renal cell carcinoma
Pancreatic cancer
Pleural effusion
Cytogenetics
Lumbar puncture
Ovarian cyst
Biopsy
Vitrectomy
Lesion
Pleurisy
Carcinoma
Soft tissue
Adenocarcinoma
Medical imaging
Mentorship
Salivary gland
Internal medicine
Ascites
Endoscopy
Bladder cancer
Genital wart
Cytopathology
Human papillomavirus
Cyst
Esophagus
Fixative
Karyotype
Artifact
Methylation
Lymph node
Lymphoma
Non-Hodgkin lymphoma
Respiratory system
Intestine
Differentiation
Ophthalmology
Urinary system
Obstetrics and gynaecology
Camera
Pneumonia
X-ray computed tomography
Multiple sclerosis
Philadelphia
Peritoneum
Stomach
Confocal
Pancreas
Infection
Thyroid
Ovary
Microscope
Microscopy
Mechanics
Chemotherapy
Chromatin
Breast
Adrenal gland
Japan
Moving
Anus
Antibodies
Pathology
Gene
Effusion
Neuraxis
Clustering
Small
Illumination
India
Assay
City
Electronic
Automation
Clientélisme (Rome)
Inflammation
Fluorescence
Baltimore
DNA
Réaction en chaîne par polymérase
Gênes
Copyright
Éthanol
Colon

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Date de parution 18 septembre 2008
Nombre de lectures 0
EAN13 9781437719628
Langue English
Poids de l'ouvrage 8 Mo

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COMPREHENSIVE
CYTOPATHOLOGY
Third Edition
Marluce Bibbo, MD ScD FIAC FASCP
Professor of Pathology and Cell Biology, Jefferson Medical
College, Director, Cytopathologym, Thomas Jefferson
University Hospital, Philadelphia, PA, USA
David Wilbur, MD
Associate Professor of Pathology, Harvard Medical School,
Director, Cytopathology, Massachusetts General Hospital,
Boston, MA, USA
S A U N D E R SCopyright
First edition 1991
Second edition 1997
No part of this publication may be reproduced, stored in a retrieval system, or
transmitted in any form or by any means, electronic, mechanical, photocopying,
recording or otherwise, without the prior permission of the Publishers. Permissions
may be sought directly from Elsevier's Health Sciences Rights Department, 1600
John F. Kennedy Boulevard, Suite 1800, Philadelphia, PA 19103-2899, USA:
phone: (+1) 215 239 3804; fax: (+1) 215 239 3805; or, e-mail:
healthpermissions@elsevier.com. You may also complete your request on-line via
the Elsevier homepage (http://www.elsevier.com), by selecting ‘Support and
contact’ and then ‘Copyright and Permission’.
British Library Cataloguing in Publication Data
A catalogue record for this book is available from the British Library
Library of Congress Cataloging in Publication Data
A catalog record for this book is available from the Library of Congress
Notice
Medical knowledge is constantly changing. Standard safety precautions must be
followed, but as new research and clinical experience broaden our knowledge,
changes in treatment and drug therapy may become necessary or appropriate.
Readers are advised to check the most current product information provided by
the manufacturer of each drug to be administered to verify the recommended
dose, the method and duration of administration, and contraindications. It is the
responsibility of the practitioner, relying on experience and knowledge of the
patient, to determine dosages and the best treatment for each individual patient.
Neither the Publisher nor the author assume any liability for any injury and/or
damage to persons or property arising from this publication.
The Publisher
Printed in China
Last digit is the print number: 9 8 7 6 5 4 3 2 1
Commissioning Editor: William Schmitt
Development Editor: Nani Clansey
Project Manager: Rory MacDonaldDesigner: Stewart Larking
Illustration Manager: Gillian Richards
Illustrator: Marion Tasker
Marketing Manager(s) (UK/USA): Kathy Neely/Lisa DamicoContributors
Fadi W. Abdul-Karim, MD, Associate Professor of
Pathology and Orthopedics, Department of Pathology,
University Hospitals of Cleveland, Cleveland, OH, USA
Tahseen Al-Saleem, MD, Director of Hematopathology
and Flow Cytometry, Senior Member, Division of
Medical Science, Fox Chase Cancer Center,
Philadelphia, PA, USA
Anniek J.M. van Aspert – van Erp, PhD CFIAC, Research
Associate, Department of Pathology, University of
Nijmegen, Ravenstein, The Netherlands
Peter H. Bartels, PhD, Professor Emeritus of Optical
Sciences, Department of Optical Sciences, University of
Arizona, Tucson, AZ, USA
Simon Bergman, MD, Associate Professor of Pathology,
Department of Pathology, Wake Forest University
Health Sciences, Winston-Salem, NC, USA
Marluce Bibbo, MD ScD FIAC FASCP, Professor of
Pathology and Cell Biology, Department of Pathology,
Anatomy and Cell Biology, Jefferson Medical College,
TJU, Director of Cytopathology, Thomas Jefferson
University Hospital, Philadelphia, PA, USA
Sandra H. Bigner, MD, Medical Director, Laboratory
Corporation of America, Burlington, NC, USA
Lukas Bubendorf, MD, Professor of Pathology, Institute
for Pathology, Division of Cytology, University Hospital
Basel, Basel, Switzerland
Johan Bulten, MD PhD MIAC, Assistant Professor of
Pathology, Department of Pathology, RadboudUniversity Nijmegen Medical Center, Nijmegen, The
Netherlands
Alicia L. Carter, MD, Fellow Cytopathology, Duke
University Medical Center, Burlington, NC, USA
Mamatha Chivukula, MD, Assistant Professor of
Pathology, Magee-Womens Hospital of UPMC, Pittsburg,
PA, USA
Luiz M. Collaço, MD, Professor of Pathology, Federal
University of Parana, Evangelica Faculty of Parana,
Bigorrilho, Curitiba, Brazil
Terence J. Colgan, MD, Professor of Laboratory
Medicine and Pathobiology, Mount Sinai Hospital,
Toronto, ON, Canada
David J. Dabbs, MD, Professor and Chief of Pathology,
Department of Pathology, Magee-Womens Hospital of
UPMC, Pittsburgh, PA, USA
Magnus von Knebel Doeberitz, MD, Professor of
Pathology, Department of Applied Tumor Biology,
Institute of Pathology, University of Heidelberg,
Heidelberg, Germany
Craig E. Elson, MD FCAP, Director of Cytopathology,
Department of Pathology, Research Medical Center,
HCA Midwest Division, Kansas City, MO, USA
Michael S. Facik, MPA CT(ASCP), Cytopathology
Laboratory Supervisor, Department of Cytopathology,
University of Rochester Medical Center, Rochester, NY,
USA
Brendan T. Fitzpatrick, MD, Attending Pathologist, Our
Lady of Lourdes Medical Center, Camden, NJ, USA
William J. Frable, MD, Professor of Pathology, Virginia
Commonwealth University, Richmond, VA, USAHugo Galera-Davidson, MD FIAC, Professor of Pathology,
Faculty of Medicine, University of Seville, Hospital
Universitario Virgen Macarena, Seville, Spain
Kim R. Geisinger, MD FCAP, Professor of Pathology,
Department of Pathology, Wake Forest University
School of Medicine, Winston-Salem, NC, USA
Ben J. Glasgow, MD, Associate Professor of
Ophthalmology and Pathology, Department of
Pathology, UCLA Center for Health Sciences, Los
Angeles, CA, USA
Katharina Glatz-Krieger, MD, Professor of Pathology,
University Hospital of Basel, Basel, Switzerland
Ricardo González-Cámpora, MD FIAC, Professor of
Pathology, Faculty of Medicine, University of Seville,
Hospital Universitario Virgen Macarena, Seville, Spain
Hans Jurgen Grote, MD, Assistant Professor, Institute of
Cytopathology, Heinrich-Heine University, Dusseldorf,
Germany
Prabodh K. Gupta, MD FIAC, Professor, Pathology and
Laboratory Medicine, Cytopathology and Cytometry
Section, Department of Pathology and Laboratory
Medicine, Hospital of the University of Pennsylvania,
Philadelphia, PA, USA
Pierre Heimann, MD PhD, Pathologist, Service
d'Anatomie Pathologique, Cytologie, Cytogénétique,
Institut Jules Bordet, Brussels, Belgium
William W. Johnston, MD FIAC, Professor Emeritus of
Pathology, Duke University, Durham, NC, USA
Ruth L. Katz, MD, Professor of Pathology, Director,
Image Analysis and Research Cytopathology,
Department of Pathology, MD Anderson Hospital,
Houston, TX, USACatherine M. Keebler, ScD(hon) CFIAC, Registrar,
International Academy of Cytology, Chicago, IL, USA
William H. Kern, MD FIAC, Clinical Professor Emeritus of
Pathology, Department of Pathology, LAC/USC Medical
Center, Pasadena, CA, USA
Larry F. Kluskens, MD PhD, Assistant Professor, Director
of Cytology, Department of Pathology, Rush University
Medical Center, Chicago, IL, USA
Savitri Krishnamurthy, MD, Associate Professor,
Department of Cytopathology, MD Anderson Cancer
Center, University of Texas, Houston, TX, USA
Oscar Lin, MD PhD, Associate Attending, Memorial
Sloan-Kettering Cancer Center, New York, NY, USA
Diane B. Mandell, CT (ASCP) CFIAC, Supervisor,
Cytology Service, Center for the Health Sciences,
University of California at Los Angeles, Los Angeles, CA,
USA
Cindy McGrath, MD, Assistant Professor of Pathology
and Laboratory Medicine, Cytopathology Section,
University of Pennsylvania Medical Center,
Philadelphia, PA, USA
C. Meg McLachlin, MD, Associate Professor, Pathology
and OBGYN, University of Western Ontario, Medical
Leader, Surgical Pathology, London Health Sciences
Centre, London, ON, Canada,
Christopher R.B. Merritt, MD, Professor of Radiology,
Thomas Jefferson University Hospital, Philadelphia, PA,
USA
Kiran F. Narsinh, MD, Fellow Eye Pathology,
Department of Pathology, JSEI,UCLA Medical Center,
Los Angeles, CA, USAJoseph F. Nasuti, MD, Attending Pathologist, Dianon
Systems, Division of LabCorp, Bridgeport, CT, USA
Ritu Nayar, MD, Associate Professor of Pathology,
Northwestern University, Feinberg School of Medicine,
Northwestern Memorial Hospital, Chicago, IL, USA
Bernard Naylor, MB ChB FIAC, Professor Emeritus of
Pathology, The University of Michigan, Ann Arbor, MI,
USA
Wai-Kuen Ng, MBBS FRCPA FHKCPath
FHKAM(Pathology) FIAC, Honorary Clinical Associate
Professor, Department of Pathology, Queen Mary
Hospital, The University of Hong Kong, Hong Kong SAR,
China
José Schalper Perez, MD MIAC, Professor of Pathology,
San Sebastian University School of Medicine, Chief,
Division of Anatomical Pathology and Cytopathology,
Concepcion, Chile
Reda S. Saad, MD PhD, Associate Professor, Department
of Pathology and Laboratory Medicine, Allegheny
General Hospital, Pittsburgh, PA, USA
Jan F. Silverman, MD FCAP, Professor and Chairman,
Department of Pathology and Laboratory Medicine,
Allegheny General Hospital, Pittsburgh, PA, USA
Lambert Skoog, MD PhD, Professor of Clinical Cytology,
Department of Pathology and Clinical Cytology,
Karolinska University Hospital Solna, Stockholm,
Sweden
Diane Solomon, MD, Division of Cancer Prevention,
National Cancer Institute, National Institutes of Health,
Bethesda, MD, USA
Theresa M. Somrak, JD CT(ASCP) CFIAC, Director,
Cytopathology Education Consortium Activities,Chicago, IL, USA
Kari Syrjanen, MD PhD FIAC, Professor, Department of
Oncology & Radiotherapy, Turku University Hospital,
Turku, Finland
Edneia M. Tani, MD PhD, Associate Professor Clinical
Cytology, Department of Pathology and Cytology,
Karolinska University Hospital Solna, Stockholm,
Sweden
Liang-Che Tao, MD FRCPC, Professor Emeritus of
Pathology and Laboratory Medicine, Department of
Pathology, Indiana University, Camano Island, WA, USA
Alain Verhest, MD PhD FIAC, Professor of Pathology
(retired), Brussels, Belgium
G. Peter Vooijs, MD PhD FIAC, Professor of Pathology,
Scientific Director, Technological Medicine, University
of Twente, Enschede, The Netherlands
Nicolas Wentzensen, MD, Department of Applied Tumor
Biology, Institute of Pathology, University of
Heidelberg, Heidelberg, Germany
David C. Wilbur, MD, Associate Professor of Pathology,
Harvard Medical School, Director, Cytopathology,
Massachusetts General Hospital, Boston, MA, USA
Moira D. Wood, MD, Assistant Professor of Pathology,
Department of Pathology, Anatomy and Cell Biology,
Thomas Jefferson University, Philadelphia, PA, USA
Bin Yang, MD PhD, Assistant Professor, Department of
Pathology, The Cleveland Clinic Foundation, Cleveland,
OH, USA
Grace C.H. Yang, MD FIAC, Professor of Clinical
Pathology and Laboratory Medicine, Papanicolaou
Cytology Laboratory, Department of Pathology andLaboratory Medicine, New York Presbyterian
HospitalWeill Cornell Medical Center, New York, NY, USA
Nancy A. Young, MD, Director, Outpatient Laboratory,
Senior Member, Division of Medical Science, Fox Chase
Cancer Center, Philadelphia, PA, USA
Maureen F. Zakowski, MD FCAP, Professor of Pathology,
Memorial Sloan-Kettering Cancer Center, Department of
Pathology and Cytology, New York, NY, USA
Lucilia Zardo, MD, Director, Section for Technology in
Cytopathology, Division of Pathology, National Cancer
Institute, Rio de Janeiro, Brazil=
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Preface
Since the publication of the second edition of Comprehensive Cytopathology in
1997 new trends in the practice of gynecologic and non gynecologic cytology have
emerged. Liquid based preparations and HPV testing have been introduced in
gynecologic cytology, immunocytochemistry, clinical cytogenetics and molecular
techniques have seen expanded applications to non gynecologic cytology, and ne
needle aspiration under sonographic, computer tomographic or uoroscopic
guidance for cytologic diagnosis has become an indispensable component of the
workup of body lesions, with more FNA's being performed by radiologists, and
endoscopists in close cooperation with cytologists.
The table of contents re ects additions and changes in topics to provide detailed
discussions of the signi cant advances in the eld. Examples include New
concepts on molecular basis of neoplasia (chapter 1), The role of genetics in
cancer development (chapter 2), Evaluation of the sample in smears and liquid
based preparations (chapter 5), FNA of Mediastinum (chapter 26), FNA of
Pediatric Tumors (chapter 29), Virtual Microscopy (chapter 33), Automation of
Cervical Cytology (chapter 34), Molecular Techniques (chapter 36). Among the
unique features of this edition is the large number of more than 1700 color
illustrations depicting the main diagnostic entities with applications of adjunct
techniques to cytopathology. New contributors from institutions around the world
(chapters 1, 2, 3, 6, 9, 10, 18, 21, 24, 25, 26, 28, 29, 33, 34, 35 & 36) have joined
the distinguished roster of authors not only to write new chapters but also to
rewrite them.
I would like to thank Dr David Wilbur for editing the chapters in the FGT
section of CC3E and for his valuable input. Thanks to all contributors for their
e orts and outstanding contributions. Special thanks to Nani Clansey,
Development Editor, for her enormous help in the development of this work and
William R Schmitt, Editor, for his support and also to Rory MacDonald, Project
Manager, Stewart Larking, Designer and to Gillian Richards, Illustration Manager
in Elsevier Ltd.
In this third edition of Comprehensive Cytopathology we have attempted, through
an international group of experts to present a state of the art work. Again we
placed in a single comprehensive book discussions on general cytology, diagnostic
exfoliative cytology of all body sites, entities and diagnostic challenges in ne=
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needle aspiration of various organs, e ects of therapy on cytologic specimens and
special methodologies and technologies in cytology. We hope that our attempt to
incorporate advances in the eld while keeping emphasis on cytomorphology will
be valuable to all professionals interested in cytopathology.
Marluce Bibbo, MDDedication
To George L Wied and Stanley F. Patten, our great mentors and friends, and our
families for encouragement and support.
Marluce Bibbo, David WilburAcknowledgments
Some of the material in the third edition is derived from chapters in the second
edition. The editors acknowledge the contributions of the following authors to the
previous edition.
George L. Wied
Karen H. van Hoeven
Dorothy Rosenthal
Torsten Lowhagen
Hi Young Hong
Lisa M. Bibb
Specific acknowledgment is made for permission to use the following figures:
Ch 2, Figs 5 & 6: Courtesy of Applied Imaging Corporation
Ch 5, Fig 12: Courtesy of Dr Zubair W Balloch, Philadelphia, PA, USA
Ch 5, Fig 14: Courtesy of Dr Yener Erozan, Baltimore, MD, USA
Ch 5, Figs 18 & 19: Courtesy of Dr Corrado Minimo, Philadelphia, PA, USA
Ch 7, Fig 11: Courtesy of Dr Belur Bhagavan, Baltimore, MD, USA
Ch 7, Figs 23 & 75: Courtesy of Dr S Bhambhani, New Delhi, India
Ch 7, Fig 25: Courtesy of Dr K Kapila and K Verma, New Delhi, India
Ch 9, Fig 16a: Courtesy of Robert H Young, Boston, MA, USA
Ch 10, Figs 18, 19, 37 & 38: Courtesy of K. Shimizu, Japan
Ch 10, Figs 20, 21, 39 & 40: Courtesy of Y Norimatsu, Japan
Ch 14, Fig 13: Courtesy of Dr Jan Silverman, Pittsburg, PA, USA
Ch 14, Fig 16: Courtesy of Dr Robert Petras, Cleveland, OH, USA
Ch 14, Fig 22: Courtesy of Josh Sickel MD, Mountain View, CA, USACh 15, Figs 3, 9 & 14: Courtesy of Dr PT Chandrasoma, Los Angeles, CA, USA
Ch 15, Fig 16: Courtesy of Dr Yener Erozan, Baltimore, MD, USA
Ch 15, Fig 44: Courtesy of Dr Demetrius Bagley, Philadelphia, PA, USA
Ch 16, Figs 2, 4, 7, 10 & 16 Courtesy of Dr Keith Volmar, Chapel Hill, NC, USA
Ch 18, Fig 49: Courtesy of Dr Syed Z Ali, Baltimore, MD, USA
Ch 19, Fig 31: Courtesy of Dr Sudha R Kini, Detroit, MI, USA
Ch 19, Fig 36: Courtesy of Dr Suzanne M Selvaggi, Madison, WI, USA
Ch 19, Fig 64: Courtesy of Dr Praboth K Gupta, Philadelphia, PA, USA
Ch 19, Fig 66: Courtesy of Ms Jamie L Covell, Charlottesville, VA, USA
Ch 19, Fig 67: Courtesy of Dr Cesar V Reyes, Mines, IL, USA
Ch 19, Fig 69: Courtesy of Ms Sharon Hicks, Martinez, CA, USA
Ch 19, Fig 72: Courtesy of the late Dr James N Landers, Detroit, MI, USA
Ch 19, Fig 94: Courtesy of Dr Andrew A Renshaw, Miami, FL, USA
Ch 19, Fig 126: Courtesy of Mr. Tarring A Seidel, Danville, PA, USA
Ch 19, Fig 137: Courtesy of Dr Dorothy L Rosenthal, Baltimore, MD, USA
Ch 19, Fig 139: Courtesy of Dr Bella Maly, Jerusalem, Israel
Ch 19, Fig 140: Courtesy of Dr Maria C Gamarra, Buffalo, NY, USA
Ch 24, Figs 9 & 10: Courtesy of Dr Janet F Stastny, Outpatient Cytopathology
Center, Johnson City, TN, USA
Ch 24, Figs 14 & 16: Courtesy of Dr Nivaldo Medeiros, Emeritus Attending, Clinical
Hospital of the University of Sao Paulo, Brasil
Ch 27, Fig 9: Courtesy of Dr Ritu Nayar, Northwestern University, Chicago, IL. USA
Ch 27, Fig 15A: Courtesy of Gary Rust, MD, Humble, TX, USA
Ch 27, Fig 36B: Courtesy of Dr Stephen Somerville, Longview, TX, USA
Ch 27, Fig 45: Courtesy of Dr Alan Heimann, Stony Brook University, NY, USA
Ch 27, Fig 56: Courtesy of Dr Bruce MacKay, MD Anderson Cancer Center, Houston,TX, USA
Ch 28, Fig 100: Courtesy of Dr Syed Z Ali of Johns Hopkins Hospital, Baltimore, MD,
USA
Ch 31, Fig 10 (right): Courtesy of Dr J Reagan, Case Western Reserve Cytopathology
Laboratory, Cleveland, OH, USA
Ch 34, Figs 4AB, 5 & 17: Courtesy of James Linder MD, Cytyc Corporation
Ch 34, Figs 10AB: Courtesy of Dr Mathilde Boon, Leiden, Netherlands
Ch 35, The authors would like to acknowledge Jeff Richmond, MD, Cytology Fellow,
University of Pittsburgh Medical Center (UPMC), Marluce Bibbo, MD, and Mary
Blumberg, MD, Surgical Pathology Fellow, UPMC, for their kind contribution of
some of the figures; and Sean Toddy, CT, American Society for Clinical Pathology
(ASCP), and Peter Atanasoff, CT, ASCP, Product Manager, Cytyc Corp, for providing
the Cellient Automated cell block system pictures.Table of Contents
Copyright
Contributors
Preface
Dedication
Acknowledgments
Part I: GENERAL CYTOLOGY
Chapter 1: The Cell: Basic Structure and Function
Chapter 2: Basic Cytogenetics and the Role of Genetics in Cancer
Development
Chapter 3: Cytologic Screening Programs
Chapter 4: Diagnostic Quality Assurance in Cytopathology
Chapter 5: Evaluation of the Sample in Smears and Liquid-Based
Preparations
Part II: DIAGNOSTIC CYTOLOGY
Chapter 6: The Bethesda System for Reporting Cervical Cytology
Chapter 7: Microbiology, Inflammation and Viral Infections
Chapter 8: Benign Proliferative Reactions, Intraepithelial Neoplasia,
and Invasive Cancer of the Uterine Cervix
Chapter 9: Glandular Lesions of the Uterine Cervix
Chapter 10: Endometrial Lesions, Unusual Tumors and Extrauterine
Cancer
Chapter 11: Vulva, Vagina, and Anus
Chapter 12: Peritoneal Washings and Ovary
Chapter 13: Respiratory Tract
Chapter 14: Alimentary Tract (Esophagus, Stomach, Small Intestine,
Colon, Rectum, Anus, Biliary Tract)Chapter 15: Urinary Tract
Chapter 16: Central Nervous System
Chapter 17: Eye
Chapter 18: Cytology of Soft Tissue, Bone, and Skin
Chapter 19: Pleural, Peritoneal, and Pericardial Effusions
Chapter 20: Fine-Needle Aspiration Biopsy Techniques
Chapter 21: Imaging Techniques
Chapter 22: Salivary Glands and Rare Head and Neck Lesions
Chapter 23: Thyroid
Chapter 24: Lymph Nodes: Cytomorphology and Flow Cytometry
Chapter 25: Breast
Chapter 26: Mediastinum
Chapter 27: Kidneys, Adrenals, and Retroperitoneum
Chapter 28: Liver and Pancreas
Chapter 29: Pediatric Tumors
Chapter 30: Effects of Therapy on Cytologic Specimens
Part III: SPECIAL TECHNIQUES IN CYTOLOGY
Chapter 31: Cytopreparatory Techniques
Chapter 32: Light Optical Microscopy
Chapter 33: Virtual Microscopy
Chapter 34: Automation in Cervical Cytology
Chapter 35: Immunocytochemistry
Chapter 36: Molecular Techniques
IndexPart I
GENERAL CYTOLOGYCHAPTER 1
The Cell: Basic Structure and Function
Magnus von Knebel Doeberitz, Nicolas Wentzensen
Contents
PART I: BASIC STRUCTURE AND FUNCTION OF MAMMALIAN CELLS
Overview
Nucleus
Contents of the Nucleus
Nuclear Morphology
Hematoxylin
Nucleoli
Nuclear Envelope and Nuclear Shape
Cytoplasm and Plasmalemma
Cytoplasmic Stain
Endoplasmic Reticulum
Golgi Apparatus
Mitochondria
Lysosomes
Cytoskeleton, Centrosome
Cell Membrane, Receptors, and Signal Transduction
Cell Junctions
Cell Growth and Division
PART II: THE MOLECULAR BASIS OF NEOPLASIA
Overview
Principles of Malignant Transformation
Cancer-related Genes
The Major Pathways of CarcinogenesisCarcinogenesis induced by Papillomavirus Infections
Basic Structure of the Virus and Its Genome
Epidemiology of HPV Infections
The Role of the HR-HPV E6 and E7 Genes
Progression of HPV-Infected Epithelial Cells to Invasive Cancer Cells
Concluding Remarks
PART I: BASIC STRUCTURE AND FUNCTION OF MAMMALIAN CELLS
Overview
Cells are the basic structural and functional units of all living organisms. The estimations
14about the total cell count of a human body vary widely; a number as large as 10 seems
conceivable. Although the principal components of all cells are very similar, the
differentiation of cells results in a wide variation of cellular morphology and function.
The smallest human cells by diameter are spermatozoas with a size of ~3 m, followed
by the anucleate erythrocytes (8 m). The largest cells are female oocytes that can be as
large as 35–40 m and are visible to the naked eye. Motor neurons are extremely long
cells, with their axons reaching from the spine to the distal extremities (up to 1.4 m
length).
Most cells can only be functional in large united structures, such as organs or
suborganic structures. Other cells, mainly of hemato- or lymphopoietic origin, are mobile
and active as single cells, although in many cases, their functionality is dependent on
interaction with other cells.
Cytopathology studies diseases on the cellular level. While in histopathology, cells are
assessed in the spatial context, in virtually all cytological applications, they are removed
from their spatial context and must be assessed isolated or as cell sheets.
Apart from the loss of the spatial information, cells can have considerably altered
morphology when taken out of the united structures. Cell–cell contacts are important
features that build the shape of a cell. Many structural elements within a cell are
connected to proteins that are attached to other cells or the basal membrane. This must
be taken into account when cells are compared in histological and cytological
assessments.
Another important di: erence between histology and cytology is based on the fact that
in histology, tissue sections that are plain two-dimensional are assessed, while in most
cytology applications, complete cells that still have some three-dimensional features,
although they might appear ; at in the microscope, are analyzed. Based on these facts,
the transfer of histological morphology to the picture seen in cytology can only be
limited.
In this section, an overview of the most important cellular structures and functions
relevant for cytopathology are presented (Fig. 1.1). We have assembled the most
important information on cellular structures by describing the regular function in brief,the relevance for cytopathology, and the morphology in normal and abnormal cells. For
more detailed information about cellular structures and functions, a cell biology textbook
is recommended.
Fig. 1.1 Schematic presentation of an epithelial cell displaying the most important
structures discussed in this chapter.
Nucleus
The nucleus contains the genomic DNA, histones, and several proteins that are
responsible for DNA replication, repair, and transcription of genetic information (Fig.
1.2A).
Fig. 1.2 Contents of the nucleus, DNA. (A) A nucleus displaying nucleoli,
euchromatin, and heterochromatin. (B) Two nucleosomes consisting of DNA coiled around
histone proteins. (C) The structure of double-stranded DNA. Organic bases are connected
to a sugar–phosphate backbone. Complementary bases (A-T, C–G) are held together by
hydrogen bonds.The assessment of a cell's nucleus is one of the most important tasks in cytopathology.
The size of the normal nucleus is highly variable, depending on the underlying cell type.
In many malignant cells, nuclei are considerably enlarged. Apart from nuclear size, the
chromatin density, the nuclear membrane, and the presence of nucleoli are important
features of nuclear morphology and will be described in detail.
The nucleus contains about 25% dry substance, of which 18% is DNA plus a similar
amount of histone proteins. The rest of the dry substance contains the non-histone
proteins, nucleoli, and the nuclear membrane.
Contents of the Nucleus
DNA
The genetic information of organisms is coded in deoxyribonucleic acid (DNA). DNA is a
long stretch of single nucleotides connected by a sugar–phosphate backbone (Fig. 1.2C).
The genetic information is stored in speciAc sequences consisting of four di: erent bases:
adenine, guanine, thymine, and cytosine (A, G, T, C). A triplet of bases is coding for an
amino acid that constitutes the basic component of proteins. Although in principle the
triplet code allows for 64 di: erent variations, only 20 protein-building amino acids exist.
Many amino acids are coded by multiple base triplets. The genetic code is degenerate,
thereby tolerating errors in the base sequence to some degree. Two DNA stretches are
combined as a double helix; one complete turn is reached after 10 bases. The DNA
stretches are not covalently bound, but attached via hydrogen bonds between
complementary bases A–T and C–G.
DNA is a very robust and stable molecule, since it must protect the genetic code of an
organism. The genetic information is transferred to the ribosomes (the protein production
machinery) by ribonucleic acid (RNA) that has 3 important features di: erent from DNA:
RNA is based on a ribose backbone, it contains uracil instead of thymidine, and it is
usually single-stranded. Compared to DNA, RNA is a rather unstable molecule.
The total DNA of a cell is separated on chromosomes. In total, 22 di: erent
chromosomes and two sex chromosomes exist. The chromosomes vary in size and in the
content of coding sequences, they are numbered in decreasing order of their size. During
the metaphase of mitosis, chromosomes are condensed and can be identiAed in light
microscopy. In transcriptionally active cells, DNA is decondensed and takes up the room
of the complete nucleus. When metaphase chromosomes are stained according to Giemsa,
a heterogenous pattern of regions with strong staining (G-bands) and regions without
staining (R-bands) can be observed. R-bands contain more genes than G-bands and are
replicated early during cell division. The banding pattern of chromosomes has been used
to determine chromosomal regions by indicating the chromosome number, the position
with reference to the centrosome (p = short arm, q = long arm), and the position of the
chromosomal banding (e.g. 3q26). In total, the human genome consists of ~3.2 billion
1bases, coding for approximately 25,000 genes.
Nuclear ProteinsHistones are basic proteins that build a structural unit together with the DNA, called the
nucleosome (Fig. 1.2B). In the nucleosome, 146 base pairs (bp) are coiled around
di: erent histone subunits. The main function of the nucleosome is the high-density
packing of DNA inside the nucleus, leading to a 50,000-fold increased compactness of
DNA as compared to unpacked DNA.
Histone acetylation reduces the aE nity between the DNA molecule and histones,
leading to increased accessibility of DNA for transcription machinery components such as
RNA polymerase and transcription factors. In general, for gene transcription, the DNA
needs to be unpacked from the histones.
Besides histones, nuclear non-histone proteins build the nuclear sca: old structure and
are involved in DNA transcription and replication.
Nuclear Morphology
Chromatin
Chromatin represents the complex structure of proteins and DNA in the nucleus of
nonmitotic cells. There is usually twice as much protein as DNA in a nucleus. Since most cells
in the human body are non-mitotic, chromatin is the morphological appearance of most
cell nuclei assessed in cytology. The chromatin distribution and organization depends on
many di: erent factors, such as cell type, di: erentiation, metabolism, proliferation status,
and, most important in cytopathology, neoplastic transformation.
Two conformations of chromatin are discriminated: euchromatin and heterochromatin.
Euchromatin contains transcriptionally active protein-coding DNA regions.
Heterochromatin represents the complex of DNA that is densely packed on histones. DNA
sections not transcribed are usually stored in heterochromatin. Heterochromatin is further
di: erentiated into constitutional, facultative, and functional heterochromatin.
Constitutional heterochromatin consists mainly of highly repetitive DNA stretches in the
centromeric region that are supposed to have structural functions but have also been
found to express microRNAs that do not code for proteins but are involved in gene
regulation. Facultative heterochromatin designates inactivated DNA regions that usually
code for proteins but are not necessary in the respective cell, e.g. inactivation of the
second X chromosome (Barr body). Functional heterochromatin contains DNA regions not
necessary for the respective differentiation of a cell.
Hematoxylin
In many cytological applications, the chromatin is stained with hematoxylin.
Hematoxylin is a basic dye extracted from the heartwood of the tree Haematoxylum
campechianum. By itself, hematoxylin is a very weak stain. Di: erent mordants, such as
potassium alum, are used to generate the typical dark blue or purple staining.
Hematoxylin strongly binds to acidic components of a cell, most importantly to the
phosphate groups of nuclear DNA; the stained structures are therefore called "basophilic"
(Fig. 1.3A).Fig. 1.3 Exemplary pictures of nuclear and cytoplasmic staining. (A) Cervical cells
stained with hematoxylin only. (B) Cervical cells stained with Hematoxylin and Eosin. (C)
Cervical cells stained according to Papanicolaou.
Based on the nuclear stain, a wide variation of chromatin alterations can be observed,
both alterations of structure and staining intensity. Structural aberrations include
chromatin margination, i.e. aggregation of chromatin to the nuclear membrane, which is
a sign of cell degeneration. Other chromatin alterations are coarsening and clumping that
is usually accompanied by chromatin thinning in other regions.
Hyperchromasia, i.e. increased staining intensity, can result from increased chromatin
loads or by a decreased nuclear volume, which inversely applies to hypochromasia. In
additions, chemical modiAcations of the chromatin (e.g. during speciAc stains or cell
treatments) can increase the stain uptake, simulating hyperchromasia.
Nucleoli
Nucleoli are small basophilic spherical bodies located in the nucleus. Usually they can be
found in the central nuclear region but may also be close to the nuclear membrane. A
nucleolus is built by a nucleolus organizing region (NOR) of a speciAc chromosome.
These regions contain the genes for ribosomal RNA subunits that build the protein
synthesis machinery. Since in a diploid human cell, in total 10 chromosomes containing
NORs exist, in principal 10 nucleoli per nucleus could be present. Usually, only one or
two nucleoli are found, since NORs from several chromosomes build a commonnucleolus. Nucleoli have two distinctive regions, the pars Abrosa that contains the
proteins required for transcription and the pars granulosa that contains the ribosomal
precursors. During mitosis, nucleoli disappear and are reconstituted in the daughter cells.
Shortly after cell division, a larger number of nucleoli that fuse gradually can be
observed.
Depending on the cell type, the presence of nucleoli is physiological or can indicate
malignant processes: liver cells that regularly produce a lot of protein can frequently
exhibit nucleoli. In reactive or regenerative cells, nucleoli can become more prominent.
In hepatocellular carcinoma, usually more than 50% of the cells show prominent,
frequently multiple nucleoli. Intestinal epithelial cells also regularly show single nucleoli.
In ageing and starving cells, a shrinking of nucleoli can be observed. In cancer cells,
nucleoli can vary substantially with regard to size and shape.
In many malignant cells, multiple nucleoli that appear disjoint, odd-shaped, and
spiculated can be observed. Proteins associated with nucleolar organizer regions can be
visualized by a simple argyrophilic staining method. The structures highlighted by this
method are called "argyrophilic nucleolar organizer regions" (AgNORs). Di: erent
distributions of AgNORs have been described between normal, dysplastic, and malignant
tissues. In several cancer entities, AgNOR aberrations were found to have independent
2prognostic significance with respect to patient survival. Increased NOR counts have been
explained by increased metabolism with a high demand of ribosomes, but also by
aneuploidy leading to increasing numbers of NOR regions in cancer cells.
Nuclear Envelope and Nuclear Shape
The nuclear envelope (NE) consists of two lipid membranes. The inner membrane is
associated with the telomeres and anchors the chromosomes, while the outer membrane
is part of the endoplasmic reticulum. The space between the two lipid layers is called
perinuclear cisterna. The nuclear envelope constitutes the nucleus and separates the
genomic material from the cytosol. During cell division, the nucleus disappears; the
nuclear envelope is broken down to vesicles and is reassembled during telophase. The
nuclear envelope builds a strong barrier between nucleus and cytosol; a number of
nuclear pore complexes regulate the traE c between both compartments. There can be
passive di: usion or active transport; in general, proteins synthesized in the cytoplasm
require a specific nuclear signal in order to have access to the nucleus.
Inside the nuclear envelope is a network of chromatin Abrils and a nuclear lamina built
from laminins. The nuclear envelope can be visible in light microscopy.
The regular nuclear shape is that of a smooth sphere or spheroid, based on the orderly
arrangement of the chromosomes and the nuclear lamina. Many factors can a: ect the
shape of the nucleus: stress, transcriptional, and synthetic activities can disturb the
arrangement of interphase chromosomes; DNA ampliAcations can lead to uneven
distribution of the nuclear material and to nuclear enlargements. At the same time,
aberrations of the nuclear envelope can lead to alterations of the nuclear skeleton,
resulting in altered chromosomal distributions. It has been assumed that changes of the
nuclear envelope occur mainly after mitosis, when the nuclear envelope is reassembled.Alterations of the nuclear envelope have been directly linked to oncogene activity: Six
hours after transfection with the ret oncogene, increasing cell counts with NE alterations
were observed in human thyroid cells, indicating that nuclear alterations may occur even
3independent of postmitotic re-assembly. NE alterations and the respective nuclear shape
are an important diagnostic feature of many malignancies, especially papillary thyroid
cancers and different types of leukemias.
Cytoplasm and Plasmalemma
The cytoplasm consists mainly of water (80–85%). The remaining constituents are
proteins (10–15%), lipids (2–4%), polysaccharides (1%), and nucleic acids (1%). The
cytoplasm is conAned to the outside by the plasma membrane, a lipid bilayer, and to the
inside by the nuclear membrane. In most cytology applications, normal cells have a
homogenous cytoplasm with occasional granules or inclusions.
Cytoplasmic Stain
Eosin is the most common dye to stain the cytoplasm in histology. It is an acidic dye that
binds to basic components of a cell, mainly proteins located in the cytoplasm. It gives a
bright pink color that contrasts that dark blue nuclear hematoxylin staining (Fig. 1.3B). A
combination of hematoxylin and eosin is the most frequently used dye in histology. In
cytology, frequently, a Pap stain is performed. It consists of a hematoxylin-based nuclear
stain and a polychromatic cytoplasmic stain, including Orange G and two polychromic
dyes, EA36 and EA50. The cytoplasmic stain results in highly transparent cells, making it
possible to assess superimposed cells in a Pap smear. Based on the cell type and the
di: erentiation status, the cytoplasm can be pink–light red (e.g. superAcial cervical cells)
or light blue–green (e.g. cervical parabasal and intermediate cells) with all variations in
between. The nuclei are dark brown or dark blue/violet and the nucleolus appears bright
red (Fig. 1.3C).
Endoplasmic Reticulum
The endoplasmic reticulum (ER) consists of a single membrane making up for more than
half of all internal membranes of the cell (Fig. 1.4A). The part of the membrane that
faces the cytosol is studded with ribosomes. This part of the ER is called rough ER; the
regions without ribosomes are called smooth ER.Fig. 1.4 Membranous organelles. (A) The rough endoplasmic reticulum. (B) The Golgi
apparatus. (C) A mitochondrion.
The main function of the ER is the packaging and delivery of newly synthesized
proteins.
Golgi Apparatus
The Golgi apparatus is part of the membrane system that also contains the ER. It consists
of stacked membrane-coated cavities, called dictyosomes (Fig. 1.4B). The Golgi
apparatus is located close to the nucleus and can be very large in secretory cells, where it
Alls almost the complete cytoplasm. The convex side facing the ER/nucleus is called
cisGolgi; the concave side facing the cytoplasm is called trans-Golgi. From the Golgi
apparatus, small vesicles transport products to other cellular sites or the exterior. Inside
the structure, complex biochemical operations are being performed most of them
resulting in post-translational modiAcations of synthesized proteins. Several secretory
mammalian cell types are characterized by a prominent polarized Golgi apparatus
located between the nucleus and the luminal surface: Goblet cells in the respiratory and
digestive tract produce large amounts of glycoproteins, pancreatic cells secrete enzymes
such as zymogen, and breast cells produce milk droplets.
Mitochondria
Mitochondria produce ATP, the universal fuel of living organisms, by oxidative
processing of nutrients. They are located in the cytoplasm and separated from it by a
double membrane (Fig. 1.4C). On average, an eukaryotic cell contains about 2000
mitochondria. Depending on age and cell type, mitochondrial size can vary between 0.5
and 10 m. The highest mitochondrial counts can be found in cells with high energy
demand, such as muscle cells, nerve cells, or metabolically active cells in the liver.
Mitochondria are inherited in non-mendelian fashion via the cytosol of oocytes. Duringcell division, mitochondria are divided between the two daughter cells. They are
genetically semi-autonomous since they possess their own circular genome, but are
dependent on a number of proteins encoded by the nuclear DNA.
The Pap stain does not color mitochondria, but iron hematoxylin or acid fuchsin does.
A more speciAc stain for mitochondria is rhodamine 123. Stained mitochondria appear as
single spheres or long, branching structures, up to 7 × 0.5 m in size. Mitochondria can
be found in large numbers in hepatocellular carcinoma, resulting in a granular
appearance of the cytoplasm. There are many other causes of granular cytoplasm; the
underlying cellular components can only be visualized by ultrastructural methods. Since
mitochondria represent the energy system of living cells, they are very important in the
malignant development. Multiple functional and structural alterations during
4carcinogenic processes have been described.
Lysosomes
Lysosomes are small vesicles derived from the Golgi apparatus; they contain up to 40
acidic enzymes (hydrolases) at a pH 5. The membrane prevents the aggressive enzymes
from destroying cellular structures. Although the contents can vary substantially, there
are basically no morphological di: erences between functionally di: erent lysosomes. The
main function of lysosomes is the digestion of internal (non-functional cell organelles)
and external (food, bacteria, leukocytes, debris) material. The processed material is either
released to the cytoplasm, secreted, or stored in lysosomes.
Several storage diseases (e.g. Hunter-Hurler-Syndrome) are characterized by a
deAciency of lysosomal enzymes. These disorders lead to accumulation of incompletely
digested mucopolysaccharides in the lysosomes.
Cytoskeleton, Centrosome
The cytoskeleton is a complex lattice of various Alaments building the cellular structure
and shape; it is responsible for dynamic activities such as movement in growth and
di: erentiation (Fig. 1.5). Although is has been thought for a long time that the
cytoskeleton is a special feature of eukaryotic cells only, it is becoming more and more
5clear that also prokaryotes have cytoskeleton-like structures.Fig. 1.5 Display of an epithelial cell with cytoskeleton and cell–cell contacts.
The Alaments are required for cell movement (cytokinesis), transport of material across
the cell surface, muscle contraction, intracellular transport, and sorting and dividing of
replicated chromosomes by the mitotic spindle.
Three main classes of cytoskeletal Alaments are distinguished: actin Alaments,
intermediate filaments, and microtubules (Fig. 1.5).
Actin Alaments have a diameter of 7 nm and are built from six di: erent actin types; in
muscle cells, actin is functionally linked to myosin. They can be found in all cells, with
especially high numbers in Abroblasts and the highest concentrations in muscle cells,
since actin is part of the contractile structures. Lamellipodia (bulges of the cell surface for
cell motility) and Alopodia (enhanced cell surface for absorption) are built from bundled
actin. Several glandular tissues such as breast and prostate have contractile myoepithelial
cells that can forcibly express the glandular contents.
Intermediate Alaments have a diameter of 10 nm, consist of one or more of 19 di: erent
cytokeratin molecules, and are the strongest Abers among the cellular Alaments. They are
mainly responsible for the structural framework of a cell and determine the cell's tensile
strength. They build rope-like polymers. Keratins belong to the group of intermediate
Alaments. In keratinizing epithelial cells, keratin Alaments accumulate and are
crosslinked by other proteins and disulAde bonds. The keratinizing process starts at the
periphery and progresses to the nuclear area. In fully di: erentiated cells, the nucleus
becomes more and more pyknotic and finally dissolves.
Other examples for intermediate filaments are desmin in skeletal muscle, glial filaments
in astrocytes, and neurofilaments in axons.
Microtubules are long, hollow tubes with 25 nm diameter assembled from microtubule
oligomers originating in a membraneless body, the centrosome. The centrosome is the
main microtubule organizing center (MTOC) of a cell and functions as an important
regulator of the cell cycle. The centrosome consists of two orthogonally arranged
centrioles surrounded by pericentriolar material and is situated between the nucleus and
the Golgi apparatus. Upon cell division, each daughter cell receives one centriole.
Although in most model organisms, a proper cell division can be achieved without a
functional centrosome, an organism requires functional centrosomes to survive in the
long term. Aberrant centrosomes are a hallmark of chromosomally instable cancer cells.
Because of aberrant formation of the mitotic spindle apparatus, these cells acquire more
and more chromosomal aberrations.
Two classes of substances can interfere with the microtubular network: Colchicine
prevents the polymerization of microtubules, while paclitaxel interferes with their
depolymerization. In a living cell, the microtubular network is continuously polymerized
and depolymerized. Therefore, both agents lead to a non-functional spindle apparatus
abrogating the cell division and are used as cytotoxins in cancer therapy.
Cell Membrane, Receptors, and Signal TransductionThe basic structure of the cell membrane is a semi-permeable lipid bilayer built from
phospholipids, glycolipids, and steroids that contains various proteins ; oating on one side
of or reaching through the complete membrane. The lipid bilayer has a gage of 6 to 10
nm and is barely visible in light microscopy. The cell membrane separates all cellular
components from the environment and it assures the spatial and functional entity of a
single cell. It allows the cell to persist in environments that would be harmful to the
cellular components, such as extreme pH conditions and ion concentrations di: erent
from the cytoplasm. The cell membrane controls what is going into and out of a cell;
thereby it regulates the import of nutrients and the export of cellular products. The
transport is organized by passive (transport via a gradient that does not require energy)
and active (transport against a gradient that requires energy) protein channels.
There are di: erent types of membrane proteins. Peripheral membrane proteins only
temporarily adhere to the respective cell membrane; they usually interact with integral
membrane proteins. Many regulatory subunits of ion channel and transmembrane
receptors, as well as enzymes and hormones, are peripheral membrane proteins. In
contrast, integral membrane proteins are permanently attached to the membrane.
Transmembrane proteins are integral proteins that span both lipid layers; they must
contain a hydrophobic part that is placed in the lipid section and hydrophilic intra- and
extracellular parts. Typical representatives are ion channels, proton pumps, and
Gprotein coupled receptors. Lipid-anchored proteins are covalently linked to lipids in the
cell membrane; the most common type are G-proteins.
The communication of a cell with the environment (i.e. other cells or the extracellular
matrix) is mediated by a wide variation of interacting molecules, usually designated as
receptors. The functional principle of receptors is based on the key–lock principle; i.e. a
speciAc receptor requires the binding of a speciAc ligand (either cell based or freely
circulating) to be activated. The transfer of information between cells may be mediated
by two di: erent classes of receptors located in the cell membrane: Ionotropic receptors
are based on speciAc ion channels that change the electric potential of cells upon
activation (Fig. 1.6A). Non-ionotropic receptors have no pores, but are based on
transmembrane proteins that stimulate intracellular proteins linked to the receptors and
thereby modulate intracellular signal cascades.Fig. 1.6 Important membrane proteins. (A) Different calcium ion channels controlling
the intracellular calcium concentration. (B) G-protein receptors; the example shows
adrenergic activation of the adenylate cyclase (AC). (C) Tyrosine kinase receptors; the
example shows EGF-mediated phosphorylation of intracellular downstream targets.
The two most important non-ionotropic receptor types are G-protein coupled receptors
and tyrosine kinase receptors. G-protein coupled receptors consist of seven
transmembrane domains, an extracellular receptor region, and an intracellular part that
binds the G-protein (Fig. 1.6B). Upon activation by an outside signal, the receptor
changes its conformation and releases the G-protein in a not yet fully resolved
mechanism. G-proteins, short for guanine nucleotide binding proteins, are the most
important proteins involved in second messenger cascades, responsible for many central
nervous (vision, olfactory system, neurotransmitters) and immune system functions.
Receptor tyrosine kinases activate downstream targets by adding phosphate groups to
intracellular proteins (Fig. 1.6C). They are frequently promoting cell growth and cell
division (e.g. receptors for insulin-like growth factor, epidermal growth factor, EGF).
Consequently, many malignant processes are linked to aberrations of G-protein coupled
receptors and especially tyrosine kinase receptors. In about 25% of the breast cancers, a
subtype of EGF receptors (ErbB2/Her2neu) is overexpressed, which leads to increased
signaling of the EGF pathway, resulting in increased cell proliferation. Meanwhile,inactivating antibodies directed against Her2neu are successfully used in breast cancer
therapy. Similarly, cetuximab is a monoclonal antibody downregulating ErbB1 signaling
that has been approved for special types of colorectal cancer.
Cell Junctions
As complex organisms are built from billions of cells, the cell-to-cell contact is a very
important parameter. Depending on the organ or the function of a tissue, cell contacts
establish adherence of functionally connected cells, build a barrier against a lumen, and
are involved in intercellular communication (Fig. 1.5).
Adherence is based mainly on spot desmosomes (macula adhaerens) consisting of
keratin Alaments that connect the cytoskeleton of individual cells. A di: erent form of
adherence is the adhesion belt (zonula adherens) that connects the apical part of an
epithelial cell to another epithelial cell. Hemidesmosomes are located at the basal pole of
the cell and attach the cell to the basal membrane.
Tight junctions (zonula occludens) build an impermeable barrier between cells and are
typical for structures that have a lumen.
Gap junctions are required for communication; small inorganic molecules and electric
signals are exchanged via 1.5-nm pores in the cell membrane. Gap junctions can be
quickly established from precursors floating in plasma membrane
The terminal bar describes a light microscopic structure that represents the sum of the
adhesion belt and actin Alament bundles as well as other protein Alaments at the apical
end of a cell.
In general, malignancy causes loss of cell-to-cell adherence. It has been shown that the
cell adhesion molecule E-cadherin is lost during the formation of some epithelial cancers.
The loss of E-cadherin is frequently accompanied by an overexpression of other
cadherins, an e: ect called the “cadherin switch.” Besides the cell adhesion, signal
transduction pathways and altered, inducing malignant transformation of the respective
cell.
Cell Growth and Division
In mammalian organisms, some cells are created early in embryogenesis and remain
unchanged throughout the whole life (e.g. lens of eyes, cells of CNS, heart muscle cells,
auditory cells of ear). However, many epithelial tissues, as well as hemato-lymphopoietic
cells, depend on a continuous replenishment of their cell pools. Other tissues have
retained a regenerative capacity, e.g. liver stem cells, that make it possible to regenerate
the organ after tissue damage. The detailed mechanisms of cell division can be
recapitulated in a cell biology textbook. In brief, the cell cycle consists of four phases, the
G1, S, G2, and M phases. Resting cells are in a constant G0 phase that has a direct
transition from the G1 phase. G phases are gap phases, S indicates the synthesis phase in
that the genomic material is doubled, and M indicates the phase of mitosis, in that two
daughter cells are created (Fig. 1.7A). In order to assure a proper cell division with equal
distribution of the genomic material in the daughter cells, several checkpoints (after G1,S, and G2) must be passed to allow for a continuation of the cell cycle. If the
requirements at a checkpoint are not met, the cell is not allowed to continue; it might
even go into apoptosis.
Fig. 1.7 The cell cycle and its morpholgical appearance. (A) Schematic presentation of
the cell cycle. (B) Typical anaphase during mitosis of plant cells. (C) Mitosis of
transformed epithelial cells in immunohistochemistry.
In light microscopy, mitotic Agures represent the M phase, i.e. the distribution of
chromosomes into the opposite poles of a cell (Figs. 1.7B, C). Mitotic Agures are rare in
normal tissue; in normal liver, about 1–2 mitoses can be observed per 10,000 cells. In
rapidly dividing cancer tissue, the frequency of mitotic Agures can be much higher,
depending on the underlying cell type.
The regulation of the cell cycle is very complex. In general, uncontrolled activation of
the cell cycle is a basic feature of all tumors. The reasons for uncontrolled cell cycle
activation can be either the loss of inhibitory gene functions (inactivated tumor
suppressor genes) or the activation of cell cycle promoting gene functions (activated
oncogenes). The detailed mechanisms of carcinogenesis are described in Part II of this
chapter.
PART II: THE MOLECULAR BASIS OF NEOPLASIA
Overview
Single cells in complex organisms work in a hierarchical order determined by their
di: erentiation state. Each single cell fulAlls its function through biochemical processes
that are highly regulated and act in well-orchestrated pathways. The outgrowth of tumors
occurs if the cells have lost the capability to follow these predetermined rules. Growth
restriction, a typical feature of normal tissues, is lost; the capability to commit cellular
suicide (apoptosis) once certain death signals have been activated is lost. Di: erentiation
pathways that permit the cell to enter irreversible cell cycle arrest become disconnected.
Mechanisms that under normal conditions are required to maintain the normal
histological architecture of an organ are erroneously activated to feed the outgrowingtumor with essential nutrients.
Tumors may be derived from more or less all human tissues, including epithelium that
may be transformed into benign adenomas and malignant carcinomas, mesenchymal
tissues that may be transformed into benign Abromas and malignant sarcomas,
hematopoietic tissues that may be transformed into lymphoma and leukemia, and Anally
even germ cells that may be transformed into benign teratomas and malignant
teratocarcinomas.
Benign lesions are partially growth restricted: They do not invade, do not metastasize,
but do grow locally. In contrast, malignant tumors usually acquire more autonomous
growth properties, and develop complex strategies to invade other organs, to disseminate
their cells to distant anatomical sites, to evade immunological attack of the host's immune
system, and to initiate from disseminated cells again autonomous proliferative metastatic
lesions. Following the concept that all these features are mediated by a complex network
of genes, the question arises what genes are regulating the process of growth control in
cancer cells and why are these genes activated or inactivated in distinct somatic cells that
thus acquire the capacity to grow out as cancer cell. Evidently the modiAcations required
to induce benign tumors are by far less complex than those required for an invasive
cancer. They usually can be achieved by minor modiAcations of the genome of the
a: ected cells. In many cases benign tumors are therefore precursors of malignant tumors
that require a substantially more complex pattern of genetic modifications (Fig. 1.8).
Fig. 1.8 Transformation of epithelial cells.
The biological phenotype of cancer cells is deAned by the expression of certain genes,
whereas other genes are not expressed. These phenotype-speciAc gene expression patternsare referred to as "gene signatures" that di: erentiate a distinct cell (e.g. cancer cells) from
cells with other biological properties (e.g. a normal stem cell that is not transformed).
Cancer cells of the same organ origin, e.g. gastric cancers, may display substantially
6di: erent biological properties that re; ect di: erent gene signatures. These changes of the
gene expression pattern of cells or tissues can be monitored by gene expression arrays
(Fig. 1.9). To convert a normal somatic cell into a full-blown cancer cell many distinct
steps are required, during which the gene expression pattern of the respective cell is
gradually modiAed and the phenotype of the cell is transformed into increasingly
neoplastic cells that are selected in an ongoing Darwinian selection process. SpeciAc
modifications of the gene expression signature trigger the next step of selection.
Fig. 1.9 Analysis of the gene expression signature of a panel of gastric cancer cells
with di: erent biological phenotypes. Genes expressed at high levels are indicated in red,those expressed at lower levels are indicated in green. By comparing the expression level
of distinct genes (or c-DNAs) among di: erent cell lines, complex di: erences among the
expression of many genes can be monitored in a large number of tissues of cell lines.
Using distinct software algorithms tissues or cell lines can be clustered in an hierarchical
order that re; ects their biological phenotype. The Agure shows an example of various
gastric cancer cell lines of which some display similar phenotypes: (B) epithelial cell
cluster; (C) B lymphocytes cluster; (D) T lymphocytes cluster; (E) Abroblast cell cluster;
(F) endothelial cell cluster. The results were visualized and analyzed with TreeView (M
Eisen; http://rana.lbl.gov).
6Data and images were taken from Ji et al.
Given the complex alterations required to achieve the signature of a full-blown cancer
cell and given the many individual selections steps required to transform a normal cells
into a cancer cell, transformation cannot be achieve by a linear selection process but
depends on higher level mechanisms that allow for major modiAcations of the genetic
code in a relatively brief period of time or restricted number of cell divisions. The
integrity of the number and structure of chromosomes, for example, is one particularly
important aspect herein. If the mechanisms that maintain the integrity of the
chromosomes fail, major genomic modiAcations may rapidly occur. Since most of these
are non-viable, most cells that experience these failures will die in a process called
genomic catastrophe. However, some cells may survive the complex modiAcations
induced by malfunction of the mechanisms that preserve chromosomal homeostasis. If
the gene signatures of the surviving cells allow for continuous autonomous growth
eventually even at distant anatomical sites, the respective cell clones may be selected and
their sustained growth may then clinically manifest itself as metastasizing cancer.
To preserve the ordered function of cells in higher order organisms a number of
redundant genome protective mechanisms that prevent consequences of genetic
catastrophes have been evolved. They primarily constitute organized suicide mechanisms
called apoptosis that become activated once major modiAcation of the cellular genome in
distinct genetically damaged cells occur. They assure that most cells that undergo genetic
catastrophes undergo apoptosis before they can grow out as transformed cancer cell.
Thus, outgrowth of a cancer cell still is the rare exception in view of the many billions of
proliferating cells that constitute the human body and the many events that trigger
genomic catastrophes in damaged cells.
Principles of Malignant Transformation
The development of a cancer cell from a normal cell goes through three basic steps:
(1) Immortalization: In contrast to normal cells, immortalized cells can divide
indefinitely, as long as they are supported with nutrients. They still have the same shape
as normal cells, and they stop growing when they reach other cells (contact inhibition).
(2) Transformation: Transformed cells are independent of tissue-specific growth factors;
they lose their contact inhibition and may grow invasively. Their shape is altered; thespecific differentiation is lost.
(3) Metastasis: Metastasizing cells acquire the potential to migrate to distant sites and
grow out to tumors.
These paramount changes occur on the level of the individual cancer cell.
3Subsequently, the establishment of tumors larger than 1–2 mm requires the
development of a vascular system that can support the growing tumor with nutrients. In
order to achieve this, tumors induce angiogenesis via angiogenic factors such as vascular
angiogenic growth factor (VEGF), Abroblast growth factor (FGF), and platelet-derived
endothelial growth factor (PDGF).
Tumor growth is based on a complex interplay between the transformed cells and the
surrounding tissue: invasive growth is associated with the expression of proteolytic
enzymes that degrade the peritumoral stroma, most importantly
matrix-metalloproteinases (MMPs). Furthermore, the immune system is involved in the local control of
growing cancers. It is estimated that the majority of malignant cell clones that develop in
the human body are eliminated by immune system components directed against the
transformed cells, a process called immune-surveillance. Invasive tumor development
needs to evade these immune control mechanisms. A number of immune evasion
strategies, including loss of antigen presentation machinery components, induction of
suppressive T cells, and induction of apoptosis in attacking lymphocytes, have been
analyzed.
Cancer-related Genes
Three major groups of genes are involved in carcinogenesis: oncogenes, tumor suppressor
7genes, and genes that are responsible for DNA repair and stability. Oncogenes are mostly
activators of the cell cycle that are strictly controlled in non-malignant cells. Activation
can occur by chromosomal translocations that bring an active promoter close to an
oncogene that is usually not expressed (e.g. BCR-ABL translocation in leukemia). Gene
ampliAcations frequently lead to overexpression of oncogenes, as it is the case for the
MYC gene. In addition, point mutations can lead to continuous activation of oncogenes,
e.g. activating BRAF or RET mutations. In general, mono-allelic activation of oncogenes
is suE cient for malignant transformation. In contrast, tumor suppressor genes (TSGs)
usually require two hits, since the una: ected allele can substitute in part for the mutated
allele. Still, in some cases, a partial e: ect (haploinuE ciency) conferred by the loss of one
allele has been described. TSGs can be altered by point mutations or by larger
chromosomal losses. Typically genes involved in cell cycle control, regulation of
preprogrammed cellular suicide (apoptosis), or the maintenance of genomic integrity
may serve as TSGs. Repair/stability genes comprise a speciAc subgroup of TSGs in that
they maintain the genetic integrity of the cell. Their loss of function is a prerequisite for
the rapid acquisition of the critical mutations required for neoplastic transformation. To
the latter group belong among others the mismatch repair genes (MMR), nucleotide
excision repair genes, base excision repair genes, and genes involved in chromosomal
recombination and segregation, such as BRCA1 and ATM. Germ line mutations of many
of the TSGs have been identiAed as the cause of inherited cancer predisposition genes infamilial cancer syndromes (e.g. hereditary nonpolyposis colon cancer (HNPCC), familiar
adenomatous polyposis (FAP), multiple endocrine neoplasia II (MENII), BRCA1 and 2 for
familial forms of breast cancer, and p53 for the Li-Fraumeni syndrome). In hereditary
cancer syndromes, a tumor suppressor gene is inactivated in the germ line and a second
hit is necessary to abrogate the function of the respective tumor suppressor gene in
individual somatic cells (two-hit-hypothesis). Frequently, the initial gene alteration
induces uncontrolled proliferation of the a: ected cells. In the course of accelerated cell
divisions, genetic errors are accumulated and Anally lead to malignant transformation of
the cells. For many cancer entities, speciAc pathways of consecutive gene alterations have
been described. Two central tumor suppressor genes are a: ected in many cancers: p53 is
a central protein in the control of programmed cell death. Inactivation of p53 seems to be
very important for malignant progression to be possible. pRb is a key regulator of cell
cycle progression by controlling the E2F protein. The majority of cancers show
inactivation of these TGSs themselves. However, speciAc cellular functions can be
abrogated by attacking di: erent components of the respective functional pathway. Thus
functional pathways that represent a complex network of di: erent gene functions may be
hit on various levels in order to promote tumor development. Many di: erent functional
pathways that explain the heterogeneity of the genes a: ected in sporadic cancers have
7been described.
The accumulation of gene mutations necessary for malignant progression cannot be
achieved at the standard mutation rates (1 mutation/million bases) observed in
proliferating cells. It seems clear now that this baseline mutation rate is not suE cient for
carcinogenesis and that some kind of genetic instability must occur in order to allow for
the necessary mutations in cancer cells. This can only been achieved if central pathways
that maintain the genetic integrity of a cell are hit. If such cells succeed to survive, they
may rapidly accumulate a suE cient number of mutations that permit the neoplastic
growth properties. Thus carcinogenesis can formally be subdivided into three major
pathways depending on the molecular mechanism that makes it possible for a suE cient
number of mutations or other genetic alterations of oncogenes and tumor suppressor
genes that initiate and maintain the neoplastic conversion and progression to be
accumulated.
The Major Pathways of Carcinogenesis
Alterations of mechanisms that maintain the genetic integrity of the cell's genome thus
constitute the least common denominator. Consequently, neoplasia emerges as the failure
of genetic functions that control either the composition of whole chromosomes, their
number, structure, and distribution during mitosis or, alternatively, the integrity of the
genetic information encompassed in the chromosome even if they do not undergo gross
numerical or structural alterations. Consequently, cancer is the result of three major
mechanisms that destroy the integrity of the genetic information:
1. Chromosomal instability (CIN)—Induced by failure of mechanisms that guarantee the
even distribution of chromosomes to the daughter cells that emerge during mitosis;?
2. Microsatellite instability (MSI)—Induced by failure of DNA mismatch repair enzymes
that proofread and repair errors that occur during the de novo synthesis of DNA in the
Sphase of the cell cycle; and finally
3. CpG island methylator phenotype (CIMP)—Induced by failure of the epigenetic control
of genes that regulate critical steps in these processes and is often associated with the
later development of MSI-induced cancers.
The vast majority of cancers occur via the CIN pathway. The major underlying
mechanisms of carcinogenesis mediated by CIN is induced by disturbances of the bipolar
character of the mitotic spindle during mitosis and the desegregation of chromosomes
8during mitosis (Fig. 1.10). During the normal M phase of the cell cycle the chromosomes
line up in a plane, the metaphase plate, and associate with spindle Abers of microtubule
proteins. The Abers together form a metaphase spindle. They are connected to the
kinetochores on the chromosomes, i.e. nucleoprotein bodies associated with the
centromeric DNA of the chromosomes, and the centrosomes at the poles of the mitotic
cell. In a normal dividing cell the spindle Abers pull each sister chromatid apart toward
the centrosomes. This ensures that each emerging daughter cell will get exactly one copy
of the sister chromatids to form exactly one new copy of the respective chromosomes in
the emerging new daughter cells. This complex mechanism is controlled by various
checkpoints that monitor that before the process proceeds to the next step exactly two
centrosomes and microtubules spindle apparatus have been formed, that each chromatid
in a pair associates with its own distinct half of the spindle. Chromatid separation is not
allowed to proceed until all pairs of chromatids are lined up in the metaphase plate. If
these checkpoint controls fail, chromosomal segregation may fail and both chromatids
may be pulled to one chromosome (non-disjunction). As consequence one of the daughter
cells may become haploid for the respective chromosome and the other triploid.
Alternatively, one chromosome is completely lost, if the attachment between
microtubules and kinetochores fails.
Fig. 1.10 In uence of centrosome aberrations on chromosomal instability. (A)
Normal centrosomal distribution with two spindle poles results in equal distribution of the
chromosomal material to the daughter cells. (B) Aberrant spindle pole formation leads to
unequal distribution of the chromosomal material; as a result, most cells die while somecan acquire genetic alterations that lead to a growth advantage and the development of
malignant cell clones.
More severe, however, is the impact of failing checkpoints that control the centrosomes
themselves. Cancer cells that arise through the CIN pathway are characterized by an
uneven number and uneven distribution of centrosomes during mitosis. This results in a
total disorder of the normally bipolar spindle apparatus and leads to complex multipolar
spindle structures that during mitosis result in disruption of the chromatids and a
complex uneven distribution of the chromosomal material to the emerging daughter cells
(Fig. 1.10).
The major checkpoints that control these processes appear to act at the transition of the
G1 phase of the cell cycle to the S phase and are controlled by cyclins and
cyclindependent kinase complexes. Interestingly, these processes are targeted by two important
viral oncoproteins encoded by high-risk human papillomaviruses (HPV), the HPV E6 and
E7 proteins, that induce and maintain transformation of cervical cells as we will learn
later in this chapter. This results in failing control of the mitotic processes and in
particular distribution of chromosomes during mitosis and severe numerical and
structural alterations of the chromosomes of the emerging daughter cells. The a: ected
cells usually commit suicide, induced primarily by p53 gene or related genes. Thus most
emerging cancer cells develop molecular mechanisms for evading this cellular suicide
mechanism. In the case of papillomavirus-associated cancers it is the HPV E6 protein that
binds to and inactivates p53. In other cancers not induced by oncogenic HPVs, p53
functions are usually abrogated by an inactivating mutation or deletion of the p53 gene
itself or related genes within the same functional pathway.
Cells that achieve to evade the suicide control may survive the genomic catastrophe
and form the initial cells of an outgrowing cancer. The emerging disproportionate
distribution of chromosomal material in these transformed cell clones induce various
important morphological alterations of the a: ected cells' nuclei that are the cornerstones
of cytopathology (Fig. 1.11). Aneuploid nuclei, coarser texture of the chromatin, changes
in the size and shape of the nuclei, hyper- and hypochromasia, and altered shape and
number of nucleoli are all immanent consequences of the desegregation of chromosomes
during mitosis of cells that have lost control over the strictly bipolar mitotic Agures,
resulting in chaotic multipolar mitotic spindle complexes (Fig. 1.10).Fig. 1.11 Comparative genomic hybridization (CGH) and spectral karyotype
hybridization (SKY). The average CGH ratio proAles for the diploid cell line DLD-1 and
the aneuploid cell line HT29 are presented in (A) and (B). Note the remarkably stable
genome of DLD-1 with only three copy number variations (chromosomes 2, 6, and 11).
HT29 shows a highly aberrant ratio proAle, with copy number alterations occurring on 13
chromosomes. The gains of 7, 8q, 13, and 20q are common aberrations in colorectal
carcinomas. SKY of metaphase chromosomes prepared from these cell lines is shown in
(C) and (D). No numerical aberrations were identiAed in the diploid cell line (C), whereas
trisomies were common in the aneuploid cell line HT29 (D). All aberrations detected by
SKY were also seen by CGH analysis. This indicates that no reciprocal, balanced
chromosomal translocations have occurred.
11Data and images were taken from Ghadimi et al.
9,10Alternatively, cancer cells may arise through the MSI pathway. Cancers that
emerges through this pathway are characterized by substantially di: erent biological
properties. They usually do not display numerical or structural changes of the
11chromosomes or the mitotic Agures. Cells of these tumors usually divide normally.
Consequently, these tumor cells do not display aneuploidy, aberrant mitotic Agures or
gross alterations of their chromosomes. They usually remain diploid without major
morphological alteration of their nuclei. However, errors emerge through a more subtle,
superAcially less brutal mechanism that in its clinical consequences may end as
disastrously as the chromosomal instable cancers. In these cases the cancer cells acquire
increasing mutations of the DNA sequence itself. After each round of DNA replication in
the S-phase of the cell cycle usually hundreds and thousands of mutations, which need to
be checked and repaired before the cell cycle proceeds to avoid very high accumulation,
occur in the replicated genetic code due to misannealing and mispairing. Hence, all cells
in nature develop a sophisticated proofreading mechanism mediated by the DNA-MMR
complex, a multiprotein complex that proofreads and Axes the mutations. If distinct
components of this repair complex are lost, the proofreading capacity declines andmutations particularly in thermodynamically sensitive DNA sequences occur. These lead
to the rapid accumulation of mutations particularly in repetitive DNA sequences that
consist of longer stretches of mononucleotides (mononucleotide repeats). Since these
sequences are also commonly referred to as microsatellites, this latter mechanism of
carcinogenesis is referred to as microsatellite instability. MSI is observed in up to 15% of
colorectal cancers, a subset of endometrial cancers, and a number of urinary tract
cancers. But it is also found in a number of endometrial and bladder cancers, leukemias
and lymphomas, and skin cancers. It is the hallmark of an inherited cancer predisposition
syndrome referred to as hereditary non-polyposis colon cancer syndrome. HNPCC
syndrome is characterized by inherited mutations of defect copies of genes that encode
components of the DNA-MMR complex. MSI may, however, also occur in sporadic
cancers without distinct inherited background. In most of these cases accidental failure of
the epigenetic regulation of the genes that encode the components of the MMR complex
may fail usually due to altered methylation of the respective promoter sequences.
The third emerging major mechanism of carcinogenesis is referred to as CpG island
12methylator phenotype. Cancer cells that emerge in frame of this molecular phenotype
initially experience alterations of the epigenetic control mechanisms that tell genes of
speciAc cells where and when they should be active or silent. In many instances this is
regulated by methylation of speciAc sites within the DNA by the addition of methyl
groups particularly to CpG sites frequently found throughout the whole genome. The
addition of these methyl groups either completely blocks the expression of the respective
gene by condensing its chromatin structure or may just modify the binding properties of
activating or inhibitory factors that activate or repress transcription of the respective
gene. The CIMP phenotype is clearly the less well-characterized cancer phenotype so far.
Because of the basic mechanism it may end up Anally in cancer cells that impress as CIN
phenotype due to the transcriptional inactivation of genes involved in chromosomal
homeostasis or alternatively as MSI cancers due to the inactivation of genes that maintain
the DNA-MMR functions; here the most pronounced example is the hMLH1 gene
frequently inactivated via the CIMP phenotype in MSI-positive sporadic colorectal
cancers.
Carcinogenesis Induced by Papillomavirus Infections
Human papillomavirus infections play the predominant carcinogenic role for cancers of
13the lower female genital tract, and in particular cervical cancer. Molecular pathways of
how these viruses contribute to neoplastic transformation of epithelial cells have to a
large extent been clariAed and thus will be considered in more detail in the following
paragraphs.
Published reports on the concept that infectious agents may be involved in the
pathogenesis of cancers of the female lower genital tract dates back to the middle of the
nineteenth century. Domenico Rigoni Stern described his observation that women with
frequent sexual contacts with various partners are at substantially higher risk to develop
cervical cancer than women who did not have sexual contacts. Since then sexually
transmittable agents that may explain this peculiar epidemiological feature of thesecancers have been extensively investigated. It lasted until the end of the 1970s when the
Arst truly important clues that made it possible to delineate the formal molecular
pathogenesis of cervical cancers were put forward. In 1976 Harald zur Hausen published
his hypothesis that cervical cancer and its precursor lesions may be caused by agents
similar to those that cause hyperproliferative lesions in the genital tract, the condylomata
14acuminata or genital warts. This hypothesis initiated an intense chase to track down
the putative agents. Genomes and viral particles of a new type of human papillomavirus
15were identified in biopsies of genital warts and labeled as HPV 6. Shortly later a related
HPV type was cloned from DNA samples isolated from laryngeal papillomas and referred
16to as HPV 11. This HPV type showed substantial homologies with HPV 6 and in
subsequent studies both HPV types were found in laryngeal as well as in genital
17papillomas.
Both HPV types were used as probes to look for related DNA sequences in DNA
extracted from tumor biopsies and cell lines derived from cervical cancers in further
hybridization experiments. These experiments led to the identiAcation of related but
clearly distinct HPV sequences in cervical cancer cells. Subsequent cloning and detailed
characterization has revealed that these sequences are indeed new types of the HPV
18,19group that have since then been referred to as HPV 16 and 18.
Basic Structure of the Virus and Its Genome
Human papillomaviruses are small viral particles that constitute viral capsids built up by
self-assembling proteins encoded by the L1 and L2 genes of the virus (Fig. 1.12). They
lack an envelope and are thus relatively resistant toward environmental hazards. Thus
viral capsids measures about 55 nm in diameter and include an about 8,000-bp-long
circular episomal genome that is highly twisted (supercoiled circular genomes). This
circular genome encompasses three major genetic and functional regions:
• Early region E, which includes about eight different genes (E1–E8);
• Late region L, which includes the two genes that encode the proteins that make up the
capsids (L1 and L2); and
• Upstream regulatory regions of the early region (URR), which includes the important
regulatory sequences of the early promoter and enhancer that mediates the highly
complex regulation of the viral gene expression pattern in their host cells that is so
important for all processes related to HPV-related carcinogenesis and that we will discuss
later.Fig. 1.12 Human papillomaviruses. Top: electron microscopy pictures of viral
particles and viral DNA. Bottom: Schematic diagram of the viral genome indicating the
most viral genes and most important functions.
A second regulatory element that regulates the expression of the late genes is included
in sequences that are part of the E7 gene (late promoter).
Papillomavirus types are di: erentiated in HPV genotypes based on distinct variations
of their nucleic acid sequences. A certain stretch within the L1 gene is used as a reference
to di: erentiate di: erent types according to an international agreement on the
20classification of HPV types.
Meanwhile more than 120 di: erent HPV types have been characterized, but is
estimated that the true number of papillomaviruses that may infect humans exceed 200.
Papillomaviruses are strict epitheliotropic viruses that exclusively infect epithelial cells of
the outer surfaces of the human body. Most of the HPV types infect the cutaneous parts of
the skin (skin types), whereas about 40 HPV types are preferentially found in lesions in21the mucosal surfaces in the lower anogenital tract (mucosa types). Among the mucosa
types two di: erent classes of HPV types are distinguished (Table 1.1): (a) the low-risk
HPV (LR-HPV) types that cause massive exophytic, hyperplastic wart-like lesions, and (b)
the high-risk HPV (HR-HPV) types associated with cancer particularly of the uterine
cervix (Fig. 1.13). The latter, however, usually cause only very minor lesions without
massive hyperplasia. These lesions rarely exceed the surface of the epithelium in the early
stages of the infection. They commonly occur and regress without the infected person
realizing the infection. Thus although these infections occur in men and women
apparently with the same incidence, clinical consequences that would trigger follow-up
usually only occur in women who have developed cervical lesions as part of a
carcinogenic process, whereas in men the infections usually occur and regress unnoticed.
Table 1.1 Correlation between phylogenetic and epidemiologic classiAcation of mucosal
HPV types
Phylogenetic
Epidemiologic classification
classification
High-risk HPV types Low-risk HPV types
High-risk HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 70
types 58, 68, 82, , , 26 53 66
Low-risk HPV 73 6, 11, 40, 42, 43, 44, 61,
types 72, 81, CP6108
The epidemiological classiAcation of these types as probable high risk is based on zero
controls and one to three positive cases.21
Putative high risk types.
Fig. 1.13 DiDerent types of HPV infection. (1) A state of very low viral activity
shortly after initial infection through microlesions of the epithelium ("latent phase"). (2)The replicative infection characterized by strong viral gene expression and viral capsid
formation in the upper di: erentiated epithelial layers. (3) The transforming infection
characterized by deregulated oncogene expression in the replication competent basal
cells. CIN, cervical intraepithelial neoplasia, grade 1–3.
Papillomaviruses infect the basal cells of the epithelium via binding to certain
cellsurface glycosaminoglycans expressed on the basal cells of the epithelium (Fig. 1.14).
Once they have entered the cell, the viral capsids are broken down and the episomal viral
genome is released in the nucleus. Viral early genes are expressed at this stage on a very
low and highly controlled level that allows for low copy replication of the genomes on the
order of 10 to 50 genomes per infected basal cell; however, massive ampliAcation of the
viral genome or even replication of the virus does not occur at this stage of infection in
basal cells. Only in certain instances not yet characterized in detail does high-level gene
expression of the viral early genes occur in di: erentiated cells of the intermediate layers
of the epithelium. This triggers ampliAcation of the viral genome. Once the cells have
reached the superAcial cell layers the early genes are shut o: and high-level expression of
the late genes (L1 and L2) occurs (early–late switch). This results in expression of the
respective proteins, packaging, and self-assembly of new mature viral particles that are
Anally released from the cells once during the normal di: erentiation process the infected
and virus-producing keratinocytes decay into keratin fragments. The replication strategy
of the human papillomaviruses is therefore tightly linked to natural di: erentiation
processes of their host cells. For the virus this has two advantages.
Fig. 1.14 Schematic representation of the diDerent phases of an HR-HPV infection:
After infection of basal cells of the epithelium the virus may persist in a latent state. Upon
di: erentiation and maturation of the cells, the viral genome may be replicated and novel
virus particles are produced and released at the surface of the epithelium. This stage is
often characterized by typical koilocytes as a morphological hallmark of virus production.
If transcriptional control of the viral genome in the basal and parabasal cells fails,
expression of the viral oncogenes in these replicating cells may induce chromosomal
instability and thus initiate transformation. Later in the progression to invasive cancers
the viral genome often becomes integrated into the host cells' chromosomes as a sign of
increasing chromosomal instability.
First, the biology of the primary host cells at the bottom of the epithelium that retain
the capacity to multiply and generate new cells is only marginally altered by the
infection. HPV infections induce no cytolysis, no in; ammation, or other tissue damage inthe basal cell compartment of the epithelium. Acute LR-HPVs induce more proliferation
of the infected basal cells and cause exophytic lesions that may clinically impress as warts
or condylomata seen with comparable incidence in men and women. Proliferation of
basal cells is induced by acute HR-HPV types to much less extent since a simple infection
almost never causes exophytic lesions. Usually these infections regress spontaneously and
clinically unrecognized.
Secondly, due to the fact that the virus is only produced on the very superficial surfaces
of the infected epithelium there is very limited contact between viral antigens and the
immune system of the infected host. Thus the acute infection induces very little humoral
immune responses and serum antibodies are only observed in low titers in some of the
infected individuals. They do not induce a protective humoral immunity. Cellular
immune responses are only weakly activated, which usually takes a long time during
which the virus can multiply and spread until reliable cytotoxic immune functions have
been activated to defeat the virus and the lesions it has caused.
The tight association of the replication strategy of the papillomaviruses with the
di: erentiation status of their host cells further allows the virus to multiply and spread
with a highly restricted amount of their own genetic information. Complex genetic
features that control the restricted expression pattern of the virus during the di: erent
di: erentiation stages are contributed apparently by the host cell and not by the virus;
thus the virus has no need to retain genes that may mediate these functions by
themselves.
Epidemiology of HPV Infections
HR- and LR-HPV infections are usually acquired in the early years upon uptake of sexual
activity. Most studies have been performed in women, but it can be extrapolated that the
infection pattern in men is not substantially di: erent from that in young women.The
22highest infection rates are seen in young women at 18 to 25 years of age. Over time the
incidence of HPV infections decreases, but there seems to be a second peak of HPV
infections in older women of more than 45 years of age. The infection clearly shows a
typical sexually transmitted pattern and depends on personal lifestyle habits, for
example, the number of sexual partners, and the frequency of sexual contact with
partners who have themselves sexual contact with other partners. Several studies have
shown that the cross-sectional incidence of HR-HPV infections ranges between 3 and 25%
of the female population. The cumulative infection in average women is calculated to be
more than 50% once in their lifetime. Thus a tremendous amount of women (and hence
also men) are infected with these agents.
The HPV types 16 and 18 are found in about 70% of all cervical carcinomas, whereas
among healthy women the remaining HR-HPV types are more common. This observation
suggests that women infected with the two types, HPV 16 and 18, may have a higher risk
to developing high-grade precursor lesions or even invasive carcinomas than women who
23are infected with the other HR-HPV types. This notion is strongly supported by a
variety of studies that indicate that women infected with HPV 16 and 18 are more likely
to develop high-grade lesions (cervical intraepithelial neoplasia (CIN) 3) in less time thanwomen infected with other HR-HPV types. Moreover, the time of progression of
highgrade lesions to invasive carcinomas appears also to depend on the HPV type that causes
23athe high-grade lesions.
HR-HPV infections usually last for several months and in most instances regress
24spontaneously without causing relevant clinical lesions. The acute HPV infection may
impress as CIN 1 lesions in histological sections in that the typical koilocytes indicate the
acute replication of the virus in intermediate and superAcial cells of the infected
epithelium. These acute infections strictly adhere to the regulatory gene expression
pattern outlined earlier in that the cellular di: erentiation stage determines the expression
of the viral genes in the epithelium. According to this, limited and highly regulated gene
expression is found in basal and parabasal cells of the epithelium. By this replication
strategy, HPVs successfully avoid expression of viral genes in proliferation competent
epithelial cells that may become transformed stem cells for a cancer. Under "normal"
conditions viral genes are only expressed in cells that are irreversibly cell cycle arrested.
HPVs thereby can multiply almost unnoticed by the host and spread to many other
individuals (Fig. 1.14).
The Role of the HR-HPV E6 and E7 Genes
The causal relationship of HR-HPV types and cervical cancer is based on four major
experimental observations:
1. In more than 99 % of cervical cancers genomes of HR-HPV types can be found;
2. Two viral gene, E6 and 7, are in all cervical cancer preserved and expressed;
3. The E6 and E7 genes can transform primary epithelial cells into neoplastic cell clones;
and
4. If the expression of these genes is inhibited cervical cancer cells stop to proliferate and
lose the neoplastic growth properties.
These facts clearly underline the central role of the viral E6 and E7 genes for the
HRHPV mediated transformation processes.
During the past 20 years basic research on the biochemical activities of both viral
proteins have revealed three major aspects that underline their oncogenic activities: the
E6 protein of the oncogenic HPV types induces premature degradation of the p53 tumor
25suppressor gene and thus interfere with its proapoptotic functions ; the E7 protein
induces destabilization of the retinoblastoma protein complex and thus allows the cell to
26evade cell cycle control at the G1/S phase transition thorough the pRB pathway ; and
both genes interfere with centrosome synthesis and function that results in desegregation
of the chromosomes during mitosis and numerical and structural chromosomal
27aberrations. As a result of chromosomal instability induced by E6 and E7, HPV
28genomes may become integrated into the host genome. Both viral oncoproteins interact
with many more proteins of epithelial cells that are summarized in Fig. 1.15. These
interactions further support that central role of E6 as anti-apoptotic protein and of E7 asproliferation inducing and cell cycle deregulating factor.
Fig. 1.15 Interactions with cellular proteins and oncogenic functions of the
HRHPV proteins E6 and E7.
Progression of HPV-Infected Epithelial Cells to Invasive Cancer Cells
The progression of pre-neoplastic lesion of the uterine cervix to invasive cancer cells is a
well-described process usually subdivided into three main stages referred to as cervical
intraepithelial neoplasia 1, 2, and 3 (Figs. 1.13, 1.14). This classiAcation is based on
histopathological criteria that are essentially describing the pathogenic e: ects of
persisting HR-HPV infection in the cervical epithelium. CIN 1 is characterized by minor
activation of the proliferation rate of the basal cells, whereas the proliferating cells are
still restricted to the lower third of the epithelium. The abundant presence of koilocytes in
the more superAcial cell layers document the massive production and release of HPV in
the more superAcial cell layers. In CIN 2 lesions the proliferating cells extend to the two
lower thirds of the epithelium. In these lesions the number of koilocytes decreases
gradually. CIN 3 lesions are characterized by proliferating cells that extend now into the
upper third of the epithelium or in case of the carcinomata in situ lesions (CIS) extend
through the full thickness of the epithelium. These diagnostic categories are very useful in
clinical practice since they make it possible to subdivide precancerous stages according to
their likelihood to progress to invasive cancer. However, since they do not make it
possible to visualize the molecular events induced by the deregulated expression of the
viral oncogenes in these cells, they do not formally make it possible to subdivide the
preneoplastic lesions according to the molecular events involved in the carcinogenic
processes.
As discussed above, the expression of HPVs is tightly regulated in basal and parabasal
cells of the epithelium. Thus, the initiation of the carcinogenic process is not the infection
of basal epithelial cells by HR-HPVs but rather the emergence of epithelial cell clones
29that fail to downregulate the expression of the viral oncogenes in the basal cell layer.According to this model, preneoplastic HPV-induced lesions can be subdivided into acute
virus-producing lesions (CIN 1, acute or replicating HPV infection) and transforming HPV
infections that emerge from CIN 1 lesions but may progress via CIN 2 Anally to CIN 3/CIS
30lesions into invasive cervical carcinomas. In terms of the relative risk that each of these
di: erent stages carries for progression, this revised molecular model of the pathogenesis
of the HPV-induced cancers strongly suggests that as long as no deregulated expression of
the viral genes has occurred in the basal cells, the risk for progression to high-grade
lesions or even cancer is comparably low. This situation dramatically changes once
individual HR-HPV infected basal cells lose the control over the expression of the viral
genes and start to express the viral E6 and E7 genes that then may initiate their
deteriorating activities and initiate the carcinogenic process. Expression of the HR-HPV
type E7 protein interferes with the regulation of the G1/S phase control of the cell cycle
and permits proliferation of cells even if the complete cell cycle machinery is not yet
prepared to replicate the DNA of the chromosomes. This results in Axation of unrepaired
mutation but also in damage of the genome by itself. In normal cells this would have
been counteracted by the activation primarily of p53 mediated control mechanisms that
either would have resulted in stoppage of cell cycle progression or, if the genomic
damage is already too severe, induction of the cellular suicide mechanisms (apoptosis)
that prevent survival and expansion of cells with damaged genomes. During the normal
life cycle this never occurs since the expression of the viral early genes is restricted to
terminal differentiated cells of the intermediate or superficial cell layers (Fig. 1.14).
Cells that display chromosomal instability under the in; uence of the papillomavirus
genes E6 and E7 rapidly accumulate numerous morphological alterations. First aberrant
mitotic Agures are seen in dividing cells, then enlarged nuclei with altered chromatin
structures appear, and Anally severe changes of the DNA content of the cells results in
aneuploidy and anisonucleosis. All these criteria are classical features that build up the
cytological classiAcation system to score the degree of abnormalities induced by HPV
infections in cervical cells.
One important aspect in the pathogenesis of HPV-induced cancers in the female
anogenital tract is its typical anatomical restriction to the transformation zone of the
uterine cervix. Although HR-HPV infection occur at multiple sites in the male and female
anogenital tract, the epithelial cells of the transformation zone of the cervix appear to be
substantially more sensitive to HPV-mediated transformation than other HPV-infected
epithelial cells in the vagina, vulva, outer surface of the cervix, and particularly the
epithelium of the penis, which only rarely become transformed. These peculiar
epidemiological features point out important molecular di: erences in these di: erent host
cells. It is highly likely that these di: erences rely on the stringency of the molecular
control that prevents the activation of the E6 and E7 oncogenes in the basal cells of the
epithelium. Given the fact that the molecular features that mediate this control are an
inherent part of the di: erentiation control system of the epithelial cells, it can be
speculated that the need for epithelial stem cells at the transformation zone of the uterine
cervix plays an important role. Stem cells of the transformation zone retain two potential
pathways: to di: erentiate under the in; uence of high estrogen levels into a single-layer
glandular epithelium, and to di: erentiate under the in; uence of low estrogen levels intoa multilayer squamous epithelium. These morphological di: erences are regulated by the
activation of di: erent sets of genes and hence di: erent epigenetic regulatory mechanisms
that become activated in either situation. There is accumulating evidence that these
epigenetic control mechanisms are also used by the papillomavirus genome to either
restrict or activate the expression of their genes. The multipolar di: erentiation capacity
of the epithelial cells at the uterine transformation zone may thus explain why these cells
are so much more sensitive to transformation by HR-HPV types than many other infected
cells of the human body.
Once the activation of the viral oncogenes in the basal cells of the infected epithelium
has occurred and the Arst transformed cells that display chromosomal instability have
expanded, the lesions may progress from a CIN 1 lesion (representing acute replicating
HPV infections) to CIN 2 and 3 lesions (re; ecting transforming HPV infections) and
finally invasive carcinomas (Fig. 1.14).
The knowledge now gathered on this important step of the HPV-associated
transformation process o: ers new targets for screening and diagnosis of cervical
precancer and cancer. Activation of the expression of the HR-HPV E6 and E7 genes in the
31-35basal and parabasal cells results in overexpression of a cellular protein, p16INK4a.
All cells that express HR-HPV proteins and retain the capacity to proliferate express
extremely high levels of the p16INK4a protein; hence p16INK4a is an interesting
surrogate marker for HPV transformed cells.
Based on these Andings it can be expected that in the near future these new techniques
will have a deep impact on revised cervical cancer screening programs.
Research over the past thirty years has thus made it possible to identify an infectious
agent (HR-HPV) as cause of a major human cancer. The experimental analysis as well as
epidemiological research has made it possible to clarify the role of these viruses in
carcinogenesis. Detailed analysis of the expression and function of viral genes has
revealed that it is not the acute infection by HPVs but rather the lack of normal cellular
control functions that suppress their expression in basal cells that initiates the
transforming infection and may result Anally in full neoplastic transformation of
individual infected cells. This research has paved the way for substantially improved
diagnostic and therapeutic tools, including novel assays for cervical cancer screening as
well as major global vaccination programs that aim to prevent the primary infection of
humans by HPV and were shown to e: ectively prevent induction of HPV-induced
preneoplastic or neoplastic lesions.
Concluding Remarks
The scientiAc analysis of cells and their components as the essential units of complex
organisms has initiated a dramatic development in biomedical research and Anally led to
the identiAcation of the essential biochemical basis of inheritance and diversity of life. It
has initiated a paradigm shift in biology from a purely descriptive science to one that has
made it possible to fully explore the construction kits of life. Genes have been recognized
as the basic units of inheritance and their activity in given cells has been realized as the
determinants of a distinct biological phenotype. The biological and chemical principlesthat control the activities of genes within cells have been explored and have revealed why
and when certain cells adopt distinct functions. Since the expression of certain sets of
genes also determines the shape of cells, functional irregularities and errors are frequently
indicated by altered morphology. In particular early changes associated with neoplastic
transformation of cells are indicated by initially discrete and later substantial and
prominent morphological alterations of cells. Early detection of cancer and many other
diagnostic applications in medicine are based on these fundamental concepts and have
built the cornerstones of pathology and cytopathology.
As achievements in understanding genes and their functions in cellular biology and
pathology have progressed, distinct markers or proteins encoded by deAned genes that
indicate speciAc alterations have been identiAed. In recent years this concept has been of
particular importance for the area of oncology, where the (over)expression of certain
proteins has been found to be associated with the initial events that Anally result in
cellular transformation and outgrowth of cancer cells. Neoplastic transformation of cells
has been realized to be a malfunction of genes that control growth and di: erentiation.
Basic principles of this malfunction have been elucidated. Desegregation of chromosomes
during cell division, lack of genome surveillance during the replication of the genetic
code, and activation of viral (e.g. papillomavirus) oncogenes in cells have been identiAed
as causes of neoplastic transformation. Based on these fundamental concepts, functional
genomics and proteomics have revealed the expression of distinct patterns of proteins in
speciAc disease conditions. Such proteins are now usually referred to as biomarkers. The
detection and evaluation of biomarkers is becoming a more and more important part of
pathology and cytopathology as it makes it possible to track down morphological
alterations of cells to the functional level of genes. The contribution of certain
carcinogenic human papillomavirus types to carcinogenesis particularly of cells at the
uterine transformation zone is a good example for this paradigm and exempliAes the
diagnostic potential that these new concepts hold for the future.
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12. Weisenberger D.J., Siegmund K.D., Campan M., et al. CpG island methylator phenotype
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13. zur Hausen H. Papillomaviruses and cancer: from basic studies to clinical application. Nat Rev
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14. zur Hausen H. Condylomata acuminata and human genital cancer. Cancer Res. 1976;36(2 pt
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15. Gissmann L., zur Hausen H. Partial characterization of viral DNA from human genital warts
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16. Gissmann L., Diehl V., Schultz-Coulon H.J., et al. Molecular cloning and characterization of
human papilloma virus DNA derived from a laryngeal papilloma. J Virol. 1982;44(1):393-400.
17. Gissmann L., Boshart M., Durst M., et al. Presence of human papillomavirus in genital tumors.
J Invest Dermatol. 1984;83(1 Suppl):26s-28s.
18. Boshart M., Gissmann L., Ikenberg H., et al. A new type of papillomavirus DNA, its presence in
genital cancer biopsies and in cell lines derived from cervical cancer. EMBO J.
1984;3(5):1151-1157.
19. Durst M., Gissmann L., Ikenberg H., et al. A papillomavirus DNA from a cervical carcinoma
and its prevalence in cancer biopsy samples from different geographic regions. Proc Natl Acad
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20. de Villiers E.M., Fauquet C., Broker T.R., et al. Classification of papillomaviruses. Virology.
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21. Munoz N., Bosch F.X., de S.S., et al. Epidemiologic classification of human papillomavirus types
associated with cervical cancer. N Engl J Med. 2003;348(6):518-527.
22. Lau S., Franco E.L. Management of low-grade cervical lesions in young women. CMAJ.
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23. Khan M.J., Castle P.E., Lorincz A.T., et al. The elevated 10-year risk of cervical precancer and
cancer in women with human papillomavirus (HPV) type 16 or 18 and the possible utility of
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23a. Vinokurova S., Wentzensen N., Kraus I., et al. Type-dependent integration frequency of
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24. Ho G.Y., Bierman R., Beardsley L., et al. Natural history of cervicovaginal papillomavirus
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28. Wentzensen N., Vinokurova S., von Knebel Döberitz M. Systematic review of genomic
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30. von Knebel Döberitz M. New markers for cervical dysplasia to visualise the genomic chaos
created by aberrant oncogenic papillomavirus infections. Eur J Cancer.
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for dysplastic and neoplastic epithelial cells of the cervix uteri. Int J Cancer.
2001;92(2):276284.
32. Klaes R., Benner A., Friedrich T., et al. p16INK4a immunohistochemistry improves
interobserver agreement in the diagnosis of cervical intraepithelial neoplasia. Am J Surg Pathol.
2002;26(11):1389-1399.
33. Sano T., Oyama T., Kashiwabara K., et al. Expression status of p16 protein is associated with
human papillomavirus oncogenic potential in cervical and genital lesions. Am J Pathol.
1998;153(6):1741-1748.
34. Wentzensen N., Bergeron C., Cas F., et al. Evaluation of a nuclear score for p16INK4a-stained
cervical squamous cells in liquid-based cytology samples. Cancer. 2005;105(6):461-467.
35. Zhang Q., Kuhn L., Denny L.A., et al. Impact of utilizing p16INK4A immunohistochemistry on
estimated performance of three cervical cancer screening tests. Int J Cancer.
2007;120(2):351-356.CHAPTER 2
Basic Cytogenetics and the Role of Genetics in Cancer
Development
Alain Verhest, Pierre Heimann
Contents
Introduction
Historical Background
Basic Knowledge of Cytogenetics
Cell Cycle
The Interphase
The Mitosis
The Meiosis
The Chromosome Structure
Methodology
The Karyotype
Fluorescent in Situ Hybridization
Comparative Genomic Hybridization (CGH)
Acquired Chromosomal Aberrations in Cancer
Introduction
Lymphomas
Sarcomas
Thyroid Carcinomas
Clinical Applications of Conventional Cytogenetics and Fish in Cytology
Introduction
FISH Strategy
Application
Concluding Remarks
Introduction
This chapter will summarize the knowledge acquired on conventional cancer cytogenetics in the second
half of the last century and introduces additional applications of ' uorescent in situ hybridization available
for the study of cancer development and evolution.
Other indications of these techniques applied on cytology samples are also described in Chapter 36.
Historical Background
As suspected by von Hansemann more than a century ago, cancers are associated with nuclear and mitotic
anomalies in their cells.
In 1914, Boveri hypothesized his theory on somatic mutations responsible for the origin and development
of malignant transformation. He stressed the acquisition of an unbalanced chromosome constitution as a
cause of cancer illustrated by mitotic asymmetry and asynchrony, and foresaw the monoclonal origin of the<
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cancer cell. It took at least 40 more years to establish the exact number of human chromosomes. The
bloodculturing method became more successful than the squash method when colcemid was discovered to arrest
the mitotic cycle in metaphase by poisoning the mitotic spindle and to prevent the centromeres from
dividing. The erroneous adjunction of a hypotonic solution to a pellet of harvested cells was an unexpected
improvement in the spread of individualized chromosomes rid of their cellular envelope, resulting in a nicer
dispersal on the metaphase spread.
In 1956 Tjio and Levan accurately reported that the human somatic cell contains 46 chromosomes,
including 22 pairs of autosomes and one pair of sex chromosomes; one X of maternal origin and the other
1chromosome—X or Y—being from the paternal source. Rarely have discoveries had such impact on
modern biology and medicine as the description of the 46-chromosome karyotype. The newborn
cytogenetic discipline investigated simultaneously the eld of inherited diseases and acquired chromosomal
anomalies in cancer cells.
Trisomies of chromosome 21 in mongolism and of other autosomes or numerical variations of sex
chromosomes proved their speci city and consequently their diagnostic value in congenital syndromes. In
1960 Nowell and Hungerford reported the rst evidence of a chromosome anomaly speci cally associated
2with a malignant disease, the chronic myelogenous leukemia. They showed the recurrent presence in
leukemic leukocytes of a deleted small chromosome that they named the Philadelphia (Ph) chromosome in
reference to the city where they were working. This proof of a genetic cause in cancer was the starting point
to new insights into the pathways of malignant initiation and progression.
Basic Knowledge of Cytogenetics
The human somatic cell contains two copies of each chromosome, one from paternal and the other from
maternal origin. Therefore the karyotype is diploid with doubled amount of deoxyribonucleic acid (DNA)
(2n) compared to the gametes (n) with a single set of 23 chromosomes.
The rst step is to review the di erent stages of the cell cycle which are essential to the acquisition of
chromosomes suitable for karyotyping.
Cell Cycle
The cell cycle is a process of successive cell divisions (mitosis) interrupted by so-called “resting” periods
(interphase). Actually, the resting cell is very active metabolically with continuous molecular interactions
between DNA, ribonucleic acid (RNA), and proteins.
The Interphase
The interphase is the period wherein the cell is in a nondividing state and can be at different stages: the first
gap (G1) is between the last mitosis and the S-phase (phase of DNA synthesis) and the second gap (G2) is
between the completion of the S-phase and the next mitosis (M). The mitotic division occupies only a short
time in the cell cycle. If the cell reaches its ultimate stage of differentiation and will not divide anymore, the
cell is said to be in phase G0 of the cycle. G0 applies also for those cells that have temporarily stopped
dividing (Fig. 2.1).@
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Fig. 2.1 Schematic representation of the cell cycle with the four sequential phases (see text). The cell
cycle checkpoints are located at the G1/S and G2/M transitions.
During the G1 phase, the cell is metabolically active and requires many organelles for protein synthesis
while acquiring the potential for the DNA-doubling process. The duration of the entire cycle depends on the
time of the G1 phase, which varies according to di erent conditions and tissue types. G1 phase may last
from only a few hours to weeks or months, depending on the mitotic rate of the tissue. The phase of DNA
synthesis (chromosome replication) has a duration of approximately 8 hours. The replication is not
homogeneous throughout the genome, and asynchronism of replication occurs, particularly in the synthesis
of the heterochromatin composing the inactivated X chromosome.
DNA replication is achieved when all the chromosomes are duplicated in two identical sister chromatids
with the consequence that the total amount of DNA is now doubled compared to the normal 2n value of the
interphase nucleus. The following phase, G2, takes about 4 hours and accumulates the cytoplasmic
organelles necessary to complete the mitosis.
This step-by-step progression is controlled by a series of checkpoints which stop the process if the
previous phase is not achieved. Di erent proteins act sequentially on the cell cycle: the cyclin-dependent
kinases (CDKs), the cyclins, and the CDK inhibitors (CKIs).
Activation of kinases by cyclins positively regulates the cycle by allowing the cell to enter the successive
phases. If the quality of DNA synthesis is impaired, CKIs would automatically stop the process and drive the
cell to apoptosis.
The Mitosis
Although the cell cycle is a continuous process, mitosis has four distinct phases (Fig. 2.2).@
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Fig. 2.2 The four di erent phases constituting the mitotic process (cytokinesis being included in the
telophase).
Prophase
Condensation and fragmentation of the chromatin into chromosomes becomes evident. The nucleolus
vanishes and the centrioles, replicated in G2, migrate to opposite poles of the cell. Each chromosome is still
attached to the nuclear membrane and composed of a double strand of sister chromatids. A constricted area
called centromere becomes apparent on the chromosomes and the nuclear membrane disintegrates.
Metaphase
The chromosomes are aligned at the equatorial plate of the mitotic spindle and attached by their
centromere to the network of microtubules. Metaphase chromosomes are composed of two sister chromatids
joined together by the centromere.
Anaphase
The centromeres are split into two parts and both strands of the sister chromatids are attracted to opposite
pole by shortening of the spindle bers. The chromosomes, pulled apart, are clustered at each pole of the
cell.
Telophase
Telophase results in the formation of a nuclear membrane. The constriction of the cellular membrane starts<
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the division of the cytoplasm (cytokinesis). The chromosomes progressively melt back into a chromatin
network. At the end, both daughter cells have the same number of chromosomes as the maternal cell.
The Meiosis
The meiosis is a more complex process by which the gonad cell undergoes two cellular divisions.
The meiosis I follows stages similar to the mitotic division. During prophase I, each chromosome is
duplicated. Chromatid exchanges occur between paired homologue chromosomes which are linked together
by their sites of junction: the chiasmas. This process, called “crossing over,” results in genetic
recombination, with the consequence that genomes between maternal and daughter cells will not be strictly
identical. Anaphase I start with the migration of homologue chromosomes to the opposite poles of the cell
without splitting of their centromere. Meiosis II arises without previous DNA synthesis and produces the
longitudinal separation of the two chromatids, thereby reducing the cell to a haploid n number of 23
singlestranded chromosomes. The fecundation of the ovule by the spermatozoid will restitute the diploid value of
somatic cells and provide a complete zygotic genome.
The Chromosome Structure
The chromosomes are composed of DNA and associated histone and non-histone proteins.
This combination, called chromatin, is individualized into visible chromosomes only during mitosis. The
double helix of DNA described by Watson and Crick is supercoiled around protein cores in a complex
structure of nucleosomes. Compacted nucleosomes constitute chromatin segments of approximately 30 nm
in diameter observable in electron microscopy. Further condensation makes it optically identi able as
heterochromatin in the interphase and as chromosomes at the late prophase. An animation on cell division
3and chromosome structure can be found at http://www.johnkyrk.com.
The extremities of the chromosomes are called telomeres. They preserve the integrity of chromosomal
extremities by allowing replication to occur without loss of coding sequences, but undergo repetitive
shortenings themselves after each cellular division. The so-called “mitotic clock” counts the number of cell
divisions that have occurred and pushes the cell to apoptosis before a critical telomeric shortening is
reached. If this should occur, chromosomes would be prone to fuse end to end, giving rise to sticky ends
that would favor mitotic aberrations and promote the accumulation of subsequent genetic rearrangements,
4possibly leading toward the first crucial steps in the development and progression of neoplasia.
Methodology
The Karyotype
In the 1950s and 1960s, human chromosomes were studied with Giemsa or Wright stains, making it
possible for these chromosomes to be counted accurately and grouped together according to their length
and the position of the centromeric constriction. The 22 pairs of autosomes and the sex chromosomes were
thus classi ed into seven groups, A to G. The largest pairs are numbered 1 to 3 in group A. The centromere
is located in the middle of chromosomes 1 and 3 and displaced in a submetacentric position in pair 2.
Group B is composed of pairs 4 and 5, both with a subtelomeric centromere. Group C is the largest and is
composed of medium-sized chromosomes including pairs 6 to 12 and chromosome X. Most of them are
submetacentric and roughly classi ed by decreasing length. Group D is composed of chromosome pairs 13–
15 and characterized by a distal acrocentric centromere. Group E contains the metacentric pair 16 and the
submetacentric 17 and 18 sets. Chromosome pairs 19 and 20 are smaller metacentric chromosomes and
constitute group F. Group G is composed of small acrocentric chromosomes arbitrarily placed in pairs 21
and 22. The small Y chromosome is included in group G.
Accurate individual classi cation of chromosomes was rendered possible by the banding techniques
5developed rst by applying ' uorescent quinacrine mustard on metaphase preparations. This ' uorescent
agent reveals transverse bright bands (Q banding) of di erent intensities along the chromosome arms.
Other procedures using trypsin digestion (which removes proteins from chromatin) and Giemsa staining<
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yield dark G bands superimposed on the bright Q bands. This led to a very precise identi cation of each
individual chromosome (Fig. 2.3). Techniques with heat denaturation in saline solution obtained a reverse
staining called R bands with optional enhancing of telomeric ends in T banding.
Fig. 2.3 G banded karyotype from a normal male showing 22 pairs of autosomal chromosomes and two
X and Y sexual chromosomes
The di erent banding pattern for each of the 23 di erent chromosomes allows for a perfect pairing of
homologues. The number of bands can be raised up to 800 by the high-resolution staining technique
obtained on prometaphase chromosomes. The dark G bands correspond to a compact conformation of the
chromatin while the clear bands are composed of rather uncoiled chromatin. The dense Q and G bands are
G+C-rich and contain repetitive inactive DNA. Active genes are supposed to be in clear bands; constitutive
heterochromatin is located in the pericentromeric regions as revealed by C banding and appears as
chromocenters in the nondividing nucleus. Chromosome Y has a unique strong ' uorescent appearance
visible in the interphase nucleus as a bright dot also visible as a dark C band. With these staining methods,
the chromosomes 21, already recognized in the prebanding era because of their known involvement in
Down syndrome, remained classi ed as such, and the minute marker of CML was consequently considered
as belonging to pair 22.
The Standardized Reporting
In 1971 at the International Conference in Paris, a nomenclature for banded chromosomes was adopted
and extensively explained in a later revised version (ISCN 1985), codifying the way in which all possible
6numerical and structural chromosome anomalies should be reported.
On each banded chromosome pair, the upper arms are designated as p arms (petit, meaning “short” in
French); the longer arms below are designated as q arms. Regions and bands are numbered starting from
the centromere.
According to this nomenclature, the Philadelphia (Ph) chromosome was revealed by J. Rowley as being a
t(9;22) translocation, hence the result of a reciprocal translocation between the q arms of chromosomes 9
7and 22, with breakpoints positioned in q34 and q11 chromosomal regions, respectively (Fig. 2.4). Rapidly
the complexity of neoplasia-associated chromosome aberrations appeared diMcult to adequate with the
current nomenclature. Two standing committees proposed in 1991 and 1995 new consensus guidelines to
suit the description of tumor karyotypes, including ' uorescent in situ hybridization (FISH) methodology.
However, the new abnormalities reported with an increasing variety of FISH probes and the new confusing
subtleties of ISCN 1995 accumulated a greater rate of syntax errors. The heterogeneity of the observations
and the variability of the banding resolution made these ISCN nomenclatures not very practical to use for
the description of cancer-associated chromosomal abnormalities, and still favors the use of personal
8simplified nomenclatures.<
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Fig. 2.4 G banded karyotype showing a t(9;22)(q34;q11) translocation corresponding to the so-called
Philadelphia (Ph) chromosome. Arrows indicate the derivative chromosomes 9 and 22 involved in the
translocation.
Karyotyping
Mitoses suitable for karyotyping are obtained more easily from lymphocyte cultures stimulated to grow by
phytohemagglutinin. Cells cultures from amniotic ' uid or biopsy of chorionic villi allow antenatal
diagnoses. Skin broblasts or fetal blood samples from the umbilical cord of stillborns are also suitable.
Direct examination of bone marrow or short-term cultures are the techniques of choice for hematological
disorders. Short-term tissue cultures took advantage of technical improvements such as methotrexate
synchronization or collagenase digestion in the analysis of lymphomas and solid tumors.
Chromosomes are counted and analyzed on slides. For decades, the better metaphase spreads were
photographed, and each chromosome was manually cut out before being classi ed on a sheet of paper.
Nowadays, on-screen karyotyping is the commonly used method for routine metaphase analysis. Once
acquired by the automated capture device, metaphases can be quickly and accurately presented for
chromosome assignment. The CytoVision system (Applied Imaging) used in our laboratory provides
classifiers for standard banding methods (Fig. 2.5).<
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Fig. 2.5 G-banding karyotype compared to the ideogram according to ISCN 1985.
Fluorescent in Situ Hybridization
The principle of in situ hybridization (ISH) is an uncoiling of the double DNA strand by heat denaturation
followed by subsequent speci c hybridization of the targeted DNA molecules with the complementary
labeled DNA probe. By this procedure, ISH detects the precise location of unique DNA sequences directly on
the chromosomes. The hybridized sequence is then revealed by two layers of ' uorescent or
chromogeniclabeled antibodies. FISH is preferred to chromogenic ISH (CISH) because of its higher sensitivity and the
greater palette of artificial colors available.
Di erent types of probes, including the centromeric, the locus speci c, and the whole chromosome
probes, are described:
• Centromeric probes are the most sensitive. They bind to highly repetitive juxtacentromeric
heterochromatin. Their strong signal remains very easily detectable on tissue sections. They are
preferentially used for detecting gains and losses of entire chromosomes, namely aneusomies.
• Locus-specific probes are designed to detect unique sequences spanning specific genomic loci. They are
used to detect specific gene amplifications, duplications, deletions, or chromosomal translocations. These
latter can be revealed by fusion of colors with the use of dual-colour probes flanking chromosomal
breakpoints involved in translocations.
• Whole chromosome probes, known as chromosome painting, reveal the whole chromosome except the
centromeric region. They are used to identify the origin of chromosomal markers such as ring chromosomes
and to refine complex chromosomal translocations.
In congenital diseases, the use of probes has advantageously circumvented the metaphase search for
frequent trisomies or microdeletion syndromes. Although banding analysis remains the standard for
identifying acquired chromosome abnormalities in cancer, FISH is now used as an easy and reliable
technical substitute to search for well-documented speci c chromosomal abnormalities in metaphase or
interphase cells. Nowadays, FISH is used on a regular basis as a complementary tool to conventional
cytogenetics, justifying the term “molecular cytogenetics.”
The FICTION Technique<
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Fluorescent immunophenotyping and interphase cytogenetics as a tool for the investigation of neoplasms
(FICTION) is a combination of ' uorescent immunophenotyping and in situ hybridization, making it
9possible to study genetic abnormalities in phenotypically selected cells. The technical principle and main
practical applications of this method will be discussed later in this chapter. SuMce to say it is currently used
to detect recurrent chromosomal abnormalities in multiple myeloma at diagnosis. This method may be
applied to any type of tumor cell displaying a speci c immunophenotype but is of little value for minimal
residual disease.
Multicolor Metaphase FISH
Multicolor-FISH includes mainly two di erent methods called spectral karyotyping (SKY) and
multiplexFISH (M-FISH). In SKY, the chromosomes are rst stained with a mixture of 24 chromosome-speci c
painting probes; each one being labeled with a di erent combination of ve ' uorochromes. The spectral
pattern of chromosomes is then classi ed using computer software to identify individual chromosomes.
MFISH uses a combinatorial labeling scheme with only ve ' uorochromes having di erent emission spectra.
Those ' uorochromes are similar to that used for SKY but the method for detecting and discriminating the
different combinations of fluorescence signals is different.
Both methods are useful in characterizing complex chromosomal rearrangements and in documenting
10ambiguous marker or ring chromosomes (Fig. 2.6).
Fig. 2.6 Multicolor FISH (SKY) allowing the identi cation of every human chromosomal pair with an
individual color using 24 di erent painting probes (22 pairs of autosomes plus the two sexual
chromosomes). Normal chromosomes are uniform in color, whereas rearranged chromosomes will display
two or more colors. This method makes it possible to detect cryptic rearrangements and marker
chromosomes in complex karyotypes as demonstrated here.
Comparative Genomic Hybridization (CGH)
This method has the advantage of circumventing the need for tumor cell metaphases. Total genomic
tumoral DNA is labeled in green and the normal reference DNA in red. Both di erentially labeled tumor
and normal DNA will be hybridized together to normal human metaphases and will compete with one
another. The ratio of the ' uorescent green and red intensities is measured along every chromosome, making
it possible to give an overview of DNA sequence copy number changes—gains and losses—in the neoplastic
cells mapped on normal chromosomes. CGH is thus able to detect ampli ed and deleted genomic regions
harboring oncogenes or tumor suppressor genes, respectively. The limitation of this method is that it can
identify DNA imbalances but not balanced chromosomal translocations (hence, without loss or gain of
chromosomal material subsequent to translocation).
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Introduction
It has long been agreed that tumor cells carry chromosomal aberrations, but their causes have only recently
11,12been more deeply explored. Until the seventies, cytogeneticists were dealing with malignant e usions
or long-term cell cultures yielding roughly recognizable multiple chromosome changes, with very large
amounts of rearranged DNA in complex aneuploidies. Consequently, this situation led to disillusion in the
literature seeded by a plethora of reports with confusing malignant karyotypes, suggesting to most scientists
that the chromosomal rearrangements observed were just epiphenomena accompanying the process of
malignancy. At that time, no method was able to show molecular changes at the gene level. Karyotype
analysis based on banding techniques renewed interest in the characterization of cytogenetic abnormalities
in malignant tumors. It appeared evident that nonrandom primary changes involved speci c chromosome
regions, and were subsequently overwhelmed by secondary more massive variations a ecting randomly all
chromosomes. This state of overall genomic instability developed during the malignant clonal progression.
Cytogenetic investigations focused initially on leukemias. They identi ed a constantly increasing number
of characteristic chromosomal patterns after the Ph/CML association was detected. The FAB classi cation of
leukemias was consequently enriched by the addition of prototypic karyotypic pro les. Beside leukemias,
other cytogenetic and molecular information emerged with studies of lymphomas and sarcomas. In those
tumors, relatively simple balanced rearrangements often appeared as ngerprints for a unique tumor type.
These speci c chromosomal abnormalities were rapidly considered as reliable diagnostic, prognostic, and
predictive parameters on daily routine. The importance of chromosomal identi cation in the diagnosis of
human leukemias, lymphomas, and mesenchymal tumors is now recognized as a component of the current
subclassi cations in the World Health Organization (WHO) fascicules dealing with classi cation of tumors
12,13of hematopoietic and lymphoid tissues and soft-tissue tumors.
Nowadays, the well-accepted opinion is that cancer is a genetic disease with two main genetic events
triggering cancer initiation: the activation or deregulation of oncogenes as a consequence of point mutation,
ampli cation, or chromosomal translocation; and the inactivation of tumor suppressor genes due to
chromosomal deletion, mutation, or epigenetic mechanisms. In malignant epithelial tumors, the prevailing
view is that they do not exhibit tumor-speci c genetic alteration but rather complex karyotypes with
multiple abnormalities shared by carcinoma of di erent histological subtypes and origins. However, single
and speci c chromosomal translocations are encountered in some epithelial malignancies such as thyroid
carcinoma, kidney carcinoma of childhood and young adult, aggressive midline carcinoma, and a
14surprisingly great number of prostate cancer.
Recurrent and speci c chromosome abnormalities can be easily investigated by FISH at diagnosis. The
method originally used on metaphase plates is also applicable on nondividing cells (interphase cells)
provided by smears, cytospins, or paraMn-embedded tissues. It has proved to be suitable for the detection
of numerical deviations on previously stained slides or fresh smears and to be feasible for improving the
15sensitivity of conventional cytology yielding “atypical cells” in cell suspensions. As it will be illustrated
below, FISH may be more sensitive than conventional cytology. FISH combined with cytology can improve
16the diagnostic sensitivity of detecting malignancy in bronchial brushing and washing specimens. FISH
has many more applications in all elds of diagnostic cancer cytology, with signi cant improvement in
tumor classi cation and a critical value in selection of patients who will bene t from targeted therapies (see
Chap. 36).
In the following sections, we will review the chromosomal abnormalities observed in lymphoma and
sarcoma with their relationship to tumor development. We will mainly focus on speci c aberrations that
can be used as diagnostic tools in complement to cytology. In the same way, the examples of chromosomal
markers in carcinoma will be limited to thyroid carcinoma. In a second part, we will discuss the
applications of FISH in the eld of cytology, again limiting our comments to lymphoma and sarcoma. The
contribution of FISH in multiple myeloma will also be mentioned because of the novel and promising
FICTION technique used to detect chromosomal abnormalities in selected nondividing plasma cells.
Lymphomas@
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Recurrent chromosomal abnormalities in lymphoma are mainly represented by balanced chromosomal
translocations that exert their tumorigenic action by two alternative molecular mechanisms (Fig. 2.7). In
the rst mechanism, the breakpoints on both chromosomes will occur adjacent to two genes and bring them
close together but will not alter the protein produced by one of the targeted genes, mainly an oncogene.
This latter is translocated close to strong promoter/enhancer elements of the other gene involved, hence the
immunoglobulin (Ig) or T-cell receptor (TCR) genes. The functional consequences are constitutive activation
of the oncogene through its overexpression driven by Ig or TCR enhancers. In the second mechanism, the
chromosomal breakpoints occur within the coding sequence of each gene, such that the two broken genes
are fused, leading to a chimeric gene translated into a new chimeric protein with dysregulated function.
The rst mechanism accounts for the majority of lymphoma diseases while the second molecular event
predominates in sarcoma.
Fig. 2.7 Main molecular mechanisms subsequent to chromosomal translocations encountered in cancer.
(A) In the rst mechanism, breakpoints on both chromosomes will spare the coding sequence of the targeted
genes. The translocation will lead to the juxtaposition of strong promoter/enhancers elements (blue lozenge)
from one gene (A) with the entire intact coding sequence of another gene (B), leading to overexpression of
this latter. In the classical example, promoter/enhancers are brought by Ig or TCR coding genes and the
targeted coding sequence is oncogenes such as BCL2 or BCL1. (B) The second mechanism is characterized by
chromosomal breakpoints occurring within the coding sequence of both genes involved in the translocation,
leading to a chimeric gene translated into a hybrid protein with altered function. White vertical bars denote
chromosomal breakpoints.
There is an abundant literature demonstrating good correlations between chromosomal abnormalities
12and di erent lymphoma subtypes. Identi cation of speci c genetic aberrations has several meaningful
implications in non-Hodgkin lymphoma's (NHL). First, it may help in accurately diagnosing NHL. For
example, identi cation of the t(11;14) translocation makes it possible to distinguish mantle cell lymphoma
(MCL) from small lymphocytic lymphoma/chronic lymphoid leukemia (SLL/CLL). The presence of the
t(2;5) translocation is the characteristic genetic feature of a subgroup of anaplastic large cell lymphoma
(ALCL). Second, demonstration of chromosomal translocations may help in prognostic assessment of NHL;
marginal zone lymphoma of MALT type with a t(11;18) is unlikely to respond to antibiotic therapy. By
contrast, the MALT-NHL negative for the t(11;18) is most often associated with Helicobacter pylori gastritis
and more often responds to antibiotic therapy. The presence of the t(2;5) translocation and its consecutive
anaplastic lymphoma kinase (ALK) overexpression in ALCL is associated with good prognosis. Third,
identi cation of genetic abnormalities in NHL may serve as markers for staging assessment and for studies
of minimal residual disease.
As Hodgkin's lymphoma does not exhibit any consistent or speci c genetic abnormality detectable by
cytogenetics or FISH analysis, the following topic will focus on non-Hodgkin's lymphoma. Within this last<
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group, we will restrict our talk to NHL subtypes exhibiting characteristic chromosomal aberrations that can
be used as diagnostic tools on a regular basis, and will organize this section according to the REAL/WHO
morphological classification, hence from small cell lymphomas to large cell lymphomas.
Follicular Lymphoma
Follicular lymphoma (FL) is characterized by the t(14;18)(q32;q21) translocation (Fig. 2.8), which
juxtaposes the B-cell lymphoma/leukemia 2 (BCL2) oncogene at 18q21 into the heavy chain
17immunoglobulin (IgH) gene locus at 14q32, leading to upregulated expression of the BCL2 protein. BCL2
is an antiapoptotic gene, and its overexpression leads to prolonged cell survival that may make the cell
more vulnerable to additional genetics events, leading to cell overgrowth and cancer. In a minority of cases,
variant translocations such as t(2;18)(p11,q21) and t(18;22)(q21;q11), which relocate the BCL2 oncogene
to the kappa light chain immunoglobulin (IgL kappa) gene locus and lambda light chain immunoglobulin
(IgL lambda) gene locus, respectively, have also been observed.
Fig. 2.8 Karyotype of follicular lymphoma showing the balanced t(14;18)(q32;q21) translocation
(arrows). The gains for chromosomes 3, 12, 15, and X as well as deletion 13q are additional abnormalities
associated with clonal evolution.
This translocation t(14;18) and its variants are observed in up to 85% of FL which are mainly represented
by histological grades 1, 2, and 3A. The remaining 15% cases do not exhibit a t(14;18)(q32;q21)
18,19translocation and are essentially constituted by FL grade 3B. Among them, a minority (~30%) exhibit
BCL2 overexpression on immunohistochemistry, resulting from a non-Ig-related mechanism. The origin of
this BCL2 gene overexpression is still unknown but could be due to duplication of chromosome 18 as
19observed in some karyotypes, or could involve other unknown mechanisms favoring BCL2 overexpression.
The clinical outcome of this subgroup seems to be similar to that of follicular lymphoma with t(14;18). The
major subgroup (~70%) does not show any BCL2 overexpression but presents a recurrent translocation of
the 3q27 chromosomal region, resulting in a disruption of the B-cell lymphoma/leukemia 6 (BCL6)
oncogene located at this breakpoint. This abnormality is also observed in di use large B-cell lymphomas
(DLBCL), a feature that will be discussed later. Of interest, these 3q27+ FL grade 3B show peculiar
20clinicopathologic features distinct from their t(14;18)+ counterparts : a stage III/IV disease as well as a
−bulky mass are less frequently observed, and they usually disclose a CD10 phenotype. Finally, this genetic
subgroup seems to have a better survival rate and have clinically more in common with de novo 3q27+
19,20DLBCL. These ndings indicate that the search for BCL2 and BCL6 rearrangement status by genetic
analysis may be clinically warranted for all cases of follicular lymphoma.
Although the t(14;18) translocation is an early event and is critical for lymphomagenesis, it is by itself
insuMcient to produce FL. As said before, the prolonged cell survival provided by BCL2 overexpression
21allows the acquisition of further genetic events that contribute to the development of FL. These genetic
events occur as a series of chromosomal gains and losses that can be detected at diagnosis as complex and
heterogeneous karyotypes. It is not the karyotypic complexity but rather the type of abnormalities exhibited
that underlies the varied clinical outcome observed in FL. Recurrent cytogenetic aberrations that have been<
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noted to correlate with a more aggressive disease include chromosomal gains such as +7, +12 or gain of
12q13-14, +18 and chromosomal losses including del 6q, del(9)(p21), and del(17)(p13), the two last
aberrations corresponding to loss of tumor suppressor genes p16 and p53. Beside a complex karyotype,
3q27/BCL6 translocations can subsequently occur in t(14;18)+ FL, less frequently in low than in high
22grades, and have been shown to correlate with a risk of transformation to diffuse large B-cell lymphoma.
Mantle Cell Lymphoma
According to the REAL/WHO classi cation, the diagnosis of mantle cell lymphoma should be based on
12clinicomorphological but also cytogenetic or molecular features. The genetic hallmark of MCL is the
t(11;14)(q13;q11) translocation (Fig. 2.9) that juxtaposes part of the IgH locus on chromosome 14q32 to
the entire coding sequence of BCL1 oncogene, also named Cyclin D1 or PRAD1, located on chromosome
2311q13. BCL1 gene is thus brought under the control of an IgH enhancer, leading to overproduction of
cyclin D1 protein, a mechanism similar to that observed for the BCL2 oncogene in FL. Cyclin D1 is one of
the key regulators of the cell cycle, and complexes with CDK-4 and -6 in order to promote the G1/S-phase
transition of the cell cycle. Increased Cyclin D1 production in MCL will dramatically induce cells to enter
the S-phase and, therefore, tumor cell proliferation, by inhibiting the cell cycle inhibitory e ects of the
24retinoblastoma (Rb) and CDK inhibitors p27kip1 proteins. Concurrent disruptions of other cell
cycleassociated genes contribute also to the pathogenesis of MCL. In particular, homozygous deletions of the CDK
INK4 INK4inhibitor p16 were observed in aggressive variants of MCL. p16 is an inhibitor of CDK-4 and -6
INK4and thus maintains the Rb protein activity by preventing its phosphorylation. p16 deletion and an
increased level of Cyclin D1 may therefore work together in promoting the G1/S-phase transition in MCL
cells.
Fig. 2.9 Karyotype of mantle cell lymphoma displaying the t(11;14)(q13;q11) chromosomal
translocation (arrows), associated with multiple additional abnormalities such as interstitial deletion
involving one chromosome 13, loss of the normal chromosome 14, and marker chromosome (A). This pro le
is observed in aggressive cases.
The t(11;14) translocation is very speci c to MCL among other B-NHL and is detected by conventional
cytogenetics in 60–75% of MCL cases, but this number rises to nearly 100% with the use of FISH.
Beside the presence of t(11;14) translocation, the study of the overall cytogenetic pro le brings
prognostic meanings. Normal karyotype or karyotype with a single t(11;14) is associated with the typical
form of MCL and is a good prognostic factor. In the majority of aggressive cases, t(11;14) is associated with
a complex karyotype including numerous structural and numerical alterations of chromosomes 1, 2, 3, 9,
2511, 13, 17 as well as unidenti ed chromosomal aberrations (markers). Also, near-tetraploid karyotypes
(hence ±92 chromosomes) seem to be characteristic for the blastoid variant MCL. These karyotypic
features occurring in aggressive MCL cases re' ect the existence of alterations in both the DNA damage
response pathways and mitotic checkpoints that may constitute another important pathogenetic mechanism<
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in this lymphoma subtype. Indeed, one of the most frequently additional cytogenetic aberrations observed
in MCL is deletion in the 11q22-23 chromosomal region where the ATM (ataxia-telangiectasia mutated)
gene is located. ATM gene plays a key role in genomic stability by activating gatekeeper and caretaker
26genes such as p53 and BCRA1 in response to DNA damage. ATM inactivation in MCL is associated with a
high number of chromosomal alterations, suggesting that it may, at least in part, be responsible for the
26chromosomal instability in these lymphomas.
Marginal Zone B-cell Lymphoma
Several chromosomal abnormalities are encountered in marginal zone B-cell lymphomas (MZL) and are
distributed according to the three different subtypes: extranodal MZL of MALT type, nodal MZL, and splenic
MZL
In MALT lymphoma, four main recurrent chromosomal translocations have been observed and
demonstrate a site-speci city in terms of their incidence: t(11;18)(q21;q21), t(1;14)(p22;q32), t(14;18)
(q32;q21), and the recently described t(3;14)(p14.1;q32) (Table 2.1). The latter, limited to MALT
lymphoma of the thyroid, skin and ocular adnexa regions, leads to the juxtaposition of the transcription
27factor Forkhead box-P1 (FOXP1) next to the enhancer region of the IgH gene. This molecular event
results in FOXP1 gene overexpression but the pathogenetic relevance of this translocation is still not known.
Table 2.1 Four main recurrent chromosomal translocations observed in MALT lymphomas
The three other translocations a ect a common signaling pathway, resulting in the constitutive activation
of the nuclear factor- B (NF- B), a transcription factor which plays a major role in cellular activation,
28proliferation and survival. The t(1;14)(p22;q32) is detected in approximately 5% of MALT lymphoma,
arising in localizations such as stomach, intestine, and lung. This translocation results in overexpression of
the BCL10 gene (chromosome 1p22) due to its juxtaposition with the IgH gene enhancer. The t(14;18)
(q32;q21) translocation, cytogenetically identical to the t(14;18)(q32;q21) involving BCL2 gene in follicular
lymphoma, is observed in more or less 20% of MALT lymphoma, especially in non-gastrointestinal
localizations such as liver, lung, salivary glands, skin, and ocular adnexa. This translocation brings the
mucosae-associated lymphoid tissue (MALT1) gene, also involved in antigen-receptor-mediated NF- B
activation, under the control of the IgH enhancer region, with subsequent MALT1 overexpression. The
t(11;18)(q21;q21) represents the most common translocation, accounting for 15–40% of cases, and is
observed in stomach, intestine, and lung MALT lymphoma cases. It results in the reciprocal fusion of the
API2 and MALT1 genes. API2 (cellular inhibitor of apoptosis protein 2) gene is believed to be an apoptosis
inhibitor by inhibiting the biological activity of caspases 3, 7, and 9.
The pathogenesis of those three translocations sharing the same molecular pathway is beginning to be
28,29understood. NF- B activation is driven by stimulation of cell-surface receptors, such as B- or T-cell
receptors. In unstimulated lymphocytes, NF- B proteins are bound with inhibitory B (I B) proteins and
sequestered in the cytoplasm. Phosphorylation of the I B proteins by the I B kinase (IKK) heterodimer leads
to ubiquitylation and degradation of I B, allowing NF- B to migrate to the nucleus and transactivate genes
involved in cellular activation, proliferation and survival, and induction of e ector function of
lymphocytes.
In MALT lymphoma with t(1;14) translocation and BCL10 overexpression, BCL10 is able to complex with
MALT1 and trigger aberrant NF B activation without the need for upstream signaling. With the t(14;18)
translocation causing MALT1 overexpression, MALT1 interacts and stabilizes BCL10, leading to its
cytoplasmic accumulation. Both proteins in high cellular concentration will then synergistically favor a<
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constitutive NF- B activity. In t(11;18) positive MALT lymphoma, the API2-MALT1 chimeric protein
activates NF- B through self-oligomerization, and bears a gain of function when compared to wild type
MALT1. This higher activation is also due to the API2 protein partner. Indeed, wild-type API2
downregulates BCL10 expression by ubiquitylation and degradation, a mechanism used to regulate BCL10
activity after antigen receptor stimulation. The API2–MALT1 protein is no longer able to ubiquitylate it and
high BCL10 expression will synergistically increase API2–MALT1's intrinsic capacity for NF- B activation,
independently of any antigen-receptor activation.
Because of their speci city, the identi cation of these chromosomal translocations can be of interest for
diagnostic purposes. They have also an immediate impact on treatment decisions, at least for two of them.
Indeed, a causal relationship between H. pylori infection in the stomach and development of gastric MALT
lymphoma has been clearly demonstrated, and 75% of these lymphomas can be successfully treated with
28appropriate antibiotics targeting H. pylori. However, the presence of either the t(11;18) or t(1;14)
translocation de nes patients who will not respond to H. pylori eradication. At the opposite, gastric MALT
lymphoma without these chromosomal translocations, sometimes carrying trisomies of chromosomes 3, 12,
and 18, can be e ectively treated by antibiotic treatment, at least at their early stages. However, they can
progress, become H. pylori-independent and transform into high-grade tumors following the acquisition of
additional genomic alterations (such as TP53 and CKN2A inactivation). Intriguingly, t(11;18) positive
MALT lymphomas will rarely develop into high-grade tumors, unlike their t(1,14) counterparts. These
clinical features indicate that chromosomal abnormalities in some MALT lymphoma can also serve as
prognostic parameters.
In splenic marginal zone lymphoma (SMZL), cytogenetic alterations include mainly partial or complete
trisomy 3, and interstitial deletion of chromosome 7q involving segments of variable size, usually centered
around the 7q31q32 region (Fig. 2.10). Recent gene expression pro ling revealed that genes mapping to
the 7q31 chromosomal region were consistently downregulated, among which three were found to be very
30SMZL-specific: ILF1, Senataxin, and CD40.
Fig. 2.10 Karyotype showing an interstitial deletion of chromosome 7q as observed in splenic marginal
zone lymphoma. In the present case, the deletion involves the 7q22q32 chromosomal segment.
Nodal marginal zone lymphoma (NMZL) is a very rare disease. However, local regional lymph node of
MALT lymphoma is virtually indistinguishable from NMZL, requiring clinical information and, in some
respect, cytogenetic data to diagnose it. NMZL is characterized by frequent trisomies of chromosomes 3, 7,
12and 18, but the characteristic translocations of MALT lymphoma are never seen.
Small Lymphocytic Lymphoma
The histology, immunophenotypic and cytogenetic features of small lymphocytic lymphoma are
12indistinguishable from the more common CLL. Chromosomal aberrations observed in SLL include thus@
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trisomy 12, 11q, and 17p deletions—all of them being poor-risk cytogenetic parameters—and a 13q14
deletion which is considered as a marker of good prognosis. A t(14;19)(q32;q13) translocation occurs
infrequently in SLL and juxtaposes the BCL3 gene located on chromosome 19 next to the enhancer region of
the Ig-heavy-chain gene, leading to BCL3 overexpression. When present, it confers a more aggressive
31behavior.
Lymphoplasmacytic Lymphoma
Lymphoplasmacytic lymphoma (LPL) is a rather uncommon entity but its diagnosis remains challenging for
most pathologists. Cytogenetic investigations had previously considered the t(9;14)(p13;q32)—juxtaposing
the PAX5 transcription factor with the Ig-heavy-chain gene enhancer—as characteristic of LPL, but more
recent studies question the accuracy of this association. Firstly, no PAX5 rearrangement was detected in a
32series of 13 LPL. Secondly, PAX5/IgH rearrangement was observed in other types of lymphoma including
T-cell-rich B-cell lymphoma, post-transplantation di use large B-cell lymphoma, and some cases of
33SMZL.
Diffuse Large B-cell Lymphoma
Di use large B-cell lymphoma is a very heterogeneous clinicopathologic entity, displaying numerous and
disparate chromosomal aberrations. In this section, we will only focus on the most frequent cytogenetic
aberrations observed in DLBCL, hence chromosomal translocations involving BCL6, BCL2 and C-MYC
oncogenes.
The translocations involving the 3q27 chromosomal region are the most characteristic and frequent
12cytogenetic aberrations, detected in 30 to 40% of DLBCL (Figs 2.11A and 2.11B). The 3q27 breakpoint
involves the BCL6 gene, which is required for germinal center (GC) formation and the B-cell immune
response. The gene partners of the BCL6 chromosomal translocations are multiple. They most often involve
the Ig-heavy- or -light-chain ( and λ) genes on chromosome bands 14q32, 2p11 and 22q11, but more than
3420 non-Ig partners have also been described, a phenomenon termed “promiscuous translocation”.
Whatever the partner is, the chromosomal translocation brings the entire coding sequence of BCL6 under
the control of a replaced promoter that will cause its deregulated expression during B-cell differentiation.
Fig. 2.11 (A) Karyotype of a di use large B-cell lymphoma exhibiting a characteristic t(3;14)(q27;q32)
chromosomal translocation (arrows). Other abnormalities such as additional material of unknown origin
attached to the 1p36 chromosomal region of one chromosome 1, to the 6p24p25 chromosomal regions of
both chromosomes 6, and deletion of the 7p21 segment of one chromosome 7 are additional aberrations
re' ecting clonal evolution. (B) Metaphase FISH with the use of a dual-color (green and red) break-apart
probe speci c to the BCL6 gene. The yellow signal (juxtaposition of green and red colors) identi es the
normal BCL6 gene, whereas splitting of the green and red signals indicates a disruption of the other BCL6
gene subsequent to the t(3;14) chromosomal translocation.
BCL6 plays a key role in the generation of a germinal center by B cells. It encodes a transcriptional<
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repressor protein that downregulates the expression of the B-lymphocyte-induced maturation protein 1
(BLIMP1) gene necessary for plasma cell di erentiation, and also the expression of p27kip1, cyclin D2, and
35P53 which control the cell cycle, apoptosis, DNA repair, and maintenance of genomic stability. In a
normal situation, BCL6 expression is tightly regulated during B-cell ontogenesis, being restricted to B cells in
the GC. In contrast, the heterologous Ig and non-Ig promoters exhibit a broader spectrum of activity in
Bcell ontogenetic stages and will prevent BCL6 downregulation in post-GC cells. A block in the normal
downregulation of BCL6 might thus favor di erentiation arrest, continuous cell proliferation, survival, and
genetic instability, all of which allowing neoplastic transformation. Indeed, the 3q27/BCL6 rearrangement
is suMcient in itself to produce lymphoma as demonstrated by transgenic mice studies. In addition and
independently of BCL6 translocations, point mutations and small deletions of BCL6 have been reported in
35approximately 70% of DLBCL, leading also to its deregulated expression.
The clinical relevance of BCL6 gene translocations has been initially a subject of controversy with studies
reporting improved survival in patients with BCL6 translocation, and other failing to show any statistically
36signi cant impact of such rearrangements on the clinical outcome of DLBCL. More recently, a cDNA
microarray analysis demonstrated that DLBCL patients with the germinal center B-cell-like (GCB) gene
expression pro le had a better overall survival than those with the activated B-cell-like (ABC) expression
37pattern. As BCL6 is a marker of the GCB-type signature, its mRNA and protein levels were correlated to
clinical outcome of DLBCL patients: high-level expression of BCL6 was associated with signi cantly longer
36overall survival and shown to be a predictor of a favorable treatment outcome in cases of DLBCL.
In some cases, 3q27/BCL6 translocation coexists with other translocations in a single clone, including
t(14;18)(q32;21) and t(8;14)(q24;q32), involving BCL2 and c-MYC oncogenes, respectively. This
coexistence of two to three chromosomal translocations seems not necessarily to have a signi cant impact
38on the clinical features. Finally, it must be added that around 20% of DLBCL exhibit a t(14,18)(q32;q21)
similar to that associated with follicular lymphoma and mutually exclusive of BCL6 rearrangements.
Burkitt's Lymphoma
Burkitt's lymphoma (BL) and its leukemic equivalent, the L3 variant of acute lymphoblastic leukemia, are
characterized in nearly 90% of cases by a reciprocal chromosomal translocation that juxtaposes the c-Myc
oncogene (chromosome 8q24) to one of the immunoglobulin genes located on chromosome 14q32 (IgH),
chromosome 22q11 (Igλ), or chromosome 2p12 (Ig κ) (Fig. 2.12). All three chromosomal translocations lead
to overexpression of the c-Myc gene product. C-Myc gene is a transcription factor that regulates a very large
39number of genes through heterodimerization with the partner protein Max. The genes targeted by the
cMyc/Max heterodimer complexes are involved in cell proliferation, di erentiation, and apoptosis. Such
global transcriptional regulatory function may explain why c-Myc overexpression is suMcient in itself to
promote lymphoma diseases as demonstrated in transgenic mice studies.
Fig. 2.12 Karyotype showing a t(8;14)(q24;q32) chromosomal translocation (arrows) characteristic of<
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Burkitt's lymphoma (or ALL L3). Segmental duplication of chromosome 1q and loss of chromosome 17p are
recurrent additional chromosome aberrations in this type of lymphoma.
The so-called “Burkitt-like” form is characterized by three cytogenetic categories: one with an 8q24/
cMYC translocation, a second with associated 8q24/c-MYC and 18q21/BCL2 translocations, and a third with
miscellaneous rearrangements, frequently including an 18q21/BCL2 chromosomal translocation.
Recurrent chromosome aberrations associated with the 8q24 translocations include duplications of the
1q21q25 chromosomal region, 6q11q14 and 17p chromosomal deletions, and trisomies for chromosomes 7,
8, 12, and 18. A recent cytogenetic and CGH study on BL has demonstrated that the presence of
abnormalities on chromosome 1q (demonstrated either by cytogenetics or by CGH) and gains of 7q
40(ascertained only by CGH) were associated with adverse prognosis.
Anaplastic Large Cell Lymphoma
Anaplastic large cell lymphoma is a CD30+ T-cell NHL that can be divided in two majors groups according
to the WHO classi cation: (1) systemic nodal ALCL and (2) primary cutaneous ALCL. As this latter group
does not exhibit speci c chromosomal alteration, it will not be pursued further in this review. In this
section, we will only focus on systemic nodal ALCL, more particularly on anaplastic lymphoma
kinasepositive ALCL where a characteristic t(2;5)(p23;q35) translocation is observed in approximately 60% of
cases. This translocation fuses the nucleophosmin (NPM) gene on chromosome 5q35 to the ALK gene on
41chromosome 2p23, leading to the NPM-ALK chimeric gene. It is present in approximately 75% of ALCL
with ALK gene rearrangement. In the remaining approximately 25% of cases, 2p23/ALK locus translocates
41with various partner genes. The common molecular features of all ALK rearrangements is the fusion of
the ALK tyrosine kinase domain to the 5′ region of partners which provide a strong promoter and most
likely an oligomerization motif allowing constitutive activation and aberrant expression of the ALK kinase.
ALK gene encodes for a receptor tyrosine kinase normally expressed in fetal and mature nervous systems
but not in lymphoid cells. As any receptor tyrosine kinase and in normal situation, ALK protein will activate
signaling pathway and cell cycle after oligomerization induced by binding with its ligand. In ALK
rearrangements, the partner gene brings to ALK the ability to self-associate in a ligand-independent fashion,
leading to its constitutive activation. In addition, the gene partner brings a strong promoter, driving
illegitimate and high levels of ALK receptor tyrosine kinase fusion gene expression in lymphoid cells. The
functional consequence is to exaggerate and dysregulate otherwise normal downstream signals which will
41promote cell growth and inhibit apoptosis. Clearly, ALK activation is a critical step in the development of
ALCL of T cell origin. As ALK gene is not expressed in normal lymphoid cells, the immunodetection of ALK
protein in a lymphoid tumor represents a highly sensitive test for identi cation of lymphoma with ALK
rearrangement, correlating in nearly 100% of cases with the presence of such abnormality.
Regardless of other clinical and biological prognostic parameters, the outcome for patients with
ALKpositive ALCL is signi cantly better than that for patients with ALK-negative ALCL with the 5-year survival
42rates ranging between 79 and 88% and 28 and 40%, respectively. Additional information on lymphomas
is found in Chapter 24 Lymph Nodes and Flow Cytometry.
Sarcomas
Although sarcomas are relatively rare neoplasms in adulthood, they represent the most frequent malignant
tumors in childhood and young adults. Abundant genetic studies have revealed that a signi cant number of
sarcoma are associated with speci c chromosomal abnormalities (mainly chromosomal translocations) that
13,14,43,44can be used as practical diagnostic markers in histological equivocal cases. A typical example is
the so-called “small round blue cell” undi erentiated pattern shared by disparate tumor entities such as
embryonal or alveolar rhabdomyosarcoma, Ewing's sarcoma, neuroblastoma, and lymphoma.
Two major genetic groups distinguishable at the cytogenetic level are observed in sarcomas. One group is
characterized by a near-diploid karyotype with a single or few chromosomal abnormalities, whereas the
second exhibits complex karyotype with numerous aberrations that re' ect severe disturbance in genomic<
stability. Sarcoma with genetic abnormalities not detectable by conventional cytogenetics and/or FISH
means—such as GIST and its specific c-KIT mutation—will not be discussed in this section.
Sarcomas with Single Karyotypic Abnormalities
This group is characterized by karyotype harboring single and tumor-speci c chromosomal translocations
(Table 2.2). Most of these translocations lead to fusion genes encoding aberrant transcription factors but a
small subset creates aberrant chimeric genes related to growth-factor signaling pathway.
Table 2.2 Translocations associated with sarcomas
Translocation Genes Type of fusion gene
EWING'S SARZCOMA
t(11;22)(q24;q12) EWSR1-FLI1 Transcription factor
t(21;22)(q22;q12) EWSR1-ERG Transcription factor
t(7;22)(p22;q12) EWSR1-ETV1 Transcription factor
t(17;22)(q21;q12) EWSR1-ETV4 Transcription factor
t(2;22)(q33;q12) EWSR1-FEV Transcription factor
CLEAR-CELL SARCOMA
t(12;22)(q13;q12) EWSR-ATF1 Transcription factor
DESMOPLASTIC SMALL ROUND-CELL TUMOR
t(11;22)(p13;q12) EWSR-WT1 Transcription factor
MYXOID CHONDROSARCOMA
t(9;22)(q22-31;q11-12) EWSR-NR4A3 Transcription factor
MYXOID LIPOSARCOMA
t(2;16)(q13;p11) FUS-DDIT3 Transcription factor
t(12;22)(q13;q12) EWSR1-DDIT3 Transcription factor
ALVEOLAR RHABDOMYOSARCOMA
t(2;13)(q35;q14) PAX3-FKHR Transcription factor
t(1;13)(p36;q14) PAX7-FKHR Transcription factor
SYNOVIAL SARCOMA
t(X;18)(p11;q11) SYT-SSX Transcription factor
DERMATOFIBROSARCOMA PROTUBERANS
t(17;22)(q22;q13) COL1A1-PDGFB Growth factor
CONGENITAL FIBROSARCOMA
t(12;15)(p13;q25) ETV6-NTRK3 Transcription factor-receptor
INFLAMMATORY MYOFIBROBLASTIC TUMOR
2p23 rearrangements TMP3-ALK; TMP4-ALK Growth factor-receptor
ALVEOLAR SOFT-PART SARCOMA
t(X;17)(p11.2;q25) ASPL-TFE3 Transcription factor@
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The Ewing's family of tumors, which includes Ewing's sarcoma and primitive neuroectodermal tumor
(ES/PNET), are characterized by a t(11;22)(q24;q12) translocation leading to the EWSR1-FLI1 fusion gene
and observed in nearly 90% of cases of ES/PNET (Fig. 2.13). The remaining cases show alternative
chromosomal translocations fusing the EWSR1 gene (chromosome 22q12) with partner genes other than
FLI1 and that belong to the same ETS family of transcription factors. EWSR1 gene is also involved in
chromosomal translocations arising in several other tumoral entities such as the intra-abdominal
desmoplastic small round-cell tumor (DSRCT), myxoid chondrosarcoma, and clear cell sarcoma. However,
EWSR1 gene fused in each case with gene partners not encountered in the Ewing's family of tumors, giving
13,43,44rise to specific fusion genes suitable for diagnostic purposes.
Fig. 2.13 Karyotype of an Ewing's tumor with the characteristic t(11;22)(q24;q12) chromosomal
translocation (arrows) leading to the EWS-FLI1 fusion gene. Secondary recurrent chromosomal abnormalities
such as monosomies 6 and 15 and trisomies 2 and 14 are also observed.
Some new data indicate that soft-tissue tumors can no longer be classi ed only on basis of their site of
43,45origin but also according to their genetic aberrations. Congenital brosarcoma and mesoblastic
nephroma were thought to be unrelated tumors until cytogenetic analysis revealed a common aberration,
hence the t(12;15)(p13;q25) translocation with subjacent ETV6-NTRK3 fusion gene, indicating that they
are simply the same tumoral entity that develops in di erent locations. Another similar example is
illustrated by the t(X;17)(p11.2;q25) translocation shared by the alveolar soft-part sarcoma (ASPS) and a
46cytogenetic subset of childhood papillary renal cell carcinoma (PRCC). Although this translocation is
cytogenetically unbalanced in ASPS and balanced in PRCC, it gives rise at the molecular level to the same
ASPL-TFE3 fusion transcript in both tumoral types. Therefore, some fusion genes can exert their oncogenic
properties in more than one target cell type and seems not to play any role in cell di erentiation. On the
other hand, in vitro experiments showed that fusion proteins such as EWS-FLI1 contribute to the phenotypic
features of ES/PNET by subverting the di erentiation program of its neural crest precursor cell to a less
di erentiated and more proliferative state. In synovial sarcoma, the SYT gene on chromosome 18q11 can
fuse with various members of the SSX cluster located on chromosome Xp11 (Fig. 2.14). The SSX2
translocation partner is more likely observed in monophasic synovial sarcoma, whereas SSX1 is much often
associated with the biphasic forms, indicating that this latter gene partner may drive epithelial
di erentiation in synovial sarcoma. Finally, some data support the hypothesis that the gene fusion occurs in
an already established lineage that imposes constraints such that the target cell selects the fusion gene. In
contrast, other observations suggest that this fusion will modulate the phenotypic features of the
45undifferentiated precursor harboring this fusion gene.<
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Fig. 2.14 Karyotype of a synovial sarcoma with the speci c t(X;18)(p11;q11) chromosomal
translocation (arrows). Losses and gains of other chromosomes represent additional secondary changes.
Sarcoma-associated chromosomal translocations and/or their respective fusion genes may have some
14,43prognostic impacts. In Ewing's sarcoma, several molecular variants are observed in the EWS-FLI1
fusion gene due to various breakpoint junctions. The most common, designated type 1 (linking exon 7 of
EWS with exon 6 of FLI1) is associated with a better prognosis than other variants. The SYT-SSX fusion type
in synovial sarcoma appears to be a signi cant prognostic factor since patients with the SYT-SSX2 variant
have an improved overall survival when compared with SYT-SSX1 positive patients, independent of the
histological type. Patients with metastatic alveolar rhabdomyosarcoma having the PAX7-FKHR fusion gene
show a substantially better prognosis than those with the PAX3-FKHR translocation. These variations in
behavior could be due to subtle di erences in the biochemical activities of the variant fusion proteins, with
a better prognosis associated with variants having a less transcriptional activity.
The close association between speci c translocations and distinct sarcoma types indicates that they are
early events in tumorigenesis but their exact role in tumor development remains often diMcult to assess. In
the small subset of translocations with aberrant chimeric genes related to growth-factor signaling pathways
(see Table 2.2), the pathogenesis arises through cell cycle activation although this is probably not suMcient
per se to induce full transformation. The great majority of chromosomal translocations in sarcoma involve
transcription factors without obvious putative oncogenic properties at rst sight. Transcription factors are
proteins that directly interact with the DNA strand of their target genes, and regulate the expression of these
genes by binding their promotor regions upstream of RNA transcription sites. A translocation will lead to
aberrant gene fusion composed of the DNA (or RNA)-binding domain of a transcription factor fused with
the transactivation domain of another transcription factor. The functional consequence is that the
transcriptional activity of the latter will be deviated toward downstream genes targeted by the DNA-binding
domain provided by the transcription factor partner. Moreover, most of these chimeric proteins show
enhanced transcriptional activity compared with their constitutive normal protein, providing eventually a
gain of function mechanism. It is thus believed that these phenomena lead to dysregulation of gene
expression, accounting for the tumoral properties of fusion genes in sarcoma.
This general opinion can be illustrated by the t(2;13)(q35;q14) and t(1;13)(p36;q14) translocations
arising in alveolar rhabdomyosarcoma and corresponding to the PAX3/FKHR and PAX7/FKHR fusion genes,
43,47respectively. Both translocations fuse the DNA-binding domain of PAX3 or PAX7 to the transactivation
domain of FKHR. PAX genes activate myogenesis, and fork head in rhabdomyosarcoma (FKHR) is though to
have pro-apoptotic activities. The resulting PAX-FKHR fusion gene is a highly potent activator with a
transcriptional activity 10 to 100 times as high as that brought by wild-type PAX gene. This enhanced
transcriptional activity is further ampli ed by mechanisms of PAX-FKHR fusion genes ampli cation. As the
DNA-binding of FKHR is lost in the PAX-FKHR fusion, any DNA-binding speci city of the fusion gene is
directed by the PAX sequence, leading to dysregulated expression of downstream target genes of PAX genes.<
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Consequently, PAX3/FKHR will be able, rstly, to inhibit cellular apoptosis through a PAX3 target gene, the
anti-apoptotic protein BCL-XL and, secondly, to activate c-MET, PDGFαR, or c-RET oncogene, downstream
targets of PAX3 involved in migration and proliferation of myogenic precursors.
Other sarcoma-associated fusion genes have been shown to get tumoral properties by activating growth
factor receptors. MET oncogene has been recently shown to be a direct transcriptional target of the
ASPLTFE3 fusion gene. Induction of MET by ASPL-TFE3 results in strong MET autophosphorylation and
48activation of downstream signaling in the presence of hepatocyte growth factor.
Another question that remains a matter of debate is whether these chromosomal translocations are
14,43suMcient for neoplastic transformation. Although expression of certain gene fusions can induce
sarcoma in primary mesenchymal progenitor cells, secondary mutations are likely to be required for full
malignancy as observed in the context of hematological disorders. Loss of tumor suppression genes
expression (such as P16 and RB) is observed in more than 50% of various sarcoma. Activation of common
growth-factor pathways not directly due to chromosomal translocation is described in sarcomas including
the insulin-like growth factor 1 (IGF1) pathway in alveolar RMS, the platelet-derived growth factor receptor
(PDGFR) in DSRCT, and the c-KIT receptor pathway in Ewing's tumors. Parallel to the situation observed in
49childhood leukemia, it is possible that some chromosomal translocations associated with childhood
tumors arise during fetal development, leading to a “pre-malignant state” preceding the sarcomatous
transformation induced by additional genetic aberrations.
Sarcomas With Complex Karyotypes
This group of sarcoma does not exhibit any speci c and recurrent chromosomal translocation but rather
complex karyotypes with multiple numerical and structural aberrations characteristic of severe genetic and
43chromosomal instability (Table 2.3) (Fig. 2.15). The underlying genetic mechanisms frequently include
alterations in cell-cycle genes such as P53, INK4A, and RB1 as well as genes directly involved in DNA-repair
pathways. Oncogene ampli cations occur in cytogenetically complex sarcoma. MDM2 and MYCN gene
ampli cations are well-known examples. MDM2 ampli cation is observed in liposarcoma (other than
myxoid) and malignant brous histiocytomas. MDM2 is a p53 inhibitor and its ampli cation will lead to
inability of p53 to induce apoptosis in cells with DNA damage, which, in turn, will induce genomic
instability. MYCN ampli cation (Fig. 2.16) is used as a genetic parameter for better therapeutic
strati cation of patients su ering from neuroblastoma, one of the most frequent malignant tumors in
50childhood. MYCN is a member of the MYC family of proto-oncogenes which are transcription factors
promoting cell proliferation and inhibiting terminal di erentiation. In view of its function, MYCN is
involved in the genesis of a wide range of cancers including neuroblastoma, small cell lung carcinoma,
some cases of medullary thyroid carcinoma, retinoblastoma, and breast cancers. A forced expression of
MYCN in central nervous system cells in mouse leads to the development of a subgroup of neuroblastomas,
indicating that it is suMcient for malignant transformation. Additional information on sarcomas is found in
Chapter 18.
Table 2.3 Sarcomas with complex karyotypes
Type of sarcoma
Fibrosarcoma (other than congenital)
Leiomyosarcoma
Malignant fi brous histiocytoma
Osteosarcoma
Chondrosarcoma (types other than extraskeletal myxoid)
Liposarcoma (types other than myxoid)<
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Embryonal rhabdomyosarcoma
Malignant peripheral nerve-sheath tumour
Angiosarcoma
Neuroblastomaa
a Neuroblastoma is quoted in this table as it belongs to the “small round blue cell tumours” group.
Fig. 2.15 Typical example of a complex karyotype as observed in embryonal rhabdomyosarcoma
showing multiple numerical and structural abnormalities. The latter (isochromosome 17q and two
chromosome markers) are marked with arrows.
Fig. 2.16 Interphase FISH demonstrating ampli cation of the MYCN oncogene (red signals) in a case of
neuroblastoma. The two green spots correspond to centromeric probes speci c for the chromosome 2 and
used as control for diploid status assesment of the analyzed cell.
Thyroid Carcinomas
Among epithelial malignancies, two histological types of thyroid carcinoma, namely the papillary and
follicular thyroid carcinoma, deserve to be mentioned as they exhibit speci c genetic aberrations that
represent reliable diagnostic parameters.
Papillary Thyroid Carcinoma
Papillary thyroid carcinoma (PTC) is characterized by rearrangements of the RET oncogene, a receptor@
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tyrosine kinase (RTK) gene located on chromosomal region 10q11.2. These activating rearrangements,
called RET/PTC, are caused by either paracentric inversion of chromosome 10 or balanced translocations
51involving chromosome 10 and various chromosome partners (Table 2.4). The molecular consequences
are fusion of the tyrosine kinase domain of RET with the 5′ part of the various gene partners with
subsequent release of the extracellular ligand-binding and juxtamembrane domains of RET receptor. As the
juxtamembrane domain negatively regulates RET mitogenic signaling, its deletion contributes to RET/PTC
52activation, which is further enhanced by dimerization potential brought by the gene partner. This leads
to ligand-independent activation of the RET kinase, signaling pathway stimulation and cell-cycle activation;
a well-known oncogenic process in tumoral cells harboring RTK rearrangements. As part of its oncogenic
e ect, RET/PTC directly modulates genes involved in in' ammation/invasion of the cell such as various
cytokines (GM-CSF, M-CS, IL6, etc.), chemokines (CCL2, CXCL12, etc.), and chemokine receptors (CXCR4).
The induction of an in' ammatory-type reaction may explain the chronic in' ammatory reaction observed in
52this type of cancer.
Table 2.4 Characteristics of different types of RET/PTC rearrangement in papillary thyroid carcinoma
The prevalence of RET/PTC in papillary thyroid carcinoma is highly variable (0–87%), depending on age
of patient, geographic regions, and sensitivities of the detection methods used (polymerase chain reaction
versus FISH), particularly if the rearrangement is present only in a small proportion of tumor cells or if the
RET/PTC transcripts is expressed at low levels. The average prevalence is 20–30% in sporadic adult cases
and rises to 45–60% among tumors from children and young adults. It is higher (50–80%) in papillary
carcinoma associated with radiation exposure, and it is thought that the close association between RET/PTC
translocations and irradiation is due to spatial proximity of the participating chromosomal loci in the nuclei
of thyroid cells, providing a structural basis for radiation-induced illegitimate recombination of the
14genes. Most studies concur that RET/PTC rearrangements are rare or absent in benign adenomas, and not
observed in other types of thyroid carcinomas. They are more frequent in PTC exhibiting a classic
52architecture and in microcarcinomas. Among the di erent variants of RET/PTC translocations, RET/PTC1
53and 3 are the most frequent, accounting for more than 90% of all rearrangements.
A small subset of PTC (around 10%) is characterized by rearrangement of the NTRK gene, another
receptor tyrosine kinase, located on chromosome 1q22 and encoding one of the receptors for the nerve
growth factor. NTRK gene activation is due to chromosome 1 inversions or balanced translocations between
chromosome 1 and 10, resulting in fusion of the NTRK tyrosine kinase domain to 5′-end sequences from at
least three di erent genes: tropomyosin (TPM3) or TPR gene, both on chromosome 1, and TFG gene located
51,53on chromosome 3.
Follicular Thyroid Carcinoma
Follicular thyroid carcinomas are characterized by PPARγ (peroxisome proliferator-activated receptor γ) gene
rearrangements in 25–50% of cases, mainly under the form of a distinctive t(2;3)(q13;p25) chromosomal
53translocation. This translocation leads to the fusion of the PAX8 gene (paired box gene 8) with PPARγ@
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gene, resulting in a fusion protein designed PPFP. PAX8 is a transcription factor expressed at high levels in
thyrocytes and necessary for normal thyroid development. PPARγ encodes a nuclear hormone receptor
transcription factor whose activity is related to adipocyte di erentiation, lipid and carbohydrate
metabolism, and cellular proliferation and di erentiation. PPFP is thought to exert its oncogenic properties
through a mechanism in which it acts as a dominant-negative inhibitor of wild-type PPARγ. This results in
inhibition of apoptosis and promotion of proliferation as well as anchorage-independent growth of thyroid
follicular cells. PPARγ has mainly been observed in low-stage follicular carcinomas with vascular invasion
and has been identified at apparent lower frequency in adenomas.
Clinical Applications of Conventional Cytogenetics and fish in Cytology
Introduction
Cytological assessment of a ne-needle aspiration (FNA) specimen remains the rst-line morphological
investigation of any suspected mass but cytomorphology alone—hence, without tissue architecture—is not
54,55always sufficient for a definitive diagnosis. For example, small-to-intermediate cell lymphomas such as
MCL, FL, or MZL can show overlapping cytomorphologic features with one another as well as with reactive
lymph node hyperplasia. Limitations of FNA are also encountered in soft-tissue neoplasms, especially in the
diagnostic management of small round-cell tumors. Most of the diagnostic problems can be solved with the
help of immunocytochemistry but limitations can be encountered mainly due to immunophenotypic
56heterogeneity among small B-NHL subtypes. For examples, the intensity of CD10 expression in FL has
57been shown to be variable, and even negative in some cases. MCL and SLL can be distinguished by
58di erences in CD23 expression but CD23 can be weakly expressed in both subtypes. CD5 expression may
not systematically be used as a diagnostic criterion between MCL and SLL, and some FL can also exhibit a
59CD5 positivity. It is thus necessary that FNA examination be supplemented with ancillary methods such
as karyotype, FISH, or polymerase chain reaction (PCR). Conventional cytogenetics allows complete
karyotype analysis and, as such, remains the historic gold standard by which everything is started.
However, it is a cumbersome and time-consuming procedure requiring adequate fresh tissue and special
cell culture techniques. PCR and interphase FISH (I-FISH) methods are more practical in that they can
bypass the need of cell culture. They have both their own advantages and disadvantages, and must be
considered as complementary rather than competing with one another. It is therefore not surprising that
60-62both have been included in a combined diagnostic algorithm proposed in the literature. However,
IFISH remains a less sophisticated laboratory technique than PCR and o ers a greater qualitative sensitivity
in studies of tumor-associated chromosomal abnormalities as will be illustrated later. This technique is
advantageous for FNA specimens because it requires only a few cells. It allows also retention of cellular
morphology, which permits simultaneous evaluation of morphology and chromosomal alterations.
Moreover, recent studies have demonstrated the feasibility of FISH on Papanicolaou-stained archival
63-66cytology slides, highlighting the good ' exibility of such method. These advantages probably explain
why FISH is becoming more and more popular in cytopathology laboratories.
FISH Strategy
Interphase FISH requires simple material such as cytospins from FNA specimens. Cytospin is an optimal
preparation for I-FISH because the monolayer allows excellent hybridization results. Cytospin preparation
can be made by Ficoll-Hypaque gradient-separation technique and then xed in methanol-glacial acetic
acid (3:1) for 20 minutes at −20°C. The slides will be then air-dried and stored at −20°C prior current
FISH procedure. Specimen handling is thus very simple, but it is critical to avoid delays in specimen
processing in order to prevent possible degradation of the target DNA and subsequent poor hybridization
67results. Subsequent FISH steps can be then easily performed without further manipulation of the
samples, with the use of commercially available kit sets including the premixed probes, and according to
the protocol recommended by the manufacturer. At least, 200 nonoverlapping and intact nuclei per case
and at least two di erent areas on the same slide should be scored before giving a result. The great
advantage of working on an interphase cell can nevertheless be a source of interpretative pitfalls in that<
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random chromosome colocalizations occur not infrequently in normal nuclei and can mimic chromosomal
translocations. Although most of the commercial probes have been designed to limit the risk of
falsepositive pro les, it remains critical to determine the frequency of such false-positive cells in order to de ne
a cuto level. Normal lymphocyte nuclei can be used as negative control to assess hybridization eMciency,
and the cuto level for positivity should be set at the mean (%) ± 3 standard deviations. Beside this pitfall,
other good practice recommendations are needed and must be known by the user. Such guidelines are
68detailed in an excellent overview recently published that we highly recommend to the reader.
The commercial probes are usually several hundred kilobases in length and yield large, bright and easily
detectable signals. They are currently available to detect many of the relevant chromosomal abnormalities
69described in the previous section and are known to be highly sensitive. For detection of chromosomal
translocations, three di erent kinds of probes are available, including the dual-fusion probes, the
singlefusion extra-signal probes, and the break-apart probes, all being dual-color probes (Figs 2.17A and 2.17B).
Dual-fusion and extra-signal probe sets are made of two di erentially labeled (green and red) DNA
segments, each of these segments identifying one of the chromosomal loci involved in the translocation. For
the dual-fusion probes, an abnormal pattern will be represented by one red and one green signal
(representing the normal homolog) and by two fusion or colocalization signals corresponding to the
chromosomal translocation and its reciprocal (“2F,1R,1G” pattern). Typical examples are probes designed
to detect lymphoma-associated chimeric genes subjacent to translocation such as the BCL2-IgH or BCL1-IgH
in follicular or mantle cell lymphoma, respectively (Fig. 2.18A). Such probes make it possible to
signi cantly reduce the risk of false positives as the possibility that two overlapping signals are due to
random spatial proximity of the participating chromosomal loci remains very low. The abnormal pattern
for extra-signal translocation probes will be represented by a single fusion (corresponding to one derivative
chromosome) plus a small extra signal representing the residual portion of one of the loci involved in the
translocation. Again, the probability that such pattern is observed in a normal nucleus is very low. A
wellknown example is the probe used to detect the BCR-ABL chimeric gene in chronic myeloid leukemia. Such a
probe has not been designed for detection of recurrent chromosomal translocations in lymphoma or
sarcoma and will not be illustrated here. Dual-color break-apart probes are made of di erentially labeled
(green and red) DNA segments located on either side of a breakpoint cluster region within a target gene.
The separation of green and red signals indicates break between the 5′ and 3′ regions of the rearranged
gene. In normal cells, the two probes colocalize to produce two yellow fusion signals (corresponding to two
copies of nonrearranged genes), whereas in the case of translocation involving one of the two genes, one of
the fusion signal splits, resulting in a characteristic 1 red–1 green–1 yellow fusion (“1R1G1F”) signal
pattern. The break-apart strategy o ers the advantage of detecting in a single experiment all recurrent
rearrangement of a gene involved in translocations with di erent gene partners. A typical example is the
EWSR1 gene which can fuse with no less than nine different gene partners (Fig. 2.18B).<
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Fig. 2.17 Schematic representation of two di erent types of dual-color probes, the dual fusion and
break-apart probes. (A) Left: Dual-fusion probes are composed of two di erentially labeled (green and
red) DNA segments, each of these segments identifying one of the genes/loci involved in the chromosomal
translocation. The probes are usually several kilobases in length and extend largely on both sides of the gene
of interest. Right: a normal pattern will show two red and two green spots, whereas a cell harboring a
chromosomal translocation will demonstrate two fusion or colocalization signals corresponding to the
chromosomal translocation and its reciprocal; the red and green spots indicate the two remaining normal
chromosomes (“2F,1R,1G” pattern). (B) Left: break-apart probes are made of two di erentially labeled
(green and red) DNA segments ' anking the breakpoint cluster region of a gene involved in chromosomal
translocations. Right: a normal cell will show two yellow fusion signals corresponding to two copies of a
normal gene. The disruption of one of these two copies subsequently to a chromosomal translocation will
lead to split of one yellow signal into two red and green signals (“1R,1G,1F” pattern).
Fig. 2.18 (A) Interphase FISH of follicular lymphoma cells with the use of a dual-fusion BCL2-IgH
probe. The two fusion/colocalization signals indicate the existence of a BCL2-IgH oncogene and its reciprocal
while the green and red signals correspond to the remaining normal IgH and BCL2 genes respectively. (B)
Interphase FISH with the use of a dual-fusion break-apart probe speci c to the EWSR1 locus. The lower
nucleus shows a normal pattern, whereas the upper one displays split of one EWSR1 gene copy as it can be@
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observed in Ewing's tumors.
Interphase FISH is also able to identify submicroscopic chromosomal deletions as well as numerical
chromosomal abnormalities such as trisomy or monosomy. The probes used to detect entire chromosomal
gains or losses are juxtacentromeric alphoid DNA sequences while submicroscopic deletions will be
identi ed with locus-speci c probes. To ensure the quality of hybridization (mainly the hybridization
properties of the tumor cells being analyzed), a control probe, labeled with a di erent ' uorophore and
identifying any other chromosome, will be cohybridized with the probe of interest. For detection of
microdeletion, the control probe will also serve to identify the chromosome harboring the deleted region.
Examples are trisomy 3 (Fig. 2.19) and deletion of chromosome 7q in marginal zone lymphoma.
Fig. 2.19 Interphase FISH using centromeric probes for chromosomes 7 (green) and 18 (red). Both cells
show three signals for each probe indicative of trisomies 7 and 18 as observed in nodal marginal zone
lymphoma.
Application
Lymphomas
Studies demonstrating the feasibility and diagnostic utility of FISH in FNA specimens have focused on the
most frequent lymphoma such as FL and, to a certain extent, di use large B-cell lymphoma. Although less
common, MCL has also been a subject of interest because of the clinical relevance and diMculties to
di erentiate it cytologically from other small cell NHLs. Among the latter, small lymphocytic lymphoma
may be difficult to diagnose when it presents as isolated lymphadenopathy.
As mentioned earlier, FISH and PCR remain complementary methods for detecting predictable
chromosomal abnormalities in lymphoma, but comparative studies on specimens such as tissue imprints,
cytospins, or smears have demonstrated a higher qualitative sensitivity of I-FISH. In follicular lymphoma,
the detection rate of the t(14;18) translocation with PCR was 70% at best, whereas a positive result could
65,66,70-73be achieved in around 90% of cases with FISH. The low detection rate encountered with the
PCR technology is due to mutation involving primer binding sequences and to the fact that the current PCR
method applicable in routine use is not able to detect breakpoints outside the known major breakpoint
region (MBR) and minor cluster region (mcr). A similar situation is encountered in MCL where the
sensitivity of FISH analysis for the direct detection of the t(11;14) translocation largely exceeds DNA-PCR
64,69,72,74,75methods; the detection rate reaching nearly 100% according to FISH studies, while it falls in
74,60the range of 40% with the second method. The lower qualitative sensitivity o ered by DNA-PCR is
mainly due to the wider variation of BCL1 gene breakpoints that are diMcult to span with primers. FISH
analysis circumvents these limitations by using IgH/BCL2 and IgH/BCL1 dual-fusion probes covering the
entire BCL2 and BCL1 gene, respectively. Moreover, these results highlight the greater applicability of FISH
since all known BCL2 and BCL1 breakpoints can be covered and detected with a single-probe set. Among
other small-cell lymphoproliferative disorders, SLL/CLLs are characterized by recurrent chromosomal
abnormalities such as trisomy 12 or interstitial deletion involving 13q14 chromosomal region. Interphase
63,76FISH can easily detect these aberrations and, together with negative results for BCL2 and BCL1
rearrangements, can help in assessing an accurate diagnosis of SLL/CLL. In FNA specimens displaying
monomorphous lymphoid population composed of medium-sized or large cells, a proper diagnosis of<
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Burkitt, di use large B-cell, or anaplastic lymphoma can be easily reached with the use of speci c
break72,77apart probes targeting C-MYC, BCL6, or ALK gene, respectively. Beside the detection of
lymphomaassociated speci c translocations, atypical patterns revealed by interphase FISH can help in better
classifying lymphoma. For example, a current IgH/BCL1 dual-fusion probe can identify hypotetraploid
74,78profiles with extra copies of BCL1 signals as observed in blastoid variants of MCL.
A recent study aimed at comparing the utility of I-FISH and ' ow cytometry immunophenotyping (FCM)
71in a series of FL and DLBCL. They found that detection of t(14;18) by FISH was a slightly more sensitive
(85%) diagnostic marker than identi cation of the typical CD19+/CD10+ immunophenotype pro le by
FCM (75%). FISH appeared to be more sensitive because it could also detect FLs with an atypical
+ -CD19 /CD10 pattern. In the same study, a BCL2 gene rearrangement was detected by FISH in 29% of
DLBCL cases, whereas FCM was able to identify a CD10+ monoclonal population in only 23% of such
lymphoma cases. I-FISH appeared thus to be slightly more sensitive than FCM in identifying germinal
center B-like DLBCL.
Sarcomas
Most (if not all) chromosomal translocations described in soft-tissue sarcomas (STS) are detectable by
IFISH. This method is thus particularly useful in diagnostically difficult cases such as small blue cells tumors.
Several studies aimed at comparing the eMciency of both reverse transcriptase (RT)-PCR and FISH
79,80techniques for a molecular diagnosis in sarcoma. Both methods were complementary and had their
own advantages and disadvantages in terms of speci city and qualitative and quantitative sensitivity.
However, the FISH break-apart approach appears to be very practical in that the use of a single break-apart
probe can recognize each speci c translocation such as the t(X;18) in synovial sarcoma, the t(2;13) or
t(1;13) in alveolar rhabdomyosarcoma, and the t(12;16) in myxoid liposarcoma. The potential
disadvantage of such an approach would be its inability to distinguish Ewing/PNET from other sarcomatous
types harboring EWS gene rearrangements (see Table 2.2) since the partner gene is not detected. In most
cases, these neoplasms are nevertheless distinguishable from each other on the basis of clinical data or
immunocytochemical differences.
81,82 83-85Several studies have demonstrated the usefulness of cytogenetics or I-FISH as an adjunct in
making a de nitive diagnosis of sarcoma by FNA. As our knowledge about the speci c chromosomal
abnormalities associated with sarcoma is constantly increasing, there is good hope that I-FISH will allow
accurate diagnosis on more cases investigated by FNA, obviating open surgical biopsy preceding therapy.
Multiple Myeloma
Multiple myeloma (MM) is characterized by numerous chromosomal abnormalities which have been shown
86to signi cantly impact survival in patients with such disease. The most relevant alterations include
hyperdiploidy, monosomy 13/deletion 13q14, deletion 17p, t(11;14)(q13;q32), and t(4;14)
(p16;q32)translocations, giving rise to the BCL1-IgH and FGFR3/MMSET-IgH chimeric genes, respectively.
Hyperdiploidy is associated with a favorable prognosis but all other abnormalities represent unfavorable
parameters, among which the t(4;14) and deletion17p appear to be the most important. Their
identi cations have implications for the design of risk-adapted treatment strategies. Historically, testing for
abnormalities in MM was based on conventional chromosomal analysis performed on bone marrow, but
results were often falsely normal since the actively normal myeloid cells were analyzed rather than the
monoclonal plasma cells, which infrequently enter mitosis. Standard FISH studies were thus employed to
detect the classical abnormalities associated with MM. Again, erroneously normal results were most often
obtained since this method is not able to distinguish between normal cells and small clones of monoclonal
plasma cells. A novel FICTION method, which is a combination of ' uorescent immunophenotyping and in
9situ hybridization, has thus been developed. First, antibodies against the cytoplasmic immunoglobulins λ
or are applied in order to speci cally identify the plasma cells thanks to their cytoplasmic ' uorescence.
Second, FISH probes will be hybridized to all cell types, but only speci cally target plasma cells will be
analyzed. This FICTION method is thus capable of detecting chromosomal abnormalities in bone marrow
specimens even when few plasma cells are present (Fig. 2.20A and 2.20B).<
<
<
<
Fig. 2.20 Illustration of the FICTION method on multiple myeloma cells. The plasma cells are detected
with the use of antibodies directed against the cytoplasmic immunoglobulins λ or . These antibodies are
colored with ' uorochrome AMCA (blue color). (A) Two plasma cells with deletion of the p53 locus
(chromosome 17p13) demonstrated by the absence of one red signal, whereas the existence of both
chromosomes 17 is con rmed by a speci c chromosome 17 centromeric probe (green signals). (B) Plasma
cell harboring the FGFR3/MMSET-IgH fusion genes corresponding to the t(4;14)(p16;q32) translocation. The
two yellow fusion signals are due to red and green signals relocating next to each other, and indicate the
FGFR3/MMSET-IgH fusion gene and its reciprocal. The green and red signals correspond to remaining
normal IgH and FGFR3/MMSET genes respectively.
Concluding Remarks
Over the past two decades, conventional cytogenetics has made it possible to identify nearly all
chromosomal abnormalities associated with speci c histological subtypes of lymphoproliferative disorders
and soft-tissue tumors. These chromosomal aberrations made it possible, in a second step, to pinpoint the
underlying oncogenes and to study the pathogenesis of tumors bearing such abnormalities. In addition to
their role in fundamental research, these alterations rapidly appeared to be powerful diagnostic and
prognostic parameters relevant to use on a regular basis. The constant emergence of commercial probes
yielding large, bright, and easily detectable signals made the FISH method a reliable tool for detecting
speci c chromosomal abnormalities on nondividing cells provided by cytology specimens such as smears,
cytospin, or liquid-based samples. At present time, there is enough evidence in the specialized literature
demonstrating that I-FISH, in conjunction with other ancillary tools such as immunocytochemistry and
molecular biology, constitutes a suitable complementary approach in the cytological diagnosis of cancers
detailed in this chapter.
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CHAPTER 3
Cytologic Screening Programs
Luiz M. Collaço, Lucilia Zardo
Contents
Principles of screening
Cervical Cancer and Screening
Cervical Cancer Incidence and Mortality Worldwide
Efficacy of Screening
Design of Screening Programs
Features of Successful Screening Programs
Limitations of Screening Programs
Screening Programs and HPV Vaccine
Screening Programs and HPV DNA Test
The Role of Laboratory in Screening Programs
Early Detection of Cancer in Other Sites
New Developments in Cytological Screening
Liquid-Based Cytology (LBC)
Automated Cytology
Concluding Remarks
Principles of Screening
Screening of diseases gained signi cance in medicine at the end of the nineteenth century, when public
health authorities emphasized the importance of screening methods for certain diseases. An example is the
1radiological screening of immigrants, searching for infectious diseases such as tuberculosis in the USA.
The idea of screening for early detection of cancer was accepted in the 1920s after the development of
2 3exfoliative cytological techniques initiated through the work of Babes and Papanicolaou. In 1941 George
Papanicolaou demonstrated a test for the early detection of cervical cancer, contributing toward the
4,5creation of screening programs. Prevention and early diagnosis are major factors in reducing morbidity
6and mortality resulting from neoplasia.
Screening of diseases presumes a test or examination that can detect the existence of a particular disease
in a high-risk population, asymptomatic or with minimum symptoms of the disease. Systematic screening of
diseases requires a series of elements with the objective of decreasing mortality from a particular disease.
7For this reason the World Health Organization lists certain principles to guide the screening systems:
1. The condition to be evaluated must be an important cause of morbidity or mortality.
2. The natural history of the disease must be known as well as forms of intervention in the pre-clinical
stage or with the disease installed.
3. The test used for screening must have a high level of sensitivity and specificity.
4. The test to be applied must be low risk, with good acceptability by the target population and the
scientific community.+
+
+
+
5. In the case of positive tests, diagnostic methods for confirming screening finds should be possible.
6. The test must be shown to be efficient in reducing morbidity or mortality caused by the disease.
It therefore follows that screening of a particular disease requires a precise test, easy to do, at a low cost,
and the capability of detecting the presence of a lesion. In principle this is not a test for a de nitive
diagnosis, although it can in some situations serve to indicate subsequent therapy.
Cervical Cancer and Screening
Cancer of the uterine cervix is an important cause of morbidity and mortality among women worldwide
and a leading public health problem. It is the second most common cancer in women, but the most
8common in developing countries.
Because of the phases that precede the lesion in the natural progress of invasive cervical cancer, and
because they can be easily discovered and treated, the disease is well suited to screening programs. The
Papanicolaou test is an established method for examining the cells collected from the cervix to determine
whether they show signs of pre-neoplastic differentiation.
Cytologic screening programs have led to a large decline in cervical cancer incidence and mortality in
developed countries. However, cervical cancer remains largely uncontrolled in high-risk developing
9countries because of ineAective or no screening. Approximately 85% of new cases of cervical cancer
(estimated at 493,000 worldwide) and deaths from cervical cancer aAect women in developing countries
10each year.
Cervical cytology, originally perceived useful in the detection of pre-invasive disease and not just for
identi cation of invasive cervical cancer, came to be seen as a technique destined to prevent cervical
cancer.
In the 1960s, its use spread among developed countries; meanwhile the concept that invasive squamous
cell carcinoma of the cervix arises from a spectrum of intraepithelial precursor lesions appeared. This
concept changed with the evolution of scienti c knowledge on the central role of human papillomavirus
11(HPV) in pathogenesis of cervical cancer and its precursor lesions. Although this morphology-based
model of a continuum has now been supplanted by a more discrete theory of multistage carcinogenesis, the
cervical intraepithelial neoplastic scale still merits consideration as the current basis of clinical
12management.
Cervical cancer screening is an example of success in the prevention of cancer. Unfortunately the
majority of women who develop cervical cancer live in countries where there is a lack of infrastructure to
support the organization and management of programs, or where other obstacles such as social and cultural
questions make their participation diD cult. Permanent eAorts to nd new and more eAective strategies will
be necessary to expand the access and participation of these women, optimizing resources and modifying
the mortality statistics for the disease, mainly in these areas.
Cervical Cancer Incidence and Mortality Worldwide
Currently cervical cancer is potentially curable, but still continues to be the second most frequent cause of
13death by neoplasia in women and the survival rate in 5 years varies from 44 to 66%.
The highest incidence occurs in Latin America, the Caribbean, Africa (tropical sub-Sahara), and South
8and Southeast Asia (Fig. 3.1). Around 80% of the cases occur in developing countries and just 20% in
developed countries. Socioeconomic and cultural aspects are a factor in this unequal distribution of this
neoplasia around the world. However, a preponderant factor in the areas of low incidence is the level of
information from the feminine population regarding the disease and the continual screening of this
population. On the other hand, in developing countries, the low level of awareness of the problem, the lack
of interest of the sanitary authorities, and the use of opportunist screening favors the continuance of this
unfavorable situation and indicates the urgent need for the public health authorities to find a solution.+
+
+
Fig. 3.1 Age-standardized (world) incidence rates of cervical cancer 2002.
Reproduced with permission of Parkin et al. Vaccine 2006;24(Suppl 3):12.
An important number of risk factors for cervical carcinoma have been identi ed and can therefore be
controlled, avoiding the progress of pre-neoplastic lesions. These factors are early start to sexual activity,
multiple partners, the number of partners a man has, infection by oncogenics HPV, precarious genital
hygiene, and smoking.
Histologically the largest number of cases is of squamous cell carcinoma; however, the incidence of
cervical adenocarcinoma has gradually increased over the past decades, particularly in young women,
14where it has doubled. A larger number of adenocarcinomas are being identi ed, either by control of
cervical cancer in developed countries or by association with HPV infection, above all the type 18.
Programs applied in Scandinavian countries and in Canada demonstrate that with continuous screening,
it is possible to reduce mortality from cervical cancer by almost 75%. However, the reduction of the
mortality rate is necessarily related to the real eAorts by doctors and population, the frequency and quality
of the specimen collection, the examination and diagnostic analysis, adequate communication between the
15specialists, and the efficacy of the system for management of the patients.
Efficacy of Screening
The eD cacy of cytological screening for cervical cancer depends on the possibility of modifying the course
of the disease through identi cation of women with high-degree precursor lesions and invasive initial
lesions. With this it is possible to distinguish the woman apparently not aAected from the woman who could
have the disease.
Even though the eD cacy of cytology screening has never been proven through randomized trials, it is
generally agreed that the marked reduction in the incidence and mortality from cervical cancer before and
after the introduction of screening programs in a variety of developed countries has been interpreted as
17,18strong non-experimental support for organized cervical cancer screening programs.
The best known studies are those that compare incidence and mortality in Iceland and in the four Nordic
19-21countries (Fig. 3.2). Before screening was installed in Iceland, mortality had been on the increase but
fell 50% in the period of 10 years from introducing screening. In the Nordic countries, the decline in
cumulative incidence rates over a 15-year period, between 1966–70 and 1981–5, was related to the
coverage and extent of the organized programs. In Norway, where only 5% of the population had been
screened opportunistically, the incidence rates fell by 20% in comparison to Finland, with a national
population-based program, where incidence fell by 65%.+
+
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Fig. 3.2 Incidence and mortality rates of cervical cancer in the Nordic countries, 1958–97 (mortality
available up to 1996). Whole female population, adjusted for age to the world standard population (Laara
et al. (1987); Engeland et al. (1993); Hristova and Hakama (1997); Parkin et al. (1997); Moller et al.
(2002); EUROCIM (European Network of Cancer Registries) database).21,74-77 Reproduced with permission
of IARC—International Agency for Research on Cancer.12
In a study of invasive cervical cancer in British Columbia, approximately half of the new cases diagnosed
22had no previous cytology or the last examination had been made more than 5 years ago.
Two important parameters traditionally used to measure the validity of screening tests are sensitivity and
the speci city. The sensitivity means the percentage of positive cases reported as being positive. It relates to
the ability of disease detection and it can be calculated using the formula
The speci city means the percentage of negative cases reported as being negative. It relates to the ability
of disease exclusion and it can be calculated using the formula
A third criterion is the positive predictive value that measures the probability of the disease to be present
in the patients whose test was reported as positive, and it can be calculated using the formula
Glandular lesions are much less frequent than those originating from squamous epithelium and the
diagnosis of the intraepithelial forms is the principal objective of the screening programs. In relation to the
prevention of cervical adenocarcinoma, the Papanicolaou test is potentially a powerful weapon, but in
comparison to the diagnosis of squamous lesions, the diagnosis of cervical adenocarcinoma in situ has
shown a signi cantly higher rate of false-negatives, not being so eAective in the prevention of invasive
23glandular lesions.
In 2004 a working group at the International Agency for Research on Cancer (IARC) of the World Health
Organization (WHO) met to evaluate the eD cacy of prevention of cervical cancer in reducing mortality
caused by the disease. They concluded that the programs of prevention based on the Papanicolaou test
continue being the mainstay for prevention of this type of cancer throughout the world, there being
24sufficient evidence that screening of cervical cancer diminishes mortality caused by the disease.
Despite the knowledge of the eD cacy of cytopathologic tests in contributing to the reduction of cervical
cancer through organized programs by their characteristics of simplicity, acceptability, and low cost,
studies have shown major variations in the estimates of sensitivity and speci city of the test. A
metaanalysis to estimate the accuracy of the Pap test in which data from 59 studies were combined reported+
+
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25estimates of sensitivity and speci city ranging from 11 to 99% and 14 to 97%, respectively. A systematic
26review reported sensitivity and specificity ranging from 30 to 87% and 86 to 100%, respectively.
Sensitivity and speci city are important parameters for the evaluation of the accuracy of the screening
test. However, it is important to bear in mind that the eD cacy of screening is not restricted to the
performance of the test used. Special emphasis must be given to the need to develop organized programs
that have a systemic approach, that are well integrated into the existing health system, and which consider
social, cultural, and economic aspects. A meta-analysis of social inequality and the risk of cervical cancer in
57 studies revealed that both cervical infection with HPV and a lack of access to adequate cervical cancer
screening and treatment services are likely to be important in explaining the large cervical cancer incidence
27rates observed in diAerent socioeconomic groups. An estimated 100% increased risk of invasive cervical
cancer was found for women in low social class categories when compared with women in high social class
categories.
In an analogous way, this diAerence occurs in developing countries and those developed where the
inequality in the access is the consequence of the inequality in the quality of the services. Past failures of
cervical screening in developing countries are attributable to failures in program quality, rather than to
28,29technological limitations of the screening test.
It is very important to evaluate the eD cacy of the programs in reducing the disease, and also whether the
screening approach chosen is cost eAective, before considering extending implementation to large
populations. A screening program is justi ed if a prior diagnosis that permits a cost-eAective and
measurable reduction of the disease is made.
Design of Screening Programs
Cervical cancer can be avoided when there is an early diagnosis of the precursor lesions, without local or
systemic compromise. The implementation of a systematic program of prevention of gynecological cancer
among women in British Columbia in 1949 reduced both the incidence and the mortality caused by this
neoplasia. Among the methods available for early detection of cervical cancer, exfoliative cytology, or the
Pap test, is recommended worldwide for mass screening, because the eD cacy in the detection of
premalignant lesions, associated with the social role of the method, permits minimization of costs with
1,4,6-8,13,15,30curative medicine.
From that stated above, a routine of procedures essential to the success of a program of prevention may
be obtained. The basic integrated actions include: (1) care with collection, (2) processing of the smears, (3)
31screening and interpretation of the specimens, (4) follow-up of the patients, and (5) quality control.
1. Care with collection—The majority of false-negatives arise from problems with collection of specimens,
and for this reason this stage should be systemized and there should be training and recycling of the
personnel responsible for taking the samples. The smears must be well identified, slim, uniform, and
without contaminants, and contain samples from the transformation zone, where in the majority of cases
the cervical cancer develops. There should be a minimum of blood, mucus, or other obscuring material
such as lubricating gel. It is also important at this moment to adequately fix the material so as not to
compromise subsequent stages.
2. Processing the specimens—In general prevention programs cover a large number of tests, so laboratories
should have guidance regarding the systemizing of the processing and the recording of a large volume of
specimens. One of the characteristics of the Pap test is that it consists of various stages. Each stage should
be monitored so as to minimize the possibility of error. The condition on arrival of the slides, and the
number of slides per case, must be verified. Special care should be taken with the flow of the tests, with
adequate numbering and balanced coloration with control of the number of cases colored in each set. The
end product of this stage will be fundamental to a good result with the rest.
3. Screening and interpretation of the specimens—The screening should be done in as little time as possible,
depending on the basic requirements of each program, by trained and qualified personnel. Care should+
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also be taken with excess workloads for cytopathologists and cytotechnicians, and also with refresher
courses and recycling. The report on the tests should be systemized and use a unique nomenclature, of
which all involved in the preparation and interpretation of the results should be fully aware.
4. Follow-up of patients—The prevention program should include reference, contra-reference, and active
search services. The mere detection of the lesions will not determine the impact on the natural history of
the disease. For this reason the treatment of lesions in a pre-invasive stage is fundamental. Outpatient
treatment centers for the more simple cases, and others for more complex cases should be integrated into a
service network for the program. Mechanisms for finding and managing patients who did not return after
the initial test and who show alterations are fundamental for a prevention program to function well.
5. Quality control—Quality is fundamental in gynecological cytopathology. One of the greatest problems in
mass cytology is the false-negative cases. Cytopathology labs must have mechanisms for internal quality
control with the objective of avoiding false-negative and false-positive tests. These mechanisms should
include measures relating to the screening and interpretation of the specimens, a review of 10% of the
cases seen by the cytotechnician, grouping the technicians according to hierarchy. External quality control
must be included in the design of the prevention program, conducted by an accredited entity and with
interlab action with the objective of guaranteeing the homogeneity and quality of the laboratory
32procedures. It should also function as a detector of eventual problems and could indicate a need for
redirectioning continuous education efforts within the program. For additional information on quality
assurance in cytopathology see Chapter 4.
Cancer screening may be oAered to a population either as an organized program or opportunistically, or
as some combination of the two. Opportunistic screening is spontaneous and initiated either by the
individual or healthcare provider during routine healthcare encounters. It is often associated with low
coverage of people at high risk and excessive repetition of procedures at frequent intervals, high costs, and
a small bene t at the population level. Systematic or organized screening programs refer to planned and
concerted public health application of early detection and treatment in de ned populations, operating
33under precise protocols and guidelines.
Some countries with organized screening programs can reduce the incidence of cervical cancer by up to
around 80% in areas with high-quality screening, good coverage, and a reliable follow-up. Organized
programs with systematic call-up, recall, follow-up, and vigilant systems have shown more expressive
34effects with less resources than less organized programs. Various alternative screening strategies are being
researched for developing countries, although the challenge in less-developed countries is surpassed by the
35complex array of problems that go far beyond the introduction of simplified technology.
Features of Successful Screening Programs
The success of cervical cancer screening is shown by its ability to reduce the incidence of cervical cancer
and the resulting mortality, in a cost-eAective way. To be successful it is fundamental that the program is
organized and broad-based, developing along the line of care for cervical cancer. All the stages involved in
the nding of the women, the collection of material for the cytological test, transport and processing of the
slides, identi cation of lesions, and nally the delivery of results, treatment, and follow-up of the women
with alterations should happen in sequence, synchronized and with the highest quality. Any failure in one
of these stages can compromise the impact of the screening on the health of the population. The following
are some aspects of successful screening programs:
• Government policy: Planning within a governmental policy and national planning for cancer control. This
includes the definition of the age range of the population to be prioritized and the frequency (interval) of
screening, apart from production of instructions to guide the process, including recommendations
regarding nomenclature and the therapeutic action for the lesions identified.
• Coverage: Measures to guarantee good coverage, with special attention to identification of women in the
target population. Education of these women regarding cervical cancer screening can contribute to
increased attendance and confidence in the procedure, apart from facilitating understanding of the results+
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of the cytological test.
• Integrated system: The different levels of healthcare in the program should be integrated like a network,
with the capacity to ensure continuity of the care within the different levels.
• Health professionals: Good results can be achieved by educating and training the health professionals,
improving the attention given to the women, the quality of samples collected, the quality of the screening
and the results of the tests, and also the research and follow-up of the patients with lesions needing
treatment.
• Quality of the diagnosis: Efficient and high-quality laboratory service, which should preferably be
centralized; quality control of cytology reading.
• Infrastructure of health services: Adapting the services to give the treatment needed, with the capacity for
attending to the planned demand, in relation both to equipment, installations, and material and to the
human resources available. It is of fundamental importance to guarantee the supply and the accessibility of
the health services.
• Information system: An integrated information system linking the different elements of the program,
permitting identification of each woman and the exchange of management information, and monitoring
and referring women with results showing alterations to the respective health services, with a view to
ensuring that these patients receive appropriate diagnosis and treatment, should be achieved.
• Indicators: Monitoring and evaluating cervical cancer prevention programs is essential for effective,
efficient planning and service organization, as well as for patient management. Indicators created to
evaluate performance at the different stages of the program should be monitored regularly, using
25information generated preferably through the routine information system.
• Leadership: Leadership, management skills, attention to linkages at all levels of the program, and
8budgeting skills are essential.
Limitations of Screening Programs
Limitations of prevention programs can be related to diAerent factors, such as errors and failures in the
program as well as socioeconomic and cultural problems.
Errors and Failures in the Program
The rst limitation refers to the Pap test. Although it is the most eAective screening test in oncology, it
shows failures with low sensitivity where the false-negatives vary between 3 and 13%, and high speci city,
with false-positives less than 5%.
Achieving the ideal coverage is another problem that limits screening programs. For a prevention
program to diminish cervical cancer mortality it must achieve a coverage estimated at around 80% of the
women, so this must be the target. For this the community must be mobilized and informed in order to
make the women realize the causes and consequences of cervical cancer and so submit to the tests. The
warnings should be spread through all means of communication including explanatory folders and
pamphlets. Lack of knowledge becomes one of the main allies of the ineD ciency of screening programs.
Strategies must be established to encourage regular participation of women in the program and the return
36of women with abnormal results. Around 29% do not return after taking the test. Some measures that can
help are assistance with transport, slides/ lms, and personal letters with folders. It is very important to
individualize the incentives according to the socioeconomic level of the patients.
Other causes of failures in the programs include inadequate collection of material to be examined, errors
of interpretation in the cytopathology lab, absence of adequate follow-up, and failures in the treatment of
precursor lesions. It becomes fundamental to keep the multidisciplinary teams who work with prevention
stimulated and up to date, aware of the importance of each stage and its role in achieving the nal result,
which is to avoid death by cervical cancer.+
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Socioeconomic and Cultural Problems
Limitations that extrapolate the limits of the program and for this reason can be considered as extrinsic also
occur. In this group are political limitations, as mainly in developing countries other questions could be
prioritized and oppose the prevention of cervical cancer just as with survival, infectious/contagious diseases
such as tuberculosis or AIDS, vaccination, or the fight against hunger and bad nutrition.
Sociocultural and behavioral questions can also be a negative in uence in the running of programs and
should be detected and minimized. This group includes the level of education of the women that could
in uence directly actions and response to the programs, religious aspects, and the decisive in uence of the
companion on the woman, contributing to diminish women's adhesion to the screening programs.
Finally it is important to those countries or regions installing screening programs to pay attention to the
37following points :
1. Try to screen the largest possible number of women—with at least one test during their lifetime.
2. Focus on the workup for those women who had a bad diagnosis in the primary screening such as
highdegree lesions or invasive carcinomas.
3. Design the programs remembering that it will not be possible to detect all the carcinomas.
4. Try to make the women, medical staff, politicians, and legal authorities understand that the Pap test is
not a perfect test, and that there are advantages and disadvantages in its use. Because of this they should
make an effort to optimize results and even add methods to help the prevention program achieve its
targets.
Screening Programs and HPV Vaccine
HPV is a sexually transmitted infection recognized as necessary for the development of cervical cancer, and
11this strong association is currently seen as causal. Oncogenic types of HPV DNA are detected in virtually
all cervical cancers and recognition of this crucial role has stimulated the investigation and development of
HPV vaccines in both prophylactic and therapeutic settings. The natural history of this cancer oAers two
opportunities for intervention to interrupt the course of the disease: primary and secondary prevention.
Primary prevention refers to measures to prevent infection by HPV using vaccines. Secondary prevention,
which is detection and treatment of precursor cervical cancer lesions, traditionally uses cytopathology as a
screening test.
The prophylactic HPV vaccines are prepared from empty viral capsids called virus-like particles (VLPs)
composed of the capsid proteins L1 and L2. These particles do not contain viral genetic material and thus
are unable to multiply, which means they are non-infectious. Early studies showed that L1 protein has the
intrinsic capacity to assemble into empty capsid-like structures whose immunogenicity is similar to that of
38infectious virions. Studies of HPV16 L1 VLP vaccine have indicated that they were well tolerated and
39highly immunogenic, generating high levels of antibodies against HPV16.
Two studies provided convincing evidence that the prophylactic vaccine is eAective in preventing new
and persistent infections of the genital tract with the two types of HPV most commonly associated with
cervical cancer and its precursors.
The rst published randomized, clinical trial of prophylactic vaccines developed to determine whether an
HPV16 L1 VLP vaccine could prevent HPV16 infection in women reported that the vaccine had a 100%
eD cacy in the vaccine group when compared to the placebo group. In this study, women who received the
vaccine had the titer of HPV16 antibodies 58.7 times as high as the titer among women with serologic
40evidence of natural HPV16 infection.
A second study on a randomized, double-blind, controlled trial to assess the eD cacy, safety, and
immunogenicity of a bivalent HPV16/18 L1 VLP vaccine showed that the vaccine was eAective in
prevention of incident and persistent cervical infections with these two virus types, and associated
cytological abnormalities and precancerous lesions. This study reported that the vaccine eD cacy was+
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4191.6% against incident infection and 100% against persistent infection with HPV16/18.
So far there exist two vaccines with types HPV16 and 18, more commonly associated with cervical
cancer, which protect against both new and persistent infections. One of the vaccines also includes types 6
42and 11, with a protector eAect against genital warts. With this it is hoped to prevent around 70% of
cervical cancer cases worldwide among women who have never been exposed to the high-risk types of
virus. Bearing in mind that the prevalence of types 16 and 18 varies from country to country, it is believed
that the vaccine that would include the seven types of most common HPV in the world (16, 18, 45, 31, 33,
4352, 58) would be able to prevent about 87% of all cases, with small regional variations. However, the
addition of multiple new types of VLP in one unique vaccine could present technical obstacles for the
44manufacturers.
Although the results of studies with vaccine are promising, some questions still need to be answered for
an eD cient implementation. The rst of them refers to viral types other than 16 and 18 strongly associated
18with cervical cancer. Screening and treatment services will still be required, because the vaccines only
prevent about 70% of cervical cancer cases and because it will be years, if not decades, before we see the
45full bene t of vaccination in terms of a reduction in the incidence of cervical cancer. EAorts to develop
multivalent vaccines that could control most HPV infections associated with cervical disease will be
necessary.
Furthermore, duration of the antibody response and protection from vaccination remains to be
determined and a long-term follow-up of vaccinated women is required before the impact can be fully
18identified. Trial data for both vaccines suggest they oAer a minimum of 4–5 years eD cacy, of close to
46100%, in preventing persistent infection by the vaccine genotypes. There are no predictions regarding
prevention of cancer among women who have already experienced an infection. The vaccines are not
designed to treat people who have already been infected with these genotypes. Moreover, it is not known
42whether effective coverage against some genotypes could favor the emergence of more pathogenic types.
Some subjects are particularly relevant for programs, and service delivery strategies still remain under
study. There are several open issues as well as the performance in Africa where chronic malnutrition, HIV,
and other infections diseases may compromise the immune response, the cross-protection, vaccine
compatibility, and the safety and eD cacy in speci c populations, such as pregnant women and
47immunocompromised patients. There is still a lack of data on infants and young children.
While the development of a prophylactic HPV vaccine may be the ultimate solution to the prevention of
cervical cancer, it is unlikely that the vaccine will be widely available in low-resource settings within the
next decade. In the meantime large numbers of women already infected with risk types of HPV remain at
18risk of developing cervical cancer. To prevent the disease in women already infected or in those infected
with other types of virals not included in the vaccines, it will be necessary to maintain and improve cervical
cancer control actions, principally in developing countries where the programs are less effective.
To measure the clinical bene ts and cost-eAectiveness of HPV vaccination, a computer-based model of
various cancer prevention policies has been developed. The most eAective strategy was one that combined
vaccination and triennial conventional cytologic screening strategies. This approach would reduce the
absolute lifetime risk of cervical cancer by 94% compared with no intervention, in a cost-eAective
48manner. HPV vaccine will be an additional tool in the strategies for reducing morbidity and mortality
from cervical cancer but will not replace screening and early treatment, and will be a component of a
44comprehensive strategy with the long-term goal of eliminating cervical cancer.
Screening Programs and HPV DNA Test
Cervical-vaginal cytology continues to be the method of choice for mass prevention of cervical cancer.
However, there are discussions regarding its limitations with regard to sensitivity, speci city, and ability to
49reproduce.
One of the rst applications of the HPV test in clinical practice has been in women referred for a+
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50colposcopy after an abnormal pap smear. The combination of methods has been proposed in an attempt
to improve the sensibility of the Pap test. Among these the association of cytology with the molecular test
for HPV using hybrid capture (HC) has been highlighted. This technology presents high sensitivity for
highdegree intraepithelial lesions and age-dependent speci city. In young women the speci city of HC-2 is
lower than cytology and in women over 35 the speci city is equal to cytology. Studies show that the
woman with negative HC-2 for HPV with normal cytology is at low risk for developing cervical cancer in
50the next 10 years. Other studies have shown an improvement in sensitivity in cervical intraepithelial
neoplasia (CIN) 3 cases.
The FDA approved HC-2 for HPV DNA as an assistant method for cytology in women over the age of
2430. Apart from the improvement in sensitivity in the detection of lesions the use of HC will bring about a
larger spacing of time between screenings and virtual reduction in the number of consultations in a
screening program.
However, some studies have shown evidence that the HPV test could be potentially superior in screening
51,52when compared with cytology.
The Role of Laboratory in Screening Programs
The laboratory can make an important contribution to the structuring and organization of cervical
screening programs based on the Papanicolaou test. It should be included in the global planning, with a
logical structure of hierarchy, regionalization, and above all integration with the healthcare system.
Data collected through the laboratory, apart from producing epidemiological and administrative
information on the results of the tests, permit the creation of indicators to help in monitoring and
evaluating, not just the quality of the laboratory activities, but also the quality of programming, thus
helping to generate pertinent and useful investigations which contribute toward the improvement of
53eAectiveness and eD ciency of the program. Using laboratory data it is possible to achieve some of the
required guarantees in cervical screening programs, such as to con rm that women in the target population
are being screened and are receiving appropriate management, and to con rm the target geographic group
29coverage (Table 3.1).
Table 3.1 Confirming that women in target demographic groups are screened
Monitoring of the diAerent steps in the program can provide valuable information for the identi cation of
problems and planning of respective measures for improvement. Care in the setting of parameters and+
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indicators is fundamental in the monitoring of each stage of the program, in order to take into account local
diAerences. The indicators can provide useful data both for the laboratory and for the local and/or regional
manager in programming suitable action.
The lab, when integrated into a screening program, should have among its objectives top quality
production, training, and updating of personnel and the guarantee of a secure place of work, where risk
53factors are under control and the environment is protected.
In the area of laboratory governance, it is possible to contribute toward an atmosphere of monitoring and
evaluation, which helps in decision-making and guarantees attention to quality. The system of internal
monitoring of laboratory quality includes a set of actions, which should be developed and disseminated in a
coordinated way, involving the various stages in the work process, from collecting a sample to issuing the
report. The system aims to accompany and evaluate the cyto- and histopathologic diagnostic procedures in
the laboratories, thus helping to determine areas where improvements can be planned and implemented,
and also evaluate the impact of these actions and the incorporation of new practices.
In some situations, the indicators measured in the lab can show evidence of problems in previous stages,
as for example in the collection of material or in the use of an inadequate xer. At these times, it is the role
of the lab, acting as an integral part of the network, to inform the health units that send the material for
examination to improve quality in a team eAort, assisting in the planning and implementation of corrective
measures and improvements.
It is recommended that laboratories have a system that permits monitoring of test quality, establishing
evaluation criteria and maintaining records of the results found. This is a simple and low-cost measure that
re ects technical advances of the staA involved, improving the relationship between the clinic and the lab,
and in the last analysis improving care to the patients.
Early Detection of Cancer in Other Sites
As mentioned at the beginning of this chapter, screening refers to the use of simple tests across a healthy
population in order to identify individuals who have disease, but do not yet have symptoms. Early
diagnosis, a concept diAerent from screening, refers to the detection of early clinical stages of disease in
54symptomatic subjects.
Evaluations of the potential of the Pap test to be a practical screening test for endometrial cancer have
shown both sensitivity and positive predictive value too low. It is likely that the early detection of some
endometrial cancers are mainly incidental. However, abnormal endometrial cells on the Pap test may be
markers for increased risk, especially when they are present in the secretory phase of the menstrual period,
55or when the patient is postmenopausal. When unexpected bleeding occurs, evaluation becomes
diagnostic rather than screening and the woman should undergo an endometrial biopsy and/or other
56diagnostic tests as appropriate.
Prospective studies of lung cancer screening have not demonstrated persuasively that screening for lung
56,57cancer with sputum cytology in combination with chest radiography saves lives. Although none of the
studies showed fewer deaths in the experimental group than in the control group, none of the studies
compared disease outcome in a group oAered screening with a group strictly not invited to, or encouraged
to, have screening. Sputum cytology was believed to have potential for the early detection of lung cancer,
but showed little added advantage over chest X-ray in the NCI cooperative trials, and was not associated
56with any reduction in deaths from lung cancer. However, sputum cytology was eAective in identifying
roentgenographically occult lung carcinoma in its early and occult stages, particularly in patients at high
risk for this disease, where lung carcinoma was suspected on the basis of symptoms, smoking, or air ow
58obstruction.
Esophageal cancer has a very poor prognosis, mainly because most tumors are asymptomatic and do not
receive medical attention until they are unresectable. Early detection is the key to the treatment. At the
current time, screening and surveillance for esophageal cancer is still controversial and screening is not
recommended except for very selected subgroups. The presence of risk factors such as long-term tobacco or+
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59alcohol use, achalasia, or squamous head and neck cancers may identify selected groups for screening.
Another indication is in high-incidence populations, such as those found in northern China where
60esophageal cancer is endemic. Since the late 1950s, Chinese scientists have performed many studies to
develop early detection strategies for this disease. The principal early detection technique developed was
esophageal balloon cytology. With this technique it was possible to collect exfoliated cells and scrape the
surface of the esophageal mucosa. Although the comparison of cytological diagnoses with concurrent
histological ndings showed low (14–36%) sensitivities for the cytological detection of biopsy-proven
61cancers, cytologic screening could decrease the mortality by facilitating early detection of the disease.
Positive cytology must be verified by endoscopy and biopsy.
Urine cytology is an inappropriate test for screening the general population. Because of the low
prevalence of bladder cancer, the positive predictive value of the tests is low. There is inadequate evidence
to determine whether screening for bladder and other urothelial cancers would have any impact on
62mortality.
Urinary cytology can be helpful in the follow-up of patients exposed to carcinogenic agents by
contributing to early diagnosis of urinary tract tumors. By far the greatest known environmental risk factor
in the general population is tobacco, especially cigarette smoking, with individuals who smoke having a
four- to sevenfold increased risk of developing bladder cancer compared to individuals who have never
62,63smoked. Bladder cancer was rst linked to an occupational exposure. An increased risk of bladder
cancer has been identi ed for a variety of occupations including chemical and rubber workers, leather
64,65,66workers, painters, dye workers, truck drivers, and garage and gas station workers.
Urine cytology is primarily used for detection of cancer in patients like those exposed to industrial
chemicals and metals, cigarette smokers, and those with schistosomiasis, associated with increased risks of
bladder cancer. Furthermore, it is also used for diagnosis of symptomatic patients and follow-up of patients
with a history of urinary tract neoplasia.
The use of cytology as a screening test for cancer has demonstrated discouraging results in sites other
than uterine cervix. In asymptomatic persons it can be more harmful than bene cial for the possibility of
false-positive results, leading to unnecessary expense and morbidity from follow-up procedures. However,
in a general way, it has had an important role in the diagnosis of early clinical stages of disease when
already symptoms are proven or when there has been clinical suspicion. Early detection of cancer greatly
increases the chances for successful treatment, education being one of the most important elements for
recognizing possible warning signs and taking prompt action that will lead to early diagnosis. Increased
awareness of possible warning signs of cancer can have a great impact on the disease.
New Developments in Cytological Screening
Liquid-Based Cytology (LBC)
New technology for alternative and complementary forms of screening alterations in the cervix has recently
been proposed, and one of these is known as liquid-based cytology (LBC). In this method, the cervical cells
are immersed in a conserving liquid before being xed on the slide, avoiding desiccation and reducing the
quantity of obscuring material. Liquid cytology can be prepared by manual or automated methods. LBC
methods have been used routinely in laboratories in the majority of developed countries, whereas
67-69developing nations more frequently use manual liquid cytology methods. Although the cost is higher,
various studies have shown the advantages of using LBC. Special attention has been given to the use of
residual material in the vial, which can be used for:
(a) Preparation of additional slides;
(b) Molecular testing of infectious agents;
(c) DNA cytometry; and+
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(d) DNA ploidy analysis.
An additional sample can be potentially useful in clarifying diagnoses in cases showing undetermined
70,71nuclear atypia, questionable graduation of the lesion, excess or scarcity of cells, blood, or exudates.
Since their introduction and approval as a method of detection of lesions on the cervix, the various LBC
70methods have been the object of diverse studies that emphasize the lower number of interpretative errors,
72more eAective diagnosis of lesions, and greater speed of analysis due to the ease of reading the slides.
Emphasized also is the reduction of unsatisfactory cases due to collection of samples and the increase in the
number of cases diagnosed with low- or high-degree lesions. Because of these ndings the FDA approved
LBC in 1996 as a screening method, and today in the USA around 80% of cervical cytology tests use the
24liquid base.
However, despite the bene ts shown by liquid cytology, studies point out the need to evaluate the cost–
bene t of using one or other type of cytology, as the cost of LBC is still high when compared with the
conventional method. A recently published review showed that the principal automated techniques, used in
70the USA, have a questionable cost-effectiveness ratio. Scotland adopted the method and the UK's National
Health Service (NHS) is recommending the introduction of the method in the screening program.
Automated Cytology
Automation of cytology has been studied for many years with the purpose of introducing methods that
reduce errors caused by human fatigue, and that can detect lesions when the sample contains a lesser
number of abnormal cells. Automated methods available can be used with conventional cytology or with
LBC. Despite technological development and the emergence of automatic apparatus, studies have shown
that automated screening would not improve the outcome of cervical cytology. For additional information
on automated systems see Chapter 34.
One of the advantages of this methodology is the possibility of testing a large number of cases with a
73minimum possibility of error. Its association with LBC would also provide a still reduced number of
unsatisfactory cases. Nevertheless the high cost of equipment and the implementation of the technology
makes its use difficult principally in developing nations.
Concluding Remarks
Cytologic screening is an important method for certain diseases, especially cervical cancer, and an example
of successful prevention of this disease. The majority of cervical cancer occurs in developing countries. The
success of cervical cancer screening is shown by its ability to reduce the incidence of cervical cancer and
the resulting mortality. The integration of procedures is essential for a successful screening program.
Recently new technologies for alternative and complementary forms of screening such as liquid-based
cytology and automated cytology have been proposed. A combination of methods has been proposed in an
attempt to improve the sensibility of the Pap test. Among these, the association of cytology with the
molecular test for HPV using hybrid capture has been highlighted. Automated cytology may be used for the
purpose of reducing human errors caused by human fatigue, and to detect lesions with a lesser number of
abnormal cells in the sample. HPV vaccine will be an additional tool in the strategies to reduce morbidity
and mortality from cervical cancer and will be a component of a comprehensive strategy with the long-term
goal of eliminating the disease. Cytologic screening can also be performed in selected high-risk populations
for lung, esophageal, and bladder cancer.
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Eur J Cancer Prev. 2002;11(Suppl 1):S1-S96.CHAPTER 4
Diagnostic Quality Assurance in Cytopathology
Marluce Bibbo, Catherine M. Keebler
Contents
Introduction
Quality Assurance Measures
Laboratory Directors
Cytotechnologists
Physical Laboratory Facilities
Safety Precautions
Equipment
Specimen Collection
Preparation, Fixation, and Staining Procedures
Slide Evaluation Workload
Cytologic Terminology
Laboratory Records, Logs, and Files
Internal Quality Assurance Mechanisms
CLIA 88 Update
Rapid Re-evaluation
Computer-Assisted Quality Assurance Mechanisms
External Quality Assurance Mechanisms
Continuing Education Practices
Concluding Remarks
Introduction
Cytopathologists are concerned about and committed to quality assurance and
quality control in their laboratories. These practices include, among others, the use
of intralaboratory and extradepartmental consultations, case reviews, correlation of
cytologic and histopathologic specimens, hierarchic review of cytopathology, and
review of completed diagnostic reports. Most of the quality assurance techniques
1-18,20,21are well described.
In the past, formal organization and mandatory documentation of these quality
assurance e0orts may have been limited or de1cient. Formal rules may be di2 cult
to apply, because laboratories and screening programs vary depending on the
volume and type of cytodiagnostic material received and on the size and
experience of its sta0 members. Even though the detailed design of a quality
assurance program emanates from the cytopathology laboratory director, basic
quality control and quality assurance principles of structure, organization,
documentation, and systematic review must be in place.
The enactment of the Clinical Laboratory Improvement Amendment of 1988 by
22the US Department of Health and Human Services, the convening of two
national conferences on cytologic quality assurance by the Centers for Disease
23-25Control, Atlanta, Georgia, the publication of the Quality Assurance Manual by
26the College of American Pathologists (1988), the American Medical Association
27Committee Report on the subject, the publication of the Compendium on Quality
Assurance, Pro ciency Testing and Workload Limitations in Clinical Cytology by the
28Tutorials of Cytology, and the proposal by the National Cancer Institute
29Workshop on the Bethesda System of reporting cytologic 1ndings, as well as
30editorials, letters to the editors, and general public interest led to the assurance
of high diagnostic standards and fostered intense activities in the quality assurance
3,24,28,31-52and quality control sectors.
Quality Assurance Measures
Cytopathology is a practice of medicine and represents a medical consultation, in
both gynecologic and nongynecologic anatomic sites. The basic principles of
quality assurance apply to all types of cytologic specimens.
The following represents several minimum quality assurance stipulations to which
most cytopathologists will probably agree.
Laboratory Directors
The laboratory should be directed by a legally quali1ed physician with a specialist
quali1cation in pathology, including special training and expertise in
cytopathology. In a case in which the current laboratory director or co-director
(associate cytopathologist) does not have board quali1cation in pathology but has
had special training in cytopathology, this situation may be approved under a
“grandfather clause.” The director or designated medical professional is responsible
for proper performance and reporting of all tests done in the cytopathology
laboratory. The director or designated cytopathologist should be physically present
in the laboratory to direct the sta0, be available for consultations, review all
reactive and abnormal gynecologic cytology samples, review 1ne needle aspirationsamples, and review all nongynecologic samples. In addition, a supervisor or senior
cytotechnologist should be assigned to review 10% of the negative cases, including
high-risk cases as designated by cytologic 1ndings, clinical 1ndings, and patient
histories. This procedure should help to detect any discrepancies in interpretation
that may occur prior to issuing the final cytologic report.
In addition the director should develop a quality assurance plan, a manual of
laboratory policies/procedures, and ensure the written policies and procedures
reDect actual laboratory practice. Issues and problems identi1ed through the
quality management process need to be addressed and resolved. Gathering of
laboratory statistics is best accomplished by collection of monthly reports during
quality assurance meetings presided over by the director.
Cytotechnologists
Cytotechnologists should meet one of the following requirements:
(1) Be certified as a cytotechnologist by either the American Society of Clinical
Pathologists or the US Department of Health, Education and Welfare; or
(2) Previously have been admitted to the practice of cytotechnology by existing
regulations under a grandfather clause.
Physical Laboratory Facilities
The laboratory should be clean, well lighted, adequately ventilated, and
functionally arranged so as to minimize problems in specimen handling,
evaluation, and reporting. The area for specimen preparation and handling should
be separate from the area where specimens are evaluated and reported.
Formaldehyde and xylene (if in use) should be carefully monitored due to the
possible presence of hazardous vapor concentrations.
Safety Precautions
Laboratory personnel must be protected against hazards (chemical, electric, 1re,
infections, or others) by using well-ventilated hoods and biologic safety hoods for
handling potentially infectious material. Fire precautions should be posted and
tested. Each employee should participate in 1re drills and should know the location
of the 1re extinguisher, blankets, emergency 1re alarm, and exits. Safety shower,
eye wash stations, and procedures to follow in case of chemical spills or splash to
the body should be posted and readily visible in the laboratory.
Equipment
An adequate number of binocular microscopes of good quality and proper working
order must be available. Laboratory instruments and equipment should be underperiodic maintenance to monitor and ensure malfunctions do not adversely a0ect
analytical results. A sample of slides from slide preparation instruments including
liquid-based technology and cytocentrifuge or 1ltration methods should be
routinely reviewed microscopically for technical acceptability.
Specimen Collection
Cytologic specimens should be accepted and examined only if requested by a
licensed medical practitioner and collected in accordance with instructions
regarding recommended collection techniques. The cytopathology laboratory
should inform the originator of the sample if the specimens are “unsatisfactory”
and detail adequacy quali1ers such as presence or absence of a transformation
29,53-56zone component or obscuring factors in “satisfactory samples.”
Preparation, Fixation, and Staining Procedures
The specimens must be identi1ed with the patient’s name and/or a unique
identi1er and must be accompanied by a requisition form with the requesting
physician’s name, address, date of specimen collection, specimen source, and
appropriate clinical information about the patient. When the specimen arrives in
the laboratory the laboratory sta0 a2 x an accession number or bar code label on
each slide for further identi1cation. The laboratory should have written criteria for
rejecting specimens. Fixation while the specimen is still wet is recommended for
conventional cell samples and rinsing of spatula and brush in preservative solution
(kit provided by manufacturers) for liquid-based specimens. The Papanicolaou
staining procedure is strongly suggested for most cytologic samples, unless
additional staining procedures are warranted. Staining solutions and chemicals
used in the cytopathology laboratory should be labeled with the time of
preparation, purchase, or both. Staining solutions should be 1ltered regularly to
avoid contamination and should be covered when not in use. E0ective measures to
prevent cross-contamination between gynecologic and nongynecologic specimens
during the staining process must be used. Separate runs followed by 1ltration or
changing of solutions or a separate staining setup is recommended.
Slide Evaluation Workload
Regulations as to the number of specimens a cytotechnologist may evaluate in a
24-hour period are currently set at 100 slides per an 8-hour day. This regulation
may not do justice to the various conditions that inDuence the quality of the slide
evaluation performance. The percentage of atypical cases evaluated versus the
percentage of negative cases in varying populations as well as screening of
nongynecologic specimens should be considered when workloads are established.
This regulation ensures that the number and type of cytologic samples evaluated do
not, through fatigue, adversely a0ect the cytotechnologist’s performance. Someslides are easier and less fatiguing to evaluate and some cytotechnologists are more
experienced than others. Other activities in which the cytotechnologist participates,
such as participation on the 1ne needle aspiration service and quality control
activities, should appropriately reduce their workload in the evaluation of cell
samples. The interpretation of the cytotechnologist should become a permanent
record and available for future review.
Cytologic Terminology
The vaginal/ectocervical/endocervical cytology sample should be interpreted
56preferably by using the Bethesda System. The nongynecologic material should be
interpreted in medical terms, i.e., conform and correspond to diagnostic reporting
systems in histopathology.
Laboratory Records, Logs, and Files
Each specimen should be recorded and a sequential accession number assigned
together with the name of the patient and the originator of the sample. Test records
must be retained for at least 2 years. The negative gynecologic cell samples should
be retained on 1le for a minimum of 5 years and negative 1ne needle aspirates for
10 years or inde1nitely if they exhibit abnormal features. The modern
cytopathology laboratory should use a computerized 1le system. Such a system
permits laboratory professionals to have information on all previous cytologic or
histologic reports on a given patient available when the new cell sample is being
evaluated. Modern computerized data collection and retrieval systems are also
essential for continuing quality control and assurance mechanisms. A record of
workload and diagnostic performance should be maintained as a part of the
personnel data file for each cytotechnologist.
Internal Quality Assurance Mechanisms
CLIA 88 Update
The standard requirements of CLIA 88 a0ecting the cytopathology laboratory in the
57United States have been published in the federal register. Recent updates of CLIA
88 have added more rules and regulations to the already highly regulated 1eld of
58gynecologic cytology. In addition to the following:
1. Prospective 10% review of negative gynecologic cell samples including high-risk
cases may be performed, prior to reporting patient results, by the pathologist,
supervisor, or cytotechnologist who has had 3 years of continuous cytology
experience within the past 10-year period.
2. Retrospective re-evaluation for current high-grade squamous intraepitheliallesions (HSIL) or cancer cases with a review of negative specimens obtained within
the previous 5 years should be performed when clinically significant discrepancies
are found that will affect current patient care; notification of physician and an
amended report are required.
3. Cytology/histology correlation for gynecologic cases with a diagnosis of HSIL,
adenocarcinoma, or other malignant neoplasm should be carried out with causes
of discrepancies, such as sampling or diagnostic errors on biopsy or cytologic cell
samples, documented.
new rules require:
1. An evaluation of the case reviews of each individual against the laboratory’s
overall statistical values, documentation of discrepancies including reasons for
deviation, and, if appropriate, corrective action taken.
2. A workload limit based on individual’s performance, every 6 months, based on
review of 10% negative cases and comparison of individual’s interpretation with
the pathologist should be established. The maximum workload limit is 100 slides
in 24 hours. Pathologists who perform primary screening are not required to
include tissue pathology slides and previously examined cytology slides in the
100slide workload limit.
3. The pathologist should confirm all nongynecologic cases and interpret each
gynecologic slide as reactive or with any abnormality, including atypical
squamous cells of undetermined significance (ASCUS), atypical glandular cells
(AGC), low-grade squamous intraepithelial lesion (LSIL), high-grade squamous
intraepithelial lesion, and carcinoma. The report needs to be signed by the
pathologist to reflect review or if a computer report is generated it must reflect an
electronic signature authorized by the pathologist.
4. Reports with narrative descriptive nomenclature for all results are
recommended. Corrected reports must include the basis for correction.
5. Records of initial examinations and all rescreening results must be documented.
6. Annual statistics should include total gynecologic and nongynecologic cases, by
specimen type, and diagnosis including unsatisfactory, cytology/histology
correlation discrepancies, and cases reclassified from normal to LSIL or higher.
7. When performing evaluations using automated and semiautomated screening
devices, the laboratory must follow manufacturers’ instructions for preanalytic,
analytic, and postanalytic phases of testing, as applicable, and meet the applicable
requirements.
8. Enrollment for proficiency testing (PT) as required by Centers for Medicare and
Medicaid Services. For more details see section on External Quality Assurance
Mechanisms.
Rapid Re-evaluation
Rapid rescreening (RR) of cervical smears for internal quality control has been
59-62advocated by a number of authors. In a meta-analysis of 14 studies by Arbyn
61and Schenck, RR was a more e0ective quality control method than full
rescreening of a 10% random sample. Although RR has received great support as
the quality control method of choice in some countries, the cost-e0ectiveness of its
potential advantage is still unknown.
Computer-Assisted Quality Assurance Mechanisms
With the approval by the Food and Drug Administration (FDA) of two automated
instruments for quality assurance rescreening of gynecologic cytology, namely, the
Focal Point System by Becton Dickinson/TriPath and the ThinPrep Imaging System
(TIS) by Cytyc Corporation, automated screening of gynecologic cytologic samples
for quality assurance has become a practical reality.
Detailed proposed speci1cations for automated instruments have been
63published, and reviews of the instruments and the concepts can be found in
Chapter 34, Automation of Pap Smears, and in the Compendium on the
64Computerized Cytology and Histology Laboratory and in the Compendium on
Quality Assurance, Pro ciency Testing and Workload Limitations in Clinical
65Cytology.
The development of automated cytologic devices had to pass two milestones: the
application of the system as a quality assurance or quality control instrument, and
the application of automated devices for primary screening of gynecologic cell
66,67samples.
The AutoPap System, now named Focal Point System, showed superior sensitivity
and specificity for detection of abnormal slides with ASCUS and more severe lesions
68 69-71when compared to current practice. Other investigators had similar results.
The Focal Point System is a more e0ective way of detecting errors within a
laboratory and reduces the false-negative rate (FNR) by greater than 25%
72according to Renshaw et al.
The ThinPrep Imaging System was statistically more sensitive than manual
evaluation for detection of ASCUS or more severe lesions and statistically
73 74equivalent for LSIL and HSIL diagnoses. According to Biscotti et al. the
sensitivity of this system equals or exceeds the sensitivity of manual screening
without adversely a0ecting speci1city. Signi1cant increase in SIL detection was75 76demonstrated with the TIS by Dziura et al. A recent study by Bolger et al.
showed sensitivity and speci1city of the imager equivalent to that of primary
manual screening.
These devices may improve the rate of false-negative evaluations—but at a price.
In other words, in the quality control mode the question is not whether the use of
either instrument may be harmful to the patient but whether the cost–bene1t ratio
is such that supplemental funds will be made available by third-party payers to
support this additional quality control procedure. Currently several third-party
payers have reimbursed for these procedures and current procedural terminology
(CPT) codes are available for billing purposes. For additional information see
Chapter 34.
Before automated devices can be e0ectively applied in primary screening, one
will have to consider other major factors that preclude successful reduction of
prevalence and incidence of gynecologic precancerous and malignant lesions. To
obtain maximum results for a population-screening program, we need to educate
women to obtain periodic gynecologic examinations, to teach medical personnel to
take and 1x the specimens appropriately, to make sure laboratory personnel follow
proper staining procedures, and to ensure the patient will make herself readily
available for required repeat cytologic examinations, colposcopy, and/or biopsies.
After a certain time of intensive cytologic evaluation, prevalent cancers and
highgrade precursors will have disappeared (the “sweeping” e0ect of an e0ective
population-screening program). What remains are incident low-grade precursors
and “interval” cancers and “interval” high-grade precursors that in fact are missed
positives (i.e., false-negatives), if one does not believe in the very rapidly growing
malignant lesion.
We should also re-emphasize to cytotechnologists that their services are urgently
needed in the future, even with approval of automated devices for routine
application, because the systems either work interactively under cytotechnologic
and cytopathologic guidance or produce alarm messages on selected cases that in
fact do not contain atypia (false-positives), which will take extensive e0ort and
time by a human re-evaluator to locate and override the alarm message.
The development of a fully automated diagnostic cytology and histology system,
i.e., complete without professional interaction and involvement, was and remains a
daydream that is neither feasible nor desirable. However, interactive systems as
aids in quality assurance mechanisms and improvement of productivity are
constructive developments and major technologic accomplishments.
External Quality Assurance Mechanisms
External quality assurance mechanisms with peer review by professional
organizations or by state or federal governmental bodies are currently beingimplemented. The Center for Medicare and Medicaid Services (CMS) has approved
two pro1ciency tests by the American Society for Clinical Pathology (ASCP) and
College of American Pathologists (CAP). Some states, e.g., New York and Maryland,
have had a testing program for cytopathology laboratories enacted and operational
42,77for many years. Any external program will be welcomed by the high-quality
laboratory, but there may be problems in the funding and execution of an
unbiased, objective, and reproducible testing
19,34,38,39,44,46,47,51,78-80,82system. Members of the Cytopathology
Educational and Technology Consortium have recommended modi1cations of the
rules regarding proficiency testing including testing interval, utilizing validated and
81monitored slides, and changing the grading system.
Continuing Education Practices
The laboratory director should conduct continuing educational activities within the
laboratory; provide up-to-date reference materials, such as cytopathology
textbooks, compendia on clinical cytology, cytologic journals, visual image
teaching slide sets, cytology websites, and CDs. The sta0 should be encouraged to
participate in ongoing educational events, such as local, regional, national, and
international cytology meetings and tutorials.
Concluding Remarks
The described quality assurance stipulations represent minimum quality assurance
measures to which most laboratories adhere. In the United States laboratories are
also governed by the described standard requirements of CLIA 88. External testing
programs are a welcome component of a laboratory quality assurance but there
may be problems in the execution of an unbiased, objective, and reproducible
evaluation system. A number of professional organizations are working with the
federal government in the United States to improve the current CLIA 88 cytology
pro1ciency testing. Unacceptable errors do occur in the cytology laboratory.
Automated systems and molecular HPV testing are available and may help to
reduce the false-negative rate of the Pap test. Continuing education practices and a
creative learning environment for the cytotechnologist and cytopathologist are
necessary to improve the diagnostic results for the patient.
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Chapter 5
Evaluation of the Sample in Smears and
LiquidBased Preparations
Marluce Bibbo, Joseph F. Nasuti
Contents
Introduction
Cervicovaginal Cytology
Specimen Type
Patient Identification
Clinical Information
Microscopic Evaluation
Nongynecologic Cytology
Specimen Type
Specimen Cross-Contamination
Specimen Mishandling
Concluding Remarks
Introduction
Several factors play a role in the evaluation of the cellular sample. The method of
sample collection and xation, the laboratory procedure to process the sample, and
the integration of the morphologic features observed in the sample with the clinical
information may affect the quality of interpretation/diagnosis reached.
The ultimate goal in specimen processing is to preserve, as much as possible, in
vitro or in vivo aspects of the sample obtained. This includes the original size,
shape, and texture of the cytoplasmic and nuclear components present. Among the
desired results of a well-preserved cellular specimen is the ability to accurately
assess quantitative and semiquantitative criteria including hyperchromasia,
nuclear/cytoplasmic ratio, and chromatin patterns. The recognition of ne nuclear
details such as grooves, notches, inclusions, and pseudo-inclusions are essential for
the de nitive diagnosis of certain subtypes of thyroid, breast, urinary, and
softtissue tumors with concomitant prognostic implications. Equally important is
maximizing the number of true tissue fragments. In contrast to the screening nature




of the Pap test, where an interpretation of the sample is followed by a biopsy to
establish the nal diagnosis, ne-needle aspiration (FNA) is used to obtain a
de nitive diagnosis and as more targeted therapies are developed, expectations on
1the diagnostic performance of cytopathologists will increase.
Cervicovaginal Cytology
Specimen Type
Two types of specimen are available for cervicovaginal cytology: smear for the
conventional Pap (CP) and liquid-based preparation (LBP), which emerged as an
2alternative sampling and preparation method in the 1990s. In the United States
ThinPrep and SurePath are the rst two LBP approved by the Food and Drug
Administration. In several countries manual methods of LBP such as DNA Citoliq,
Cyto-Screen, PapSpin, and Autocyte Manual are available. The overall quality of
most LBP is surprisingly good because cell preservation is enhanced in contrast to
conventional smears, which may have thick and thin areas or air-drying
3,4artifacts.
Patient Identification
It is important to match the name of the patients on the smears or LBP vials with
the names in the requisition form, to prevent mix-ups. The use of an automated
processing system improves the accuracy of patient identi cation and ensures
patient chain of custody with bar-coded labels and etched numbers on the glass
slides.
Clinical Information
Clinical information needs to be integrated with the sample interpretation. Minimal
information required is patient age, date of last menstrual period, history of
previous abnormal Paps, the latter being more often available in the laboratory
computer system. Age and menstrual status are particularly important for the
interpretation of endometrial cells. A history of previous malignancies of the female
genital tract will alert the laboratory of the possibility of a recurrent disease or
changes secondary to treatment.
Microscopic Evaluation
In the United States most laboratories follow the 2001 Bethesda System for
5reporting cervicovaginal cytology. Other countries utilize national reporting
systems. In most laboratory settings the evaluation of gynecologic specimens is
performed by both cytotechnologists, who screen all samples, and cytopathogists
who are responsible for the final interpretation of all abnormal cases.
Sample Adequacy Assessment





?

Sample Adequacy Assessment
Over the years there has been considerable debate about what constitutes an
adequate sample. In the 2001 Bethesda conference this issue was addressed and
speci c guidelines for assessing sample adequacy emerged. Applying the minimum
squamous cellularity criteria a cellularity of 10,000 to 12,000 squamous cells is
considered adequate for conventional Paps. There is no need to count squamous
cells but rather estimate the cellularity based on density cell patterns in reference
images. In samples with cytolysis, atrophy, and cell clustering, when cell adequacy
is borderline, professional judgment and hierarchical review is recommended. The
presence or absence of endocervical cells/transformation zone component does not
a: ect sample adequacy but should be mentioned as a quality factor. Specimens
with more than 75% of squamous cells obscured should be termed unsatisfactory
assuming that no abnormal cells are present. For liquid-based preparations 5,000
squamous cells is considered adequate cellularity and estimates are reached by
counting the number of cells in ten high-power elds across the main diameter of
the preparation. Using a 40× objective and FN20 eyepiece the number of cells in
each eld should be 3 for the ThinPrep and 7 for the SurePath for adequate
6cellularity.
Unsatisfactory sample rates are variable. Centrifugation LBP have a lower rate of
unsatisfactory samples than ltration-based preparations because in the latter red
blood cells or in ammatory cells may plug the lter pores. Reprocessing is
7,8recommended to lower the unsatisfactory rate.
A meta-analysis of prospective studies comparing cytologic diagnosis and sample
adequacy showed LBP improved sample adequacy and equal or superior results in
diagnosing premalignant cervical lesions when compared with conventional
9Papanicolaou test. In a comparison study of automated versus manual LBP few
di: erences were found in the sample adequacy and cellular presentation: less
uniform distribution of cells and more artifacts were noted in the manual methods
such as DNA Citoliq and AutoCyte Prep compared with the ThinPrep method;
3sample adequacy and overall quality of all LBP were surprisingly good.
Interpretation
Detailed criteria for the interpretation of gynecologic samples are described in
Chapters 6–11 of this book. The following highlights some observations in
6,10-14LBP.
For LBP xed in ethanol-based xative the interpretation is closer to
conventional smears. When LBP are concentrated in smaller areas, with
threedimensional cell clusters above the plane of squamous cells, focusing may be
required more often.G
?


?
There are many similarities in the evaluation of LBP and conventional smears but
there are differences:
• Cell size;
• Pattern; and
• Background.
Because samples are collected in a liquid-based fixative solution, the presentation
of the cellular material appears di: erent, concentrated and evenly distributed (Fig.
5.1). Cells are smaller, dispersed, and single, although cell clusters are also present.
Cells in solution tend to round up and wet xation enhances cytoplasm and nuclear
morphology. Hyperchromasia as observed in abnormal cells on conventional
smears is not always present in liquid-based preparations, especially in
methanolbased xatives, and lack of hyperchromasia may render the interpretation of
highgrade squamous intraepithelial lesion more di cult. Variations in nuclear size and
shape and especially appreciation of nuclear contours play an important role in the
evaluation on LBP.
Fig. 5.1 Normal squamous and endocervical cells appear evenly distributed.
SurePath (Papanicolaou x LP).
The background is generally clean and debris more clumped. Blood, mucus, and
in ammation are rarely obscuring, and in ammatory cells tend to cling to
epithelial cells. Tumor diathesis has a “ratty” appearance but this type of
background can also be observed with cytolytic or inflammatory patterns.
Some cytologic entities have key features in LBP:
Key features of atrophy
• Fewer bare nuclei;• Flat sheets of parabasal cells; and
• Preserved nuclear polarity (Fig. 5.2).
Key features of trichomonads
• Smaller;
• Difficult to visualize; and
• More visible nuclei, eosinophilic granules, or flagella.
Key features of lymphocytic cervicitis
• Lymphoid cells in clusters; and
• Confused with endometrial cells (Fig. 5.3).
Key features of repair
• Cohesive cell groups;
• More rounded cytoplasmic borders;
• Less streaming; and
• Prominent nucleoli.
Key features of metaplastic cells
• Often single, smaller, and rounder;
• Confused with HSIL; and
• Paler chromatin pattern.
Key features of low-grade squamous intraepithelial lesions (LSIL)
• Large cell size;
• Koilocytosis;
• Multinucleation; and
• Nuclei show decreased hyperchromasia (Fig. 5.4).
Key features of high-grade squamous intraepithelial lesion (HSIL)
• Fewer abnormal cells;
• Single cells more common than sheets;
• Syncytial aggregates;
• High nuclear cytoplasmic ratio; and• Nuclear membrane irregularities (Figs. 5.5, 5.6).
Key features of squamous cell carcinoma
• Single cells and syncytial aggregates;
• Pleomorphic and less hyperchromatic nuclei;
• Rare nucleoli; and
• Diathesis surround cells or cling to malignant cells (Figs. 5.7, 5.8).
Key features of endocervical adenocarcinoma in situ
• Strips of cells with pseudostratification;
• Nuclear crowding; and
• Subtle appearance of feathering and rosettes.
Key features of endometrial cells
• Tight or lose cell clusters;
• Vacuolated cytoplasm;
• Enhanced nuclear detail; and
• Confused with low-grade endometrial adenocarcinoma (Fig. 5.9).
Key features of endometrial adenocarcinoma
• Papillary configurations;
• 3D groups; and
• Less prominent tumor diathesis (Fig. 5.10).Fig. 5.2 Flat sheet of parabasal cells with preserved nuclear polarity in
atrophy. ThinPrep (Papanicolaou x MP).
Fig. 5.3 Lymphoid cells appear in aggregates in chronic lymphocytic cervicitis.
ThinPrep (Papanicolaou x MP).
Fig. 5.4 Koilocytes showing perinuclear cavitation and binucleation in LSIL.
ThinPrep (Papanicolaou x MP).Fig. 5.5 Single cells with high n/c ratio and hyperchromasia in HSIL. SurePath
(Papanicolaou x MP).
Fig. 5.6 Immature cells showing irregular nuclear outlines in HSIL. ThinPrep
(Papanicolaou x MP).
Fig. 5.7 Syncytium of malignant cells in squamous cell carcinoma withvariation in nuclear size and shape, irregular chromatin distribution and clinging
diathesis. ThinPrep (Papanicolaou x HP).
Fig. 5.8 Pleomorphic cells and clean background in squamous cell carcinoma.
SurePath (Papanicolaou x HP).
Fig. 5.9 Normal endometrial cells in tight cluster and enhanced nuclear detail.
ThinPrep (Papanicolaou x MP).



?

Fig. 5.10 Cluster of malignant cells and granular tumor diathesis in endometrial
adenocarcinoma. ThinPrep (Papanicolaou x MP).
Nongynecologic Cytology
Specimen Type
The three major processing modalities for nongynecologic specimens consist of
direct smear, filtration (Millipore, SurePath, and ThinPrep) and
cytocentrifugationbased preparations (Cytospin). All three techniques share the capacity to archive a
portion of the specimen for the application of special and immunocytochemical
stains. Each modality subjects the cellular material to di: erent degrees of physical
forces and chemical in uences. This will result in certain artifact pro les that can
a: ect cytomorphologic interpretation. True tissue fragments with architectural
features similar to those of histologic specimens are present in quality direct smears
(Fig. 5.11). The use of Rapid Pap and/or Di Quik stains in conjunction with the
direct smears technique allows for on-site cellular adequacy of FNA. The
importance of on-site specimen evaluation cannot be overstated since it has been
15shown conclusively to signi cantly reduce the inadequacy rate. The preservation
of diagnostically important true tissue fragments and the three-dimensional
microtopography of the Millipore cellulose lter produces an increased depth of
eld and subsequent exquisite cytologic detail (Fig. 5.12). The ThinPrep technique
consistently produces the truest monolayer, thus minimizing the obscuring e: ects
of background elements and cellular clumping (Fig. 5.13). SurePath and Cytospin
preparations also present excellent cytomorphology (Figs. 5.14, 5.15).
Fig. 5.11 Follicular architecture in follicular thyroid adenoma FNA. Direct
smear (Diff Quik x MP).
Fig. 5.12 Nuclear grooves, chromatin clearing, margination and
threedimensional e: ect in papillary thyroid carcinoma FNA. Millipore lter
(Papanicolaou x HP).?
G

Fig. 5.13 True monolayer of benign urothelial cells in clear background in
voided urine. ThinPrep (Papanicolaou x MP).
Fig. 5.14 True tissue fragment of papillary urothelial carcinoma in voided
urine. SurePath (Papanicolaou x MP).
Fig. 5.15 Metastatic ovarian carcinoma in peritoneal uid showing clean
background and cells with good nuclear detail. Cytospin (Papanicolaou x MP).
When su cient cellularity and technical support are available cell blocks can
provide much in the way of additional diagnostic information. Included among the
possibilities are architectural and staining properties that approach what is seen in
conventional para n-embedded hematoxylin and eosin (H and E) stained surgical
biopsies. In this manner cell blocks often provide an immeasurable level of comfort
to many pathologists more accustomed to conventional histologic diagnoses. In
particular types of FNA specimens such as salivary gland cystic lesions and thyroid
nodules conventionally prepared cells blocks may provide key information to clinch
a de%nitive diagnosis. A common example is in distinguishing chronic sialadenitis
G
G

G


from Warthin's tumor in which characteristic large papillary oncocytic
lymphoepithelial tissue fragments are best appreciated and often only appear in the
16cell block pellet derived from needle rinses (Fig. 5.16). Similarly, cell blocks have
been reported to be extremely helpful in discerning malignant papillary tissue
fragments containing brovascular cores from benign papillary tissue fragments
17that for the most part lack brovascular cores. Also the characteristic Orphan
Annie-eyed nuclear chromatin pattern (felt by some to be a useful reproducible
histologic artifact) is best seen in cytology specimens that have been conventionally
18-21processed into formalin- xed para n-embedded cell blocks. In a study by
Sanchez and Selvaggi additional morphologic information derived from cell blocks
22was found to be diagnostically contributory in 31% thyroid FNAs. In addition to its
unique ability to provide essential histologic clues cell blocks o: er in theory and
often in practice the opportunity to perform the same battery of ancillary studies as
conventionally processed surgical biopsy tissue including molecular analysis and
23,24electron microscopy.
Fig. 5.16 Warthin's tumor in parotid FNA. Cell block (H&E x MP).
A few published reports document the fact that immunocytochemistry can be
performed successfully on Cytospin, ThinPrep, Millipore lter, and direct smear
25-28slides. Options are somewhat limited in terms of the number and types of
antibodies available due to the typically sparse and delicate nature of the cellular
material present in these preparations. Many articles, on the other hand, describe
the diagnostic usefulness of a variety of immunohistochemical stains on cell block
29-38material. In particular, para n-embedded cell blocks have proven e cacious
by helping to resolve benign reactive lesions from both primary and metastatic
tumors (Fig. 5.17). Such common dilemmas may often require a panel of
immunohistochemical stains. See Chapter 35 for additional information.

?


?

G
?
Fig. 5.17 Metastatic lung adenocarcinoma in pleural uid. Cell block (TTF1
immunostain positivity x MP).
Comparison of Nongynecologic Processing Techniques
There are many articles found in the contemporary literature which compare the
e cacy of the processing modalities based on costs, cellular yield, unsatisfactory
rates, artifacts, and diagnostic accuracy. Most deal with speci c types of specimens
such as urine, pleural uid, pancreatic/biliary tract, soft tissue, breast, and thyroid
FNA. The recommendations of the various authors di: er somewhat in favoring one
processing technique over the other. A thorough review of the relevant literature,
however, leads one to the conclusion that direct smears, cytocentrifugation, and
ltration techniques are worthy of routine use with comparable diagnostic and cost
parameters for most nongynecologic specimens. In the end the decision to utilize
one or more of the processing techniques depends upon weighting the service
demands against the nancial and labor resources available in a particular
laboratory.
The following highlights some observations in the literature concerning these
processing modalities:
Cellular yield was found to be superior by the ThinPrep method which retained
39three times as many cells as cytocentrifugation. There were no statistically
signi cant di: erences in unsatisfactory rates, sensitivity, speci city, or positive
predictive value in both FNA and body cavity uid specimens processed by
40ThinPrep and direct smears methods. Overall technical quality was reported to be
improved by ThinPrep processing when compared to direct smears on split FNA
41,42specimens due to cleaner background and better monolayer formation. Some
authors, however, advised caution to avoid diagnostic errors when interpreting
ThinPrep slides due to the increased incidence of following cytologic artifacts (Table
5.1): disruption of tissue fragments, formation of cell clusters, aggregation of?


G
?
lymphocytes, cellular shrinkage, attenuation of nuclear details, and exaggeration
42nucleolar prominence. In comparison to SurePath processed specimens ThinPrep
slides demonstrated increased cellular shrinkage, attening, and fragmentation of
large cellular sheets and nuclear chromatin patterns were reportedly more di cult
43to evaluate. SurePath slides were also found to contain larger branched
three43dimensional tissue fragments.
Table 5.1 Artifacts more commonly seen with nongynecologic LBP
Cellularity Nuclear Background
Cellular shrinkage Increased naked nuclei Loss of adipose tissue,
stroma, mucin, and
colloid
Disruption of tissue Decreased nuclear Colloid that appears as
fragments chromatin details dense droplets
Flattening and Attenuation of nuclear Aggregation of
fragmentation of large grooves and pseudo lymphocytes
cellular sheets inclusions
Formation of cell clusters Exaggerated nucleolar
prominence
Artificially increased
single epithelial cells
Separate studies involving FNAs of thyroid nodules, breast and salivary gland
lesions, and pancreatic and soft-tissue tumors reported somewhat con icting results
in terms of unsatisfactory rates, quality of nuclear details, diagnostic accuracy, and
relative prevalence of artifacts when ThinPrep-processed slides were compared to
44-53direct smears. Among the artifacts attributed to the ThinPrep method for FNA
specimens are the inability to assess cellularity of individual passes;
diminished/distorted extracellular and stromal elements such as mucin, stroma,
adipose tissue, and colloid that also appeared as dense droplets (Fig. 5.18);
crowded tight tissue clusters with loss of cellular preservation; increased cellular
and tissue fragment disruption; arti cially increased single epithelial cells;
numerous naked nuclei; pronounced nucleoli; decreased nuclear details; and
attenuation of nuclear grooves and pseudo-inclusions. Authors did note that
signi cantly more conventional smears were limited by air-drying artifact.
Additionally, ThinPrep slides had greater cellularity, improved nuclear detail, and
more easily recognizable myoepithelial cells relative to direct smears (Fig. 5.19).


?

An added bene t of greater suitability for immunoperoxidase staining was also
48documented for ThinPrep processing.
Fig. 5.18 D e n s e colloid droplet in thyroid FNA in goiter. ThinPrep
(Papanicolaou x MP).
Fig. 5.19 Benign ductal cells in association with myoepithelial cells. ThinPrep
(Papanicolaou x MP).
Speci c examples in which artifact-associated ThinPrep-processed FNA slides
compromised diagnostic accuracy were cited: Four out of 21 broadenomas were
correctly diagnosed on ThinPrep-processed breast FNA due to arti cially increased
47single ductal epithelial cells and a lack of background stroma. Three benign
pancreatic lesions were interpreted as atypical/suspicious due to presence of single
atypical cells with distinct nucleoli, and one mucinous pancreatic neoplasm was
51incorrectly diagnosed due to lack of background mucin.
The diagnostic value for pleural uid specimens of ThinPrep versus Cytospin wasG
G

compared by examining a large spectrum of cytologic features that would
distinguish malignant mesothelioma (e.g., peripheral cytoplasmic skirt, bubbly
cytoplasm, cyanophilic cytoplasm, and scalloped border of cell balls) from
pulmonary adenocarcinoma (e.g., two-cell population, inspissated cytoplasmic
material, cytoplasmic vacuole, angulated and indented nuclei, and smooth border
54of cell balls). Based on statistical analysis most cytologic features examined in
this study can be seen in both preparation techniques. The authors therefore
concluded that ThinPrep preparation of pleural e: usions does not appear to
provide additional diagnostic value when compared to Cytospin preparation.
A comparative analysis of urine specimens processed by both ThinPrep and
Cytospin techniques found that the cytomorphology and screening time were
55comparable with both techniques. However, in cases of transitional cell
carcinoma Cytospin was superior in terms of better preservation of architectural
features and produced less arti cial empty spaces and air-drying artifact (Fig.
5.20). A contrasting study compared cytocentrifugation and ThinPrep techniques
for cost e ciency including wages, investments in instrumentation and
consumables, overall cytomorphologic quality, and suitability for molecular
56studies. Based on their examination of 224 split urine samples the authors
reported that cytocentrifugation with disposable chambers resulted in a global
cytomorphologic quality superior to that of ThinPrep. In addition utilizing a
200specimen per month calculation a greater cost e ciency was achieved with
cytocentrifugation than with ThinPrep. In a similar subsequent study the same
authors compared cytomorphologic quality of urine specimens prepared by
ThinPrep, direct smears, Cytocentrifugation, and ThinPrep and Millipore
57filtration.
Fig. 5.20 High-grade urothelial carcinoma. Cytospin (Papanicolaou x MP).
The conclusions of the study were as follows:


?


• Direct smears show good overall results;
• Cytocentrifugation with reusable chambers should be avoided;
• Cytocentrifugation with disposable chambers (Cytofunnels or Megafunnel
chambers) gives excellent results; and
• Millipore filtration followed by blotting should be avoided due to its poor global
quality.
Contrasting results were reported when voided urine specimens processed via
58Millipore lter cytosieve technique were examined. True tissue fragments
consisting of either at sheets or three-dimensional structures were signi cantly
more common in voided urine specimens with follow-up biopsies of TCC than in
negative biopsies. A considerably lower rate of tissue fragments was reported when
voided urine specimens were processed utilizing cytocentrifugation with no
statistically signi cant correlation found between the incidence of cell groups in
59voided urine specimens and the presence of TCC in follow-up biopsies. It has
been suggested that the reason for these discrepant ndings is due to the stronger,
more disruptive centripetal force imposed on true tissue fragments by
cytocentrifugation relative to the weaker, gentler forces of gravity and suction
58encountered by the Millipore filter cytosieve technique.
Specimen Cross-Contamination
The potentially catastrophic problem of cross contamination of cytology specimens
can occur with any processing modality or in any stage of the process from xation
to coverslipping (Fig. 5.21). Fortunately steps can be taken in the specimen
processing and staining to minimize its rate occurrence in cytopreparatory
60,61laboratories.
Fig. 5.21 Cross-contamination from ovarian carcinoma (see Fig.5.15) inpancreatic cyst FNA. Cell block (H&E x MP).
Specimen Processing
A. All test requisitions, specimen cups, tubes, and slides must be identified with
their own unique accession number before processing.
B. Laboratory technicians must carefully aspirate sample into pipette tip, making
sure no sample gets sucked into the pipette barrel. If this happens the pipette is
immediately removed from production. The barrel is cleaned with alcohol and
water, dried, and tested for contamination before being placed back into
production.
C. Pipette tips are changed for every sample.
D. All cytochambers and holders, if not disposable, are soaked for at least 30
minutes and then washed in the dishwasher and dried before being used the
following afternoon.
E. All staining solutions must be either filtered or changed after every staining run.
F. All microscopists shall inform the supervisor of suspected contamination.
Immediate corrective action shall follow with appropriate documentation of each
occurrence.
Specimen Staining
A. All stains and staining solutions must be checked, filtered, and changed daily
with documentation supporting this. Discard all water rinses after usage.
B. Special staining is to be performed in separate Coplin jars with all reagents
made fresh daily.
C. Hacker coverslipping xylene wells must be filtered after each use.
D. Known positive cases should be stained separately in Coplin jars or stained in
the regular staining dishes provided that the regular staining setup is then
discarded of all solutions.
E. Any suspected floater (atypical cell contaminant seen on a different focal plane
found on a slide) identified on a slide should be brought to the attention of the
supervisor. For nongynecologic specimens, it is recommended that additional slides
be made with the leftover sediment. Again no staining should continue until all
stains, dishes, and jars are either filtered or changed and cleaned.
F. If contamination is suspected and no residual specimen sediment is available,
consideration must be given to cancellation of the test. If the test is canceled a
phone call must be placed to the requesting clinician and a hard copy of the report
with appropriate explanation shall be issued. All inquires will be documented in
the reconciled report logbook.
It is also important to remember that the possibility of cross-contamination is not
limited to the cytopreparatory laboratory. This is particularly true when cell blocks
are processed along with surgical pathology tissue utilizing the same formalin baths
and automated processors. Contamination due to shedding of malignant cells and
tissue can occur from cell block to histology tissue block and vice versa. To minimize
this type of cross-contamination the following steps should be taken:
1. The histology laboratory should be notified when cell blocks are requested from
cytology specimens likely to shed tumor cells such as ascites fluid from a known
ovarian carcinoma patient (Fig. 5.21).
2. Cassettes for cell blocks should be placed in separate formalin baths from
histology tissue cassettes.
3. Cassettes for cell blocks whenever possible should have their own separate runs
in the automated processors without surgical pathology tissue cassettes.
Specimen Mishandling
Once errors in handling the specimens are detected, a root cause analysis should
take place to help identify what, how, and why the error happened. Understanding
why a mistake occurred is the key to develop e: ective quality control measures to
prevent it from occurring again. A review of skill-based activities, if appropriate, to
ensure appropriate level of hands-on training should be provided in addition to the
training development process to ensure adequate guidance. For errors related to
sample interpretation see Chapter 4, Diagnostic Quality Assurance in
Cytopathology.
Concluding Remarks
The parameters for evaluation of smears and liquid-based preparations have been
described in this chapter. As we have seen, several factors play an important role in
the evaluation of the cellular sample and for optimal results good xation and
processing techniques, availability of clinical information, and expertise in
interpretation are required. Criteria for interpretation of gynecologic and
nongynecologic samples are described in all chapters on diagnostic cytology in this
book but observations on liquid-based preparations were highlighted here to show
di: erences in cell size, pattern background, and artifacts. A comparison of
nongynecologic processing modalities and specimen cross-contamination as well as
procedures to prevent the problem were also presented. The use of adjunct?
techniques in diagnostic cytology is already an important component of the
specimen evaluation. In the future the diagnostic potential of cytology will increase
with development of new uorescent in situ hybridization (FISH) probes and more
assays to identify genetic alterations that serve as therapeutic targets in addition to
the use of microarrays, allowing a global and integrative approach to diagnosis.
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DIAGNOSTIC CYTOLOGYCHAPTER 6
The Bethesda System for Reporting Cervical
Cytology
Ritu Nayar, David C. Wilbur, Diane Solomon
Contents
The Bethesda System: Historical Perspective
The 2001 Bethesda System
Report Format
Specimen Adequacy
Bethesda 2001 Specimen Adequacy Categories
Squamous Cellularity
Quality Indicators
Management Guidelines
Impact on Laboratory Practice
General Categorization
Interpretation/Result
Negative for Intraepithelial Lesion or Malignancy (NILM)
Endometrial Cells
Epithelial Cell Abnormalities: Squamous Cell
Epithelial Cell Abnormalities: Glandular Cell
Educational Notes/Suggestions
Ancillary Testing
Automated Review
Interobserver Reproducibility in Cervical Cytology
The Bethesda System and Reporting Anal-Rectal Cytology
Concluding Remarks
The Bethesda System: Historical Perspective*
*
*
*
Terminology forms the basis for e ective communication between the laboratory
and clinician. The clinician is expected to provide relevant patient information to
the laboratory. It is the laboratory's responsibility to report results using
terminology that clearly conveys the diagnostic interpretation of the morphologic
/ndings. The use of a uniform diagnostic terminology facilitates communication by
establishing a common language that, in theory, does not vary signi/cantly from
cytologist to cytologist or laboratory to laboratory. However, terminology is not
static over time; rather, it evolves in parallel with increased understanding of the
pathogenesis and biology of disease. The framework, therefore, must be 4exible
enough to incorporate advances in scienti/c knowledge without creating undue
confusion or complexity.
In 1988, the National Cancer Institute sponsored an open workshop—including
cytotechnologists, pathologists, clinicians, and representatives of professional
organizations—to develop a uniform descriptive terminology for cervical/vaginal
cytologic interpretation. The format that emerged became known as The Bethesda
1System (TBS).
Approximately two years later, a second meeting was convened to critique and
re/ne the terminology based on experience with the use of the system in actual
laboratory practice. Minor modi/cations were incorporated into the 1991 Bethesda
2System that streamlined the terminology. In addition, an ad hoc committee
developed criteria for specimen adequacy and Bethesda interpretive categories,
culminating in the /rst TBS atlas that outlined and illustrated the morphologic
3features. By the mid- to late 1990s, there was a signi/cant penetration of the
Bethesda System into cytopathology practice with approximately 90% of
laboratories in the United States using the Bethesda terminology for reporting of
4cervical/vaginal cytology.
Among all the changes introduced by the implementation of TBS terminology
into practice, none was more controversial than the category of atypical squamous
cells (ASCUS). At that time, the majority of abnormal Pap tests reported annually
in the United States, approximately 2.5 million, were interpreted as ASCUS and
had highly variable management at considerable cost to the healthcare system.
Another 1.2 million were interpreted as low-grade squamous intraepithelial lesion
5(LSIL). In an e ort to determine the best management strategy (e ective as well
as cost-e ective) for women with these equivocal and low-grade abnormalities, the
National Cancer Institute (NCI) sponsored the ASCUS/LSIL Triage Study (ALTS),
6which was completed in 2001. This study, as detailed below, has allowed for a
data-driven approach to management of these prevalent cervical cytologic
abnormalities.
From its inception, the fundamental aim of the Bethesda System has been tocommunicate clinically relevant information from the laboratory to the patient's
healthcare provider, using uniform, reasonably reproducible terminology which
re4ects the most current understanding of the biology of cervical neoplasia.
Advances in the understanding of the biology of cervical cancer, results from
clinical trials, the introduction of liquid-based cytology, human papillomavirus
(HPV) testing, and automated screening devices for cervical cytology led to the
decision to convene the third Bethesda workshop in April 2001.
The 2001 Bethesda System
TBS 2001 Process
Approximately eight months prior to the workshop, nine forum groups consisting of
6 to 10 individuals with a breadth of expertise in the area of cervical cancer, were
organized under the sponsorship of the NCI to formulate draft recommendations.
Internet bulletin boards were open to the worldwide cytology community for six
months during the pre-conference process of review and discussion. Over 1000
comments were considered in revising the pre-workshop drafts. The 2001 Bethesda
workshop was co-sponsored by 44 international professional organizations and
attended by over 400 individuals, including pathologists, cytotechnologists,
gynecologists, attorneys, patient advocates, and other healthcare workers involved
in women's health initiatives. The revised draft recommendations were presented
by each forum group and after open discussions and voting by all participants, the
72001 Bethesda consensus terminology was finalized and published in 2002.
Following the Bethesda workshop, the American Society for Colposcopy and
Cervical Pathology (ASCCP) held a comparable consensus workshop on patient
management in September 2001. This was also preceded by an Internet discussion,
and resulted in the development of evidence-based management guidelines for
8abnormal cervical cytology corresponding to the 2001 Bethesda reporting format.
The ASCCP management guidelines were subsequently updated at a consensus
9conference held in September 2006.
After the initial publication of the Bethesda System 2001 terminology (Table
6.1), the NCI approached the American Society of Cytopathology (ASC) to
10collaborate on publication of the second edition of the Bethesda Atlas and the
11development of an accompanying Bethesda System educational website. Images
chosen for the atlas and website underwent an extensive selection/validation
process, and included classic as well as morphologically diF cult and “borderline”
images, illustrated on both conventional and liquid-based preparations. A subset of
images chosen for the Bethesda atlas were used to assess interobserver
reproducibility in gynecologic cytology—the details of this Bethesda interobserver
12reproducibility project are described below.Table 6.1 The 2001 Bethesda System
SPECIMEN TYPE
Indicate conventional smear vs liquid-based preparation vs other
ADEQUACY OF THE SPECIMEN
• Satisfactory for evaluation (describe presence or absence of
endocervical/transformation zone component and any other quality indicators,
e.g., partially obscuring blood, inflammation)
• Unsatisfactory for evaluation … (Specify reason)
– Specimen rejected/not processed (specify reason)
– Specimen processed and examined, but unsatisfactory for evaluation of
epithelial abnormality because of (specify reason)
GENERAL CATEGORIZATION (OPTIONAL)
• Negative for intraepithelial lesion or malignancy
• Other
• Epithelial cell abnormality: see Interpretation/Result (specify squamous or
glandular as appropriate)
INTERPRETATION/RESULT
Negative for intraepithelial lesion or malignancy
(when there is no cellular evidence of neoplasia, state this in the General
Categorization above and/or in the Interpretation/Result section of the report—
whether or not there are organisms or other non-neoplastic findings)
ORGANISMS
• Trichomonas vaginalis
• Fungal organisms morphologically consistent with Candida spp.
• Shift in flora suggestive of bacterial vaginosis• Bacteria morphologically
consistent with Actinomyces spp.
• Cellular changes associated with herpes simplex virus
OTHER NON-NEOPLASTIC FINDINGS (OPTIONAL TO REPORT; LIST NOT
INCLUSIVE)• Reactive cellular changes associated with:
– Inflammation (includes typical repair)
– Radiation
– Intrauterine contraceptive device (IUD)
• Glandular cells status posthysterectomy
• Atrophy
Other
• Endometrial cells (in a woman > 40 years of age) (specify if “negative for
squamous intraepithelial lesion”)
Epithelial cell abnormalities
SQUAMOUS CELL
• Atypical squamous cells
– of undetermined significance (ASC-US)
– cannot exclude HSIL (ASC-H)
• Low-grade squamous intraepithelial lesion (LSIL) (encompassing: HPV†/mild
dysplasia/CIN 1)
• High-grade squamous intraepithelial lesion (HSIL) (encompassing: moderate
and severe dysplasia, CIS/CIN 2 and CIN 3)
– with features suspicious for invasion
• Squamous cell carcinoma
GLANDULAR CELL
• Atypical
– endocervical cells (NOS or specify in comments)
– endometrial cells (NOS or specify in comments)
– glandular cells (NOS or specify in comments)
• Atypical
– Endocervical cells, favor neoplastic
– Glandular cells, favor neoplastic
• Endocervical adenocarcinoma in situ
• Adenocarcinoma
– endocervical
– endometrial– extrauterine adenocarcinoma
– not otherwise specified (NOS)
Other malignant neoplasms (specify)
ANCILLARY TESTING
Provide a brief description of the test method(s) and report result so that it is
easily understood by the clinician.
AUTOMATED REVIEW
If case is examined by automated device, specify device and result.
EDUCATIONAL NOTES AND SUGGESTIONS (OPTIONAL)
Suggestions should be concise and consistent with follow-up guidelines published
by professional organizations (references to relevant publications may be
included).
Report Format
The basic structure of TBS includes three elements, based on communication needs
germane, but not limited, to gynecologic cytology: (1) statement of specimen
adequacy, (2) general categorization, and (3) descriptive terminology. The
specimen type—conventional smear, liquid-based preparation, or other—should
also be stated in the report (Table 6.1).
Specimen Adequacy
Reporting of adequacy was an important quality assurance measure introduced by
the Bethesda System. The 1988 Bethesda System incorporated a classi/cation of
three categories of specimen adequacy—satisfactory, less than optimal, and
unsatisfactory—into the format of the report but did not outline speci/c
morphologic criteria for evaluation of adequacy. Participants at the 1991 Second
13,14Workshop, and others in the cytopathology community, voiced the need for
developing consensus guidelines. In response, following the second workshop, a
Criteria Committee formulated the de/nitions for adequacy based on a
combination of experience and review of an admittedly sparse scienti/c database.
Three categories—“satisfactory,” “satisfactory but limited by…,” and
“unsatisfactory”—based on estimates of overall squamous cellularity, assessment of
the transformation zone component, and the presence/extent of obscuring or
limiting factors, were suggested in an initial attempt to develop a more
15standardized approach to the evaluation of adequacy. It was emphasized that
the indicated percentages should be used as general ranges, not strict numerical*
cuto s and that patient-related clinical factors and previous cytologic /ndings
should always be taken into consideration.
Bethesda 2001 Specimen Adequacy Categories
In 2001, substantial changes were made to the adequacy component of TBS. The
previously used borderline adequacy category of “less than optimal”
(1988)/“satisfactory but limited by…” (1991) was deleted in order to provide the
clinician a clearer and more reproducible indication of the adequacy of the
16specimen. The classi/cation recommended in TBS 2001 is either as “satisfactory”
or “unsatisfactory”:
10• Satisfactory —Satisfactory for evaluation (describe presence or absence of
endocervical/transformation zone component and any other quality indicators,
e.g., partially obscuring blood, inflammation, etc.). For “satisfactory” specimens,
including information on transformation zone sampling and other adequacy
qualifiers (obscuring elements, poor preservation, etc.) encourages specimen takers
to pay greater attention to specimen procurement and handling. Any factors that
compromise specimen quality can be mentioned in a note.
10• Unsatisfactory —For unsatisfactory specimens the report should indicate
whether the laboratory processed/evaluated the slide. Suggested wording is:
– Rejected specimen—Specimen rejected/not processed because (specify
reason: specimen not labeled, broken slide, etc.).
– Fully evaluated, unsatisfactory specimen—Specimen processed and
examined, but unsatisfactory for evaluation of epithelial abnormality because
of (specify reason: inadequate squamous component, obscuring blood, etc.).
Additional comments/recommendations may be made as deemed appropriate.
While unsatisfactory specimens which are processed and evaluated are not
suitable for excluding an intraepithelial lesion or malignancy, the presence of
endometrial cells in a women 40 years or older, or the presence of organisms, can
be reported in this context, since this information may prove to be clinically
relevant for patient management.
As in prior Bethesda adequacy guidelines, if abnormal cells are detected, the
specimen cannot be categorized as “unsatisfactory.”
Squamous Cellularity
TBS 1991 required that well-preserved and well-visualized squamous epithelial
cells should cover more than 10% of the slide surface. In order to address adequacy
on conventional as well as liquid-based preparations, and to improve interobserver
reproducibility, TBS 2001 went further to provide numerical estimates of what*
constitutes adequacy for squamous cellularity in cervical cytology preparations.
• Conventional smears: An adequate conventional preparation should have a
minimum of approximately 8,000–12,000 well-preserved and well-visualized
squamous cells. This minimum cell count should be estimated, not counted. The
count includes both nucleated mature and metaplastic squamous cells. The
percentage of hypocellular areas, if present, should be estimated and the fields
10 11counted should reflect this proportion. The Bethesda atlas and website
provide “reference images” of known cellularity at low (4×) magnification as a
resource for cytologists to compare with the specimen being assessed.
• Liquid-based preparations (LBP): An adequate LBP should have an estimated
10,17minimum of at least 5,000 well-visualized, well-preserved squamous cells.
Estimation of cellularity is suggested in borderline cases by performing
representative field counts. A minimum of 10 fields, usually at 40×, are assessed
along a diameter that includes the center of the preparation. The average number
of squamous cells per field is thus estimated. One preliminary study suggested that
LBPs containing 5,000–20,000 squamous cells should be considered as
18“borderline” cellularity.
10The reader is referred to the Bethesda atlas for cellularity tables and /gures.
These guidelines may change in the future based on additional data.
The TBS numeric criteria for cellularity may not be applicable to vaginal
specimens, cases with extensive cytolysis, cell clustering, and some cases of
atrophy. Cytologists should utilize clinical information and their best judgment
when interpreting such cases. At present there are no published studies speci/cally
addressing the relationship between low cellularity and false-negative rate.
Quality Indicators
Patient/Specimen Identification and Technical Interpretability
Correct specimen identi/cation is essential for evaluation and is required by the
Clinical Laboratory Improvement Amendments of 1988 (CLIA '88). In addition to
ensuring that the specimen corresponds to the correct patient, proper identi/cation
allows the laboratory to locate prior records and slides from the patient that may
influence the current evaluation.
The cellular material must be well /xed and unobscured for interpretation.
Minimal data regarding how obscuring factors a ect the interpretive reliability of a
cervical specimen are available. In order to be considered obscuring, the epithelial
cell morphology must be uninterpretable. For example, although most cervical
samples contain in4ammatory cells, moderate numbers do not generally obscure
the nuclei of squamous cells. Even a large amount of in4ammation or blood maybe acceptable if it is spread thinly such that the intermixed epithelial cells can be
easily visualized. In general, specimens with more than 75% of epithelial cells
obscured are considered unsatisfactory. A variety of factors may compromise
visualization of the cells. Heavy in4ammation, blood, and extensive cytolysis are
patient-related and independent of the sample taker. However, presence of
airdrying, thick uneven smears, or lubricant is often inversely correlated with the skill
and experience of the clinician. With liquid-based preparations a number of these
factors become less signi/cant; however, appropriate collection/rinsing techniques
need to be utilized. Clinicians who repeatedly obtain technically poor quality
specimens may bene/t from constructive feedback provided in the written report,
by telephone, or in a summary format comparing adequacy rates of peer group
clinicians.
Clinical Information
Providing pertinent clinical information may increase the sensitivity and reliability
of the evaluation by directing attention to a clinical question or by clarifying
otherwise uncertain cytologic /ndings. At a minimum, age and date of last
menstrual period should be provided. Absence of this information does not,
however, preclude evaluation; therefore, the specimen may remain categorized as
“satisfactory” in these circumstances.
Sampling of the Transformation Zone
Presence of endocervical or squamous metaplastic cells forms the microscopic basis
for the assumption that the transformation zone has been sampled. The numeric
criterion for a transformation zone component, at least 10 well preserved
endocervical or squamous metaplastic cells, did not change from TBS 1991;
however, due to the widespread utilization of liquid-based preparations, single
endocervical/metaplastic cells are acceptable and clusters are no longer required.
This de/nition applies to specimens from both premenopausal and postmenopausal
women having a cervix. In the situation of marked atrophy, where metaplastic and
endocervical cells often cannot be distinguished from parabasal cells, the
laboratory has the option of making a comment regarding the difficulty in assessing
the transformation zone component. Patient factors, such as location of the
transformation zone, age, pregnancy, and previous therapy, may limit the
clinician's ability to obtain an endocervical sample despite optimal collection
technique.
Numerous cross-sectional studies have demonstrated that smears with
endocervical and/or metaplastic cells have a signi/cantly higher frequency and
higher grade of squamous epithelial abnormality detected than do smears without
19-22such cells. Paradoxically, short-term longitudinal studies of women whose
initial negative smears lacked an endocervical component have shown no increasein abnormalities on repeat, satisfactory smears (as might be expected if the initial
23,24smears had a higher false-negative rate).
Based on the above studies, TBS 2001 does not require the presence of a
transformation zone component to categorize a specimen as satisfactory—adequate
squamous cellularity is the only criterion. The absence of a transformation zone
component is considered to be a quality indicator. With the reported increase in
25,26endocervical carcinoma, the importance of the transformation zone
component may undergo further evaluation in the future, in order to ensure
optimized screening performance in the setting of endocervical neoplasia.
Management Guidelines
The ASCCP has published management guidelines for Pap test specimen adequacy
9and quality indicators. The preferred management for unsatisfactory Pap tests is a
repeat test within a short interval of 2–4 months. Unsatisfactory cases are
unreliable for detection of an epithelial abnormality; furthermore a longitudinal
study found that unsatisfactory Pap tests are more often from high-risk patients and
have signi/cantly more SIL/cancer on follow-up than patients with satisfactory
27index Paps. The guidelines suggest a repeat Pap test in 12 months for most
women who lack a transformation zone component or whose cytology is partially
obscured unless there is a history of prior adequate/negative Pap tests. Indications
for considering earlier repeats are also outlined and depend on additional patient
28risk factors. For quality assurance, it may be prudent to have the pathologist
review unsatisfactory cases prior to /nal sign-out because of the clinical
implications of such a report and the association of obscuring blood/in4ammation
29with invasive cancers.
Impact on Laboratory Practice
The incorporation of specimen adequacy as an integral part of the cervical cytology
report has been acknowledged as one of the most important contributions of TBS.
The impact on laboratory practice has been dramatic. Surveys conducted by CAP
revealed that in 1990 only 35% of responding laboratories routinely reported
30specimen adequacy; by 1992, this /gure increased to 85%. A 1991 CAP survey
found that most laboratories reported unsatisfactory specimen rates of 0.5–1.0%.
By the year 2003, a CAP survey assessing Bethesda implementation and reporting
31rates found that 73.6% of responding laboratories had eliminated the use of the
“satisfactory but limited by…” category and the 2001 TBS minimum squamous
cellularity criteria had been adopted by 85.3%. Experts had predicted an increase
in unsatisfactory rates with the use of TBS 2001 criteria. While some studies have
32indeed reported this (up to a tenfold increase on conventional smears ), the CAP*
31 33 34survey and other reports from the United States and Europe did not show an
increase in the unsatisfactory rate after conversion to TBS 2001 adequacy criteria.
Possibilities suggested by the authors include improved sampling and preparation
methods, related predominantly to liquid-based methodology, or, alternatively,
31lack of attention to the TBS 2001 criteria.
General Categorization
The general categorization is a clerical device to aid clinicians and their oF ce sta
in triaging patients/prioritizing cases for review and to assist laboratories in
compiling statistical information.
There are 3 headings used under the general category:
1. “Negative for intraepithelial lesion or malignancy” for specimens in which an
epithelial abnormality is not identified. Organisms and other benign/reactive
cellular changes can be reported in the Interpretation under this category.
2. “Other” may be utilized for cases in which there is no clear cytologic
abnormality but the findings may warrant follow-up/investigation based on
patient risk, for example endometrial cells in a woman 40 years of age or older.
3. “Epithelial cell abnormality” may be utilized for squamous or glandular
epithelial abnormalities. Specify as far as possible which type is present.
If more than one diagnostic entity is present—for example, an infectious process
and an epithelial abnormality—the specimen should be categorized according to
the most clinically signi/cant lesion; in this example, epithelial cell abnormality.
However, the general category should not replace narrative (descriptive)
terminology for communicating the interpretation/result. Some laboratories also
extend the concept of a general or summary categorization to nongynecologic
specimens.
Interpretation/Result
The prior Bethesda system use of the term “diagnosis” was replaced by
“interpretation/result” in 2001. Workshop participants felt that cervical cytology is
a screening, not a diagnostic test, that provides the clinician with information on
morphologic /ndings that need to be integrated with the patient's other clinical
7findings for a final diagnosis and subsequent management.
Negative for Intraepithelial Lesion or Malignancy (NILM)
The Bethesda 2001 category of NILM is used to report non-neoplastic /ndings in
the absence of an intraepithelial lesion or malignancy. This term is used both as ageneral categorization and as an interpretation and incorporates the reporting of
organisms and other non-neoplastic /ndings such as reactive cellular changes (Table
6.1). The NILM category replaces the two prior Bethesda categories of “within
normal limits” (WNL) and “benign cellular changes” (BCC). The basis of this
change was to clearly communicate to the physician that despite any other
“benign” changes reported, the Pap test is “negative” or without evidence of
cervical intraepithelial neoplasia or malignancy.
Clearly, the main purpose of cervical cytology screening is the detection of
cervical squamous cell carcinoma and its precursors; however, reporting the
/ndings of organisms or reactive conditions can make an important contribution to
patient care. This documentation can facilitate patient triage, provide
clinicalcytologic correlation, and focus attention on cytomorphologic criteria during
microscopic screening and interpretation of cervical cytology.
The category of “infections” was changed to “organisms” in TBS 2001 since the
presence of some organisms represents colonization rather than a clinically
signi/cant infection. Excellent speci/city and reproducibility can be achieved for
the cytopathologic interpretation of fungal elements, Trichomonas vaginalis,
Actinomyces, and herpes simplex virus, by application of reproducible morphologic
criteria. The interpretation of Chlamydia spp. is not listed in TBS because of the
acknowledged low diagnostic accuracy of routine cytology for this organism and
because of the availability of other, more accurate detection methods. TBS lists the
organisms that should be reported; however, the laboratory is advised to discuss the
relevance of reporting organisms and other non-neoplastic /ndings with their
clinicians and come to a decision about what to report under the NILM category.
Cells manifest reactive morphologic changes in response to a variety of traumatic
insults such as infection, in4ammation, and radiation. Reparative processes,
radiation, atrophy, and intrauterine contraceptive devices are examples of entities
that induce cellular changes that may mimic intraepithelial lesions or even cancer.
Severe reactive/reparative changes are diF cult to distinguish from neoplastic
changes and such interpretations are well known to have lower reproducibility than
35classic repair. It is, however, important to recognize benign reactive features in
order to avoid overinterpretation and resulting false-positive interpretations. A CAP
report indicates that reparative changes tend to be easier to recognize on LBP,
36yielding less false positives than on conventional smears.
Keratotic cellular changes—hyperkeratosis, parakeratosis, and dyskeratosis—are
descriptive terms that do not clearly communicate a diagnostic interpretation and
are not included in TBS. The classi/cation of such changes as benign/reactive or
dysplastic should be based on the cytoplasmic and nuclear alterations present and
reported under the appropriate general category/interpretation.
Occasionally, benign-appearing glandular cells may be seen in post-hysterectomy*
patients that can have a wide variety of sources, including adenosis, metaplasia,
37,38and prolapse of the remaining fallopian tube after a simple hysterectomy.
This /nding can be communicated to the clinician under the NILM category and
9per current ASCCP guidelines does not require further follow-up. Other
nonneoplastic changes that may be reported under NILM include atrophy and tubal
metaplasia.
Details regarding the morphology of these entities are discussed elsewhere in this
book.
Endometrial Cells
TBS 1991 recommended that benign-appearing endometrial cells in
postmenopausal women be reported as an “epithelial cell abnormality” based on
the increased risk for endometrial adenocarcinoma (6%) and endometrial
3,39,40hyperplasia (12%) on a meta-analysis.
In TBS 2001, a new category was included to report the presence of
benign7,10appearing endometrial cells in women aged 40 years or older. The basis for
including this new category in TBS 2001 was twofold: (a) review of the published
literature showed an exceedingly low rate of signi/cant lesions in anyone less than
40 years of age, and (b) pathologists may lack clinical information on menstrual
dates/menopausal status, hormone therapy/tamoxifen, abnormal bleeding, and
other endometrial carcinoma risk factors. It is important to include in the
interpretation whether the cytology is “negative for squamous intraepithelial
lesion.”
Only exfoliated, intact endometrial cells should be reported under the “other”
category. As described in Bethesda 2001, the exfoliated groups of endometrial cells
may be of epithelial and/or stromal origin; morphological distinction of these two
cell types is usually not possible. Directly sampled lower uterine segment or
abraded stromal cells/histiocytes, when present alone, should not be reported
under this category. Atypical endometrial cells should be reported as an epithelial
1,10glandular cell abnormality.
The prevalence of benign-appearing endometrial cells cervical in Pap tests from
women aged 40 years or older is diF cult to assess due to di erences in study
39designs, but has been estimated to range from 1–3/100 to 1/1600 or less. After
adoption of TBS 2001, there have been many reports in the cytology literature that
have shown minimal risk associated with this interpretation, especially in
40premenopausal women. This TBS category has been controversial for clinicians
and initially resulted in an increase in endometrial biopsies.
It may be useful to add an educational note to this interpretation in order to
clearly communicate to clinicians that this interpretation has an increased risk of*
neoplasia, but the risk is low, especially in premenopausal women and those
without endometrial carcinoma risk factors, and that clinical correlation with other
risk factors and symptoms is necessary. Examples of educational notes for this
10interpretation can be found in the second edition of the Bethesda atlas.
The 2006 ASCCP guidelines provide additional guidance and suggest that for
asymptomatic women who are documented by clinical history to be
premenopausal, with benign appearing endometrial cells, endometrial stromal
cells, or histiocytes; no further evaluation is required. For documented
postmenopausal women with endometrial cells, on the other hand, endometrial
9assessment is suggested, regardless of symptoms.
Epithelial Cell Abnormalities: Squamous Cell
Squamous intraepithelial lesion (SIL) encompasses the morphologic spectrum of
noninvasive squamous epithelial abnormalities associated with HPV infection.
Since the Bethesda System was introduced in 1988, this spectrum has always been
divided into low-grade (LSIL) and high-grade (HSIL) categories. LSIL encompasses
changes referred to as “HPV e ect,” “koilocytosis,” and mild dysplasia/cervical
intraepithelial neoplasia (CIN 1). HSIL includes moderate dysplasia (CIN 2) and
severe dysplasia/carcinoma in situ (CIN 3). The basis for this bipartite classi/cation
of SIL in TBS is based on the principles that this division (a) better re4ects natural
history and clinical management and (b) has better intra- and interobserver
reproducibility than does a three-tiered reporting system.
Atypical Squamous Cells (ASC)
The term ASCUS was initially introduced into the earliest version of the Bethesda
System to re4ect the reality and limitations of light microscopy in classifying
borderline cytologic changes. The use of multiple ASCUS quali/ers such as “not
otherwise speci/ed” (NOS), “favor reactive,” and “favor SIL/dysplasia” led to
overuse of this category and by 1996, ASCUS interpretations accounted for a mean
41of 5.2% of all cervical cytology reports in the United States. ASCUS
interpretations caused dilemmas for clinicians due to the lack of standardized
follow-up and variability of outcomes.
With advances in the understanding of the biology of HPV infections and results
42 6from various natural history studies, as well as from the NCI ALTS trial, the
focus of cervical cancer screening has shifted from detecting and treating any CIN
to focusing on treating high-grade CIN. Based on this concept, in TBS 2001, the
term ASCUS was replaced by ASC, which has a narrower de/nition and only two
quali/ers: atypical squamous cells of undetermined signi/cance (ASC-US) and
7atypical squamous cells, cannot exclude HSIL (ASC-H). A subclassi/cation was
aimed at having greater clinical utility by clearly separating equivocal /ndings into*
*
*
those that are worrisome for HSIL in distinction from other types of ASC. As a
general guide, the majority of ASC interpretations should fall into the ASC-US
7,10qualifier (90–95%) with only 5–10% into the ASC-H category.
ASC is not a single biologic or interpretive entity: it encompasses a spectrum of
cellular changes re4ecting a variety of pathologic processes that for one reason or
another cannot be more de/nitively categorized. Speci/cally, ASC should be used
for changes suggestive of SIL, that are either quantitatively or qualitatively
insuF cient for a de/nitive interpretation. For a cell to be classi/ed as ASC, it
should show squamous di erentiation, an increase in nuclear cytoplasmic ratio,
10and minimal nuclear changes. In each case of ASC, the cytopathologist must
consider the summation of the morphologic abnormalities in terms of quantity and
severity within the context of the clinical information provided.
Atypical Squamous Cells of Undetermined Significance (ASC-US)
Most often, ASC-US involves nonin4ammatory changes in squamous cells with
mature, super/cial/intermediate-type cytoplasm. Nuclear enlargement is
approximately two-and-a-half to three times the area of a normal intermediate
squamous nucleus, but the chromatin remains evenly distributed without
signi/cant hyperchromasia. Nuclear outlines are smooth and regular, although
there may be variation in nuclear size. The di erential diagnosis is usually between
a reactive change versus LSIL but the change(s) quantitatively or qualitatively fall
short of establishing a de/nitive interpretation of LSIL. Round or ovoid cells that
resemble large metaplastic or small intermediate cells may also be classi/ed as
ASC-US. In liquid-based preparations, the cells may appear smaller and rounder
compared to conventional smears. The cells in question should always be compared
to “normal”-appearing intermediate cells on the same slide. In distinguishing
reactive changes, cells that demonstrate pale round nuclei and even chromatin
distribution favor an interpretation as NILM rather than ASC.
Atypical Squamous Cells, Cannot Exclude High-Grade Squamous
Intraepithelial Lesion (ASC-H)
The ASC-H category is useful for changes suggestive of, but fall short of a de/nite
interpretation of HSIL. The di erential includes HSIL and mimics of HSIL. A
variety of patterns can be recognized:
1. Small cells with a high nuclear to cytoplasmic ratio or “atypical (immature)
metaplasia. Nuclear abnormalities such as abnormal shapes, hyperchromasia, and
chromatin irregularity favor HSIL over benign metaplasia.”
2. Crowded sheet pattern or so-called hyperchromatic cell groups. Dense
cytoplasm, polygonal cell shape, and distinct cell borders favor squamous over
endocervical cells. This cell pattern includes a broad differential from normal(atrophy, endometrial cells) to neoplastic (endocervical adenocarcinoma, HSIL, or
HSIL involving glands) changes.
3. Atypical cells in the setting of atrophy, atypia seen following radiation therapy,
poorly preserved endometrial cells or histiocytes, and intrauterine device users
may all show cellular changes that are difficult to distinguish from HSIL. In such
situations, a designation as ASC-H may be appropriate.
Laboratory Reporting of ASC
Subsequent to the publication and dissemination of TBS 1988/1991, many
clinicians felt overwhelmed by ASCUS interpretations in their patient practices.
This phenomenon was not limited to the United States or to TBS; greatly increased
rates of minor degrees of abnormality have been observed in countries that do not
43use TBS. The reasons underlying this real or perceived ASCUS explosion were
twofold: (1) The constant specter of medical–legal litigation has lowered the
9,31threshold for diagnosis of cellular abnormalities in many laboratories; and (2)
atypical cases historically may have been camou4aged in vague terms such as
“in4ammatory atypia,” “benign atypia,” “borderline HPV,” and “koilocytotic
atypia.” The aggregation of all such equivocal cases under one heading highlighted
the subjective, interpretative nature of cytopathologic diagnosis, something long
understood by laboratorians but not always recognized by clinicians. Some contend
that in TBS 1991, ASCUS merely replaced the old Pap Class 2 or “in4ammatory
atypia” designations. However, a study by Sidawy and Tabbara demonstrated that
by using criteria similar to those outlined above, almost two-thirds of 88 smears
previously interpreted as “in4ammatory atypia” could be reclassi/ed as reactive;
only 3 out of 57 cases (5%) had CIN (all low grade) on follow-up colposcopic
biopsies. In contrast, among the smears that ful/lled ASCUS criteria, 61%
44correlated with colposcopic biopsies positive for CIN.
Laboratory rates of ASC will vary depending on the patient population, the
diagnostic criteria used, and the experience and skill of the microscopist(s). If used
appropriately, ASC should be an infrequent designation employed only when
cellular changes elude a more de/nitive interpretation. Although there is no
“correct” percentage rate of ASC, benchmarks were provided when ASCUS was
introduced in the Bethesda terminology. In a low-risk population, it was suggested
that the rate of ASCUS should be less than 5%. For laboratories that serve high-risk
populations (e.g., sexually transmitted disease clinics or colposcopy clinics), the
rate of ASCUS could be higher, but by 1991 guidelines should not exceed two to
three times the rate of SIL; thus the ratio of ASCUS/SIL suggested was in the range
of 2–3:1. A 1993 CAP survey focusing on laboratory utilization of ASCUS found
that 86% of responding laboratories used the term ASCUS and the median ASCUS
rate was 2.8%, with 90% of laboratories reporting rates of less than 9%. The*
median ASCUS/SIL ratio was 1.7; for 90% of laboratories, the calculated ratio was
41less than 3.6. In 2003, a follow-up CAP survey on Bethesda 2001
implementation and reporting rates showed a decrease in the average ASC/SIL
ratio (from a median of 2.0 in 1996 to 1.4 in 2002). This can be explained by
increased LSIL detection on LBP and also possibly by using Bethesda 2001 criteria
31more stringently.
Sherman and colleagues, in a study correlating cytopathologic diagnoses with
detection of HPV DNA, also found that use of TBS criteria reduced the percentage
of inconclusive “atypical” smears. Overall, a consistent relationship between
highrisk HPV detection and TBS diagnostic categories was evident. High-risk HPV types
were detected in 10% of negative smears, 30% of ASCUS, and 60% of SIL
specimens. Based on these data, the authors proposed using high-risk HPV testing
as an objective quality assurance measure to assess the performance of a
45 5,6cytopathology laboratory. These results were substantiated by ALTS.
It is well established that ASC-US is one of the least reproducible cytologic
12,46interpretations. Various quality assurance monitors may be utilized to
evaluate the laboratory's utilization of ASC. These include the following:
1. Correlation of ASC-US cases with high-risk HPV positivity rates; results from
ALTS indicate that this should be in the range of 40–60%, or in essence that
ASCUS is a 50–50 proposition between SIL (usually LSIL) and cellular changes
47,48unrelated to HPV;
2. Correlation of ASC cases with results of colposcopically directed biopsy;
3. Review of ASC cases by a second cytopathologist; and
4. Calculation of ASC/SIL ratio.
The ASC-HPV+/ASC ratio closely mirrors the ASC/SIL ratio. However, the
ASCHPV+/ASC ratio o ers the additional advantage of identi/cation of aberrant
trends where ASC and SIL are both being misinterpreted, which may allow ASC/SIL
49ratios to remain within “acceptable” ranges despite the erroneous trend. After
implementation of LBPs, many laboratories have reported an increase in SIL rates
over the increase in ASC rates, such that lower ASC/SIL ratios are being seen in
31many laboratories. The prior 2–3:1 suggested ratio for ASC/SIL may undergo
revision as future benchmarking results are gathered.
Clinical Management of ASC
Women with ASC have a low prevalence of invasive cancer, estimated at 0.1–
500.2%. The prevalence of CIN 2/3 is substantially higher in women with ASC-H
51,52 51(37–40%) than in those with ASC-US (11.6%). ASC-US/high-risk HPV-positive cases over 2-years follow-up in ALTS have the same cumulative risk of CIN
472/3, about 27–28%, as a cytologic LSIL. In contrast women in the ALTS who
were ASC-US/high-risk HPV-negative showed a very low (1.4%) absolute risk of
subsequently detected CIN 3 or worse and no cancers were detected in the two-year
study period, similar to women with negative cytology in the absence of HPV
53testing.
8,9The ASCCP consensus guidelines for ASC follow-up have seen widespread
penetration into US practice. For ASC-US, HPV DNA testing, repeat cytological
testing, and colposcopy are all acceptable methods for managing women over 20
years of age. When liquid-based cytology is used, re4ex oncogenic or high-risk HPV
DNA testing is the preferred management approach. ASC-US/high-risk HPV
positive women should be managed in a fashion similar to those with LSIL. In
adolescents (20 years and younger), follow-up with annual cytology is suggested
due to the high prevalence of HPV in this age group and the low risk of
9persistence.
ASC-H on the other hand needs more aggressive follow-up, with colposcopic
evaluation at the /rst interpretation. HPV testing is not recommended for triage of
ASC-H. However, if a CIN 2/3 lesion is not identi/ed at colposcopy, follow-up with
either HPV testing at 12 months or cytology testing at 6 and 12 months is
9recommended.
Squamous Intraepithelial Lesions (SIL)
In TBS, LSIL and HSIL encompass the spectrum of precursors to squamous
carcinoma of the cervix. Unlike CIN and dysplasia classi/cations that maintain
HPV as a separate diagnostic category, low-grade SIL incorporates changes of HPV
as well as mild dysplasia/CIN 1. High-grade SIL includes moderate dysplasia/CIN
2, severe dysplasia/CIN 3, and carcinoma in situ/CIN 3. Cytologists are of course
free to append degrees of dysplasia or CIN classifications to a SIL interpretation.
Conceptual Basis for Two-Tiered Terminology of SIL
Previous terminology classi/cations—degrees of dysplasia and grades of CIN 1 to 3
—have emphasized the morphologic continuum of squamous lesions that was
thought to re4ect a continuous process in the development of cervical cancer.
42Natural history studies and HPV research have since established that HPV
55,56infection is a necessary cause for cervical carcinogenesis; however, even most
oncogenic or high-risk HPV types cause transient low-risk lesions that regress, and
cervical carcinoma develops in a small subset of persistent/progressive HPV
57infections. It is estimated that approximately 70% of cervical cancers are
58associated with HPV 16 or 18.The two-tiered LSIL/HSIL Bethesda approach attempts to morphologically
distinguish minor from signi/cant lesions; however, morphology is an imperfect
re4ection of biologic potential. Low-grade lesions, particularly those that persist,
may progress or be associated with the development of high-grade lesions, and
57some high-grade lesions may regress. Some have questioned setting TBS division
of LSIL and HSIL at the breakpoint of CIN 1–2, or mild/moderate dysplasia,
arguing that some CIN 2/moderate dysplasias should be considered low-grade
lesions. However, because some CIN 2 lesions represent high-grade disease
processes, conservatism dictated its inclusion into the more severe TBS category to
ensure maximal sensitivity of the process.
Morphologic Features
An interpretation of LSIL based on cellular changes associated with HPV requires
nuclear as well as cytoplasmic abnormalities. Nuclear changes may include
enlargement with hyperchromasia or pyknosis, and chromatin smudging and
wrinkling of nuclear contours. Cytoplasmic changes consist of a well-de/ned
perinuclear cavity, associated with peripheral thickening of the cytoplasm or
cytoplasmic orangeophilia, and rounding of cellular contours. Specimens with
subtle changes that fall short of de/nitive LSIL may be categorized as ASC-US.
Cytoplasmic vacuolization (pseudokoilocytosis) alone, in the absence of any
nuclear atypia, is considered a benign change and should not be classi/ed as LSIL
or ASC-US.
Intraepithelial precursors of squamous cell carcinoma present a spectrum of
morphologic changes within which one is able, in most cases, to classify lesions as
LSIL or HSIL; however, occasional “borderline” cases occur. In the CAP
Interlaboratory Comparison Program in Cervicovaginal Cytology (PAP), the
discrepant rate between low- and high-grade lesions ranged from 9.8 to 15% for
59cytotechnologist, pathologist, laboratory, and all responses. Cytology and
histology may also be discrepant; 15–25% of women with LSIL cytology are found
48,59to have histologic CIN 2/3 on further work-up. Features that favor a
highgrade lesion include increased numbers of abnormal cells, higher nucleus to
cytoplasmic ratios, greater irregularities in the outline of the nuclear envelope and
nuclear chromatin distribution, and increased number of chromocenters. The
appearance of the cytoplasm may also assist in determining whether a borderline
case is low- or high-grade SIL. LSIL changes typically involve “mature,”
intermediate, or super/cial type cytoplasm with well-de/ned polygonal borders.
Cells of HSIL have a more immature type of cytoplasm, either delicate and lacy or
dense/metaplastic, with rounded cell borders. Lesions previously termed
“pleomorphic dysplasia,” “keratinizing dysplasia,” or “atypical condyloma,” which
are composed of single cells or clusters of cells with enlarged hyperchromatic nuclei
and abundant but abnormally keratinized cytoplasm, are always considered HSIL.*
HSIL, Cannot Exclude Invasion
In rare cases of HSIL, invasive carcinoma may be diF cult to exclude. Examples
include atypical keratinized cells without diathesis/necrosis in the background or
10cases in which the background is suspicious but malignant cells are not seen.
This terminology may be used in such cases to communicate the increased concern
to the clinician.
Squamous Cell Carcinoma
Squamous cell carcinoma is defined as an invasive malignant tumor with squamous
di erentiation. TBS does not subdivide squamous cell carcinoma into keratinizing
and non-keratinizing types, although the atlas does discuss the morphology
separately. On LBPs, tumor diathesis may be more diF cult to recognize; and as
such, in the United States a trend toward undercalling squamous cell carcinoma as
60HSIL, especially on LBP, has been noted.
Management of SIL
Low-grade SIL. Based on natural history studies of HPV infection, it is clear that the
42majority of cytologically detected LSIL regress within an average of two years.
After implementation of liquid-based cervical cytology, there has been a steady
increase in the rate of LSIL in the United States—in 2003 the median rate was
312.4%. Anecdotal experiences suggest that this has further increased with the use
of location guided screening. In ALTS, the HPV positivity rate in LSIL was 83%; a
61meta-analysis published in 2006 reported a 76.6% positivity rate. Thus HPV
DNA testing is not suggested for initial triage of LSIL. Initial colposcopy identi/es
prevalent CIN 2 or greater in 18% of women with LSIL; subsequent follow-up over
2 years identi/ed another approximately 10% CIN 2/3, irrespective of whether the
54,62initial colposcopy was negative or showed histologic CIN 1.
Colposcopy is recommended for managing LSIL; exceptions include adolescents,
postmenopausal, and pregnant women. If no lesion is identi/ed or colposcopy is
unsatisfactory, endocervical sampling is recommended for non-pregnant women. If
CIN 2/3 is not detected, post-colposcopically, either HPV testing at 12 months or
repeat cytology at 6 and 12 months is suggested. In adolescents with LSIL, initial
colposcopy and/or HPV testing is not recommended; they should be followed by
annual cytologic testing. Further details can be found in the ASCCP management
9guidelines.
High-grade SIL. The median percentile reporting rate of HSIL in the United
31States is estimated at 0.5%, and approximately 2% of women with HSIL cytology
63have invasive carcinoma. Follow-up of cytologic HSIL carries a signi/cant risk of
a CIN 2/3—a single colposcopy identi/es 53–66% of prevalent CIN 2/3; and CIN*
2/3 is found in 84–97% of women who proceed to a loop electrosurgical procedure
64(LEEP). Thus both colposcopy and LEEP are acceptable for management of
9cytologic HSIL.
Epithelial Cell Abnormalities: Glandular Cell
Background
Cervical cytology is primarily a screening test for cervical squamous intraepithelial
lesions and squamous cell carcinoma; cytology may have lower sensitivity for
detection of glandular lesions has lower sensitivity due to limitations in sampling
and interpretation.
Reporting Glandular Cells in TBS 2001
In TBS 2001, the term atypical glandular cells of undetermined signi/cance
(AGUS) has been eliminated to avoid confusion, particularly among clinical sta ,
with ASC-US. Abnormal glandular cells should be subclassi/ed when possible as
endocervical or endometrial; otherwise the generic term “atypical glandular cells”
should be used. It is also advisable to use the quali/ers “not otherwise speci/ed” or
“favor neoplastic” for endocervical and glandular cells to convey the level of
concern about any abnormality identi/ed. The quali/er “favor reactive” from TBS
1991 has been eliminated as follow-up studies show that results are similar to those
in the NOS category, and as such, this quali/er provides no useful predictive value.
Atypical endometrial cells are not further quali/ed due to diF culty in doing so and
lack of reproducibility of the morphologic criteria. Adenocarcinoma in situ is a
separate interpretive entity in TBS 2001, having been well described and shown to
65have moderately good reproducibilty since the 1991 TBS version.
Atypical Glandular Cells (AGC)
As with its squamous ASC counterpart, this designation applies to glandular cells
that demonstrate changes beyond those encountered in benign reactive processes,
yet which are insuF cient for an interpretation of in situ or invasive
adenocarcinoma. This interpretation should be further quali/ed, where possible, to
indicate whether the cells are thought to be of endocervical or endometrial origin.
This category includes a broad morphologic spectrum ranging from
atypicalappearing, reactive processes all the way to adenocarcinoma in situ (AIS).
Therefore, lesions falling into this category should be further subclassi/ed, if
possible, according to whether a neoplastic process is favored or the changes are
non-speci/c (NOS). Speci/c comments may be added to the interpretation if
pertinent clinical /ndings and/or history are available and relevant (polyps, IUD,
etc.).*
Atypical Endocervical Cells, NOS
Endocervical cells can show a variety of changes associated with benign/reactive
processes in the endocervical canal. Reactive endocervical cells can show some
pleomorphism of cell size as well as nuclear enlargement, multinucleation, and
prominent nucleoli; however, there is usually a honeycomb or sheet-like pattern
and nuclei remain round and the chromatin bland. Such changes are usually
recognized as NILM and not included in the AGC category. Cells that show
cytologic changes beyond those recognized easily as reactive such as signi/cant
nuclear enlargement/crowding, hyperchromasia, loss of mucin, and loss of polarity
should be considered for inclusion in the atypical endocervical cells, NOS category.
Such changes may be seen in conditions such as tubal metaplasia, radiation
therapy, endocervical polyps, and microglandular hyperplasia, and in IUD users,
but also in neoplastic conditions in a small percentage of cases. This category
therefore includes changes that are in excess of those attributable to a
reactive/reparative condition but which fall short of those seen in glandular
neoplasia.
Atypical Endocervical Cells, Favor Neoplastic
These cells are characterized by cellular strips and rosettes demonstrating
elongated, overlapping nuclei with moderately coarse chromatin and
hyperchromasia. The peripheral border of the glandular clusters may be
“feathered,” with protruding nuclei, in contrast to the smooth communal border
typical of glandular fragments. In LBPs cells are more rounded and
threedimensional. Cellular changes, while suspicious for in situ or invasive
adenocarcinoma, are quantitatively or qualitatively insuF cient for an outright
interpretation as such.
Atypical Endometrial Cells
These are usually small groups of cells with slightly enlarged nuclei, and variable
prominence of nucleoli and nuclear hyperchromasia. Their distinction from
cytologically benign endometrial cells is based primarily on the criterion of
increased nuclear size. When dealing with LBPs, it is important to keep in mind
that menstrual/shed endometrium is often well preserved and may show nuclear
size and shape pleomorphism and the presence of nucleoli. The di erential of
atypical endometrial cells is broad and may include endometrial polyps,
endometritis, IUD associated changes, hyperplasia, and carcinoma.
Endocervical Adenocarcinoma (In Situ and Invasive)
Endocervical AIS is a high-grade endocervical neoplastic lesion that cytologically
demonstrates nuclear enlargement, hyperchromasia, strati/cation, and mitotic
activity. Invasive carcinoma overlaps cytologically with AIS, but may show features*
*
*
*
of invasion, including prominent nucleoli and tumor diathesis. The possibility of a
coexisting squamous lesion should always be carefully assessed when a glandular
66-68lesion is detected, due to the high rate of coexistence of SIL in cases with AIS.
Endometrial Adenocarcinoma
The cytologic features are directly related to the histologic grade of the tumor, with
well-di erentiated cases yielding malignant cells with minimal atypia and poorly
di erentiated tumors being obviously malignant. Tumor diathesis is often diF cult
to appreciate, particularly in LBP. In general endometrial lesions yield fewer cells
than do directly sampled endocervical lesions.
Extrauterine Adenocarcinoma
A clean background and tumors whose cytologic features are not characteristic of
uterine/cervical tumors should raise the possibility of metastasis. Diathesis is
usually not seen unless there is direct extension from the rectum or bladder with
associated tissue destruction.
Diagnostic Difficulties
Criteria indicating invasion—tumor diathesis and macronucleoli—may be absent in
the majority of well-di erentiated, early adenocarcinomas. It also can be diF cult
to di erentiate SIL with gland involvement from AIS. HSIL/CIS involving
endocervical glands may yield round cell clusters with smooth peripheral contours
showing group polarity and “columnar” shape of individual cells, thus mimicking a
69glandular abnormality. Additionally, SIL and AIS may coexist in up to 50% of
cases, and at conization, a high proportion of AIS specimens demonstrate
68concurrent SIL.
Benign entities such as tubal metaplasia, directly sampled lower uterine segment
(LUS) endometrial cells, and cervical endometriosis may all morphologically mimic
AIS. Fragments of tubal metaplasia may demonstrate crowded sheets of glandular
cells with enlarged nuclei as well as cell fragments with nuclear palisading and
70nuclear overlap, mimicking some of the morphologic features of AIS. However,
rosette formation is uncommon in tubal metaplasia, and the nuclear chromatin
tends to be more /nely granular. The most helpful /ndings though, when present,
are cytoplasmic terminal bars and cilia.
Directly sampled endometrial tissue may mimic AGC or glandular neoplasia.
Inadvertent sampling of the LUS may occur because of closer approximation of the
71LUS to the cervical os following cone biopsy or with aggressive use of
endocervical brushes. In contrast to spontaneously exfoliated endometrial cells,
which typically shed as tight ball-like clusters, direct brushing of endometrial tissue
yields large cellular fragments. These fragments often recapitulate their native*
three-dimensional architecture with branching tubular glands enmeshed in stroma
72composed of round to spindle-shaped cells. Glandular cells show crowding with
overlapping round nuclei and scant cytoplasm. Peripheral palisading may be
evident. The low power recognition of branching glands and glandular-stromal
complexes is an important clue to avoid confusion with AGC or glandular
neoplasia.
Conventional smears with a diagnosis of adenocarcinoma consistently identi/ed
correctly by CAP interlaboratory glass slide program participants were signi/cantly
more likely to have more abnormal cells, larger abnormal cells, larger nuclei,
73marked atypia, and hyperchromasia than cases that performed poorly. Glandular
lesions have a slightly di erent morphology on LBPs; speci/cally the cells may be
104atter, feathering less prominent, and diathesis more diF cult to appreciate.
Details are discussed elsewhere in this book. However, as for squamous lesions,
there have been reports showing increased detection of glandular abnormalities on
68,74,75LBPs compared to conventional smears.
Management
Atypical glandular cells are estimated to be reported in only 0.2% of cervical
31cytology tests in the United States. While AGC may be associated with benign
and reactive conditions such as endocervical/endometrial polyps, it is clear from
several studies that AGC is a “high-risk” interpretation compared to ASC; the
67,68,76,77reported rate of neoplasia in follow-up of AGC ranges from 9 to 38%.
Due to the high risk of a signi/cant lesion associated with a cytologic
interpretation of AGC, colposcopy with endocervical sampling is recommended for
women with all subcategories of AGC and AIS. In women over 35 years of age,
additional endometrial sampling is recommended. Endometrial sampling is also
recommended for women under the age of 35 with clinical indications suggesting
that they may be at risk for neoplastic endometrial lesions, such as unexplained
vaginal bleeding or conditions suggesting chronic anovulation. In women with
atypical endometrial cells, both endometrial and endocervical sampling should be
done initially. In 2006 ASCCP suggested that while HPV testing alone is not
appropriate for initial triage of any subcategory of AGC or AIS, HPV DNA testing at
the time of colposcopy is preferred in women with atypical endocervical,
endometrial, or glandular cells NOS, and the results should be utilized in overall
9patient management.
Educational Notes/Suggestions
The use of educational notes/comments is optional. If these are used by the
pathologist/laboratory, it is suggested that they be concise, be phrased in the form*
of a suggestion, not a directive, and be substantiated by published guidelines from
7professional organizations. Examples can be found in the second edition of the
10Bethesda atlas.
Ancillary Testing
If ancillary testing, such as high-risk HPV, has been performed, whether the report
is issued concurrently with the cervical cytology result or as an addendum/separate
report will depend on the laboratory's information system, turnaround time for
such testing, and clinical expectations. The methodology utilized for the ancillary
test should be speci/ed. Suggestions for reporting of molecular tests are provided in
10the Bethesda atlas.
Automated Review
For cervical cytology preparations that undergo computer-only or
computerassisted review, the type of instrument used and any result should be included in
the report. In addition, if there was no “human” review of the slide, this should be
made clear in the report.
Interobserver Reproducibility in Cervical Cytology
In an e ort to improve standardization, clarity, and reproducibility of cervical
10cytology reporting, the second edition of the Bethesda atlas emphasized more
detailed morphologic criteria and had many more images, which were
11complimented by additional images on the Bethesda website. In addition, as part
of the ASC–NCI Bethesda Project, a web based interobserver reproducibility study
was designed to gauge cervical cytology reproducibility prior to publication of the
atlas and website. A range of classic and borderline images (77) were included for
interpretation; approximately 651 cytotechnologists and pathologists worldwide
participated in the study. It was apparent from the results that the morphology
presented was more important in classifying images correctly than were
professional or academic degrees, or other variables assessed. In this study, exact
agreement with the TBS panel was relatively low (57%), although agreement was
84.1% at the threshold of distinguishing NILM from non-negative. Participants
achieved a higher sensitivity for correctly classifying high-grade squamous lesions
than that for high-grade glandular lesions. The details of this study have been
12published and all the images and associated histograms of participants' responses
11are available for review on the Bethesda website.
The Bethesda System and Reporting Anal-Rectal Cytology*
Anal cancer is considered an appropriate target for cytologic screening in selected
high-risk populations. The anatomic commonality of the anal–rectal canal and the
cervical mucosa is re4ected in that both have a transformation zone. HPV is a
common risk/etiologic factor for cancers of the anus and cervix and subsequently
the morphology of cytology samples from both sites is comparable. It follows that
sampling devices, preparation techniques, and morphologic interpretation using
the Bethesda system terminology utilized for cervical cytology can readily be
applied for anal–rectal cytology screening.
Adequacy criteria for anal–rectal cytology are based, at present, on limited
personal experiences. As a guide, minimum adequacy cellularity should be in the
range of 2000–3000 nucleated squamous cells for conventional smears and for LBP
samples 1–2 nucleated cells/high-power /eld for ThinPrep (20 mm diameter)
preparations and 3-6 nucleated squamous cells/high-power /eld for SurePath (13
10mm diameter) preparations.
Normal elements seen in anal–rectal specimens include nucleated, anucleate,
and metaplastic squamous cells, rectal columnar cells, fecal matter, and mucus. A
comment should be included in the report about the presence of a transformation
zone component. Cytomorphologic criteria are quite similar to those utilized for
cervical cytologic interpretation; however, there is a higher incidence of poor
preservation, cellular degeneration, and cytoplasmic keratinization/parakeratosis,
10,78and classic koilocytes are less frequently identified.
When targeting high-risk groups, the rate of epithelial abnormalities noted is far
78,79higher than that reported for cervical cytology. Reports from the United States
suggest that anal–rectal cytology screening is sensitive but has low speci/city for
predicting the grade of the lesion, with a tendency to under-represent the grade of
squamous abnormality. While it has been shown that screening high-risk patients
by cytology is e ective, the present impediments to the success of early detection of
anal cancer by this method include limited clinical expertise and means for the
subsequent treatment/follow-up of these patients, and the high risk of
complications associated with excisional procedures at this site.
Concluding Remarks
Cervical cytology has seen many changes since its introduction in the 1960s—
liquid-based sampling techniques, automated preparation, computer-assisted
screening, HPV DNA testing, and more recently dual testing, which combines
cytology screening with HPV testing. HPV vaccines entered the market in 2006,
and are likely to further decrease the incidence of invasive squamous cell
carcinoma of the cervix and its precursor lesions. Cervical cancer screening
guidelines have undergone signi/cant changes after implementation of LBP and80HPV testing and with advances in the understanding of cervical neoplasia. It is
predicted that the number of cervical cytology tests performed will decrease
81significantly in the future if there is compliance with these guidelines.
The Bethesda System has met the goals that were conceived at the time of its
implementation in 1988. It has seen successful penetration into cervical cytology
reporting worldwide, allowing laboratories to use consistent terminology in
conveying results to clinicians and thus enabling comparison of studies across
many countries and health care systems. The use of the Bethesda ASC-US
terminology prompted the NCI-sponsored ALTS trial, the results of which have
signi/cantly impacted the management of equivocal and low-grade cervical
cytologic abnormalities. The Bethesda terminology has been updated twice—in
1991 and 2001 since its inception in 1988 in order to keep pace with the advances
in our understanding of cervical cancer and evolving technologies in cervical
cancer screening and prevention. The Bethesda System also provided the basis for
the ASCCP to develop consensus guidelines for management of cervical cytologic
abnormalities as de/ned by TBS. This process of re-evaluation and revision will
continue in the future in order to provide the most accurate, reproducible, and
relevant terminology. Optimal communication and ultimately patient care
outcomes will therefore ensue.
References
1. National Cancer Institute Workshop. The 1988 Bethesda System for reporting
cervical/vaginal cytologic diagnoses. JAMA. 1989;262:931-934.
2. National Cancer Institute Workshop. The Bethesda System for reporting cervical/vaginal
cytologic diagnoses. Acta Cytol. 1993;37:115-124.
3. Kurman R.J., Solomon D. The Bethesda System for Reporting Cervical/Vaginal Cytologic
Diagnoses: Definitions, Criteria and Explanatory Notes for Terminology and Specimen
Adequacy. New York: Springer-Verlag; 1994.
4. Davey D., Woodhouse S., Styer P., et al. Atypical epithelial cells and specimen adequacy:
current laboratory practices of the participants in the College of American Pathologists
interlaboratory comparison program in cervicovaginal cytology. Arch Pathol Lab Med.
2000;124:203-211.
5. ASCUS LSIL Triage Study (ALTS) Group. Results of a randomized trial on management of
cytology interpretations of atypical squamous cells of undetermined significance. Am J
Obstet Gynecol. 2003;188:1383-1392.
6. Schiffman M., Adrianza M.E. ASCUS-LSIL Triage Study. Design, methods and
characteristics of trial participants. Acta Cytol. 2000;44(5):726-742.
7. Solomon D., Davey D., Kurman R., et al. for the Forum Group Members and the Bethesda
2001 Workshop. The 2001 Bethesda System—terminology for reporting results ofcervical cytology. JAMA. 2002;287:2114-2119.
8. Wright T.C.Jr., Cox J.T., Massad L.S., et al. ASCCP-Sponsored Consensus Conference.
2001 Consensus Guidelines for the management of women with cervical cytological
abnormalities. JAMA. 2002;287:2120-2129.
9. ASCCP consensus guidelines. Am J Obstet Gynecol. 2007;197(4):346-355.
10. Solomon D., Nayar R., editors. The Bethesda System for Reporting Cervical Cytology,
2nd edn, New York: Springer, 2004.
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23. Mitchell H., Medley G. Longitudinal study of women with negative cervical smears
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26. Zheng T., Holford T.R., Ma Z., et al. The continuing increase in adenocarcinoma of the
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unsatisfactory Pap smear. Cancer Cytopathol. 1997;81:139-143.
28. Davey D., Austin M., Birdsong G., et al. ASCCP patient management guidelines: Pap test
adequacy and quality indicators. J Low Genit Tract Dis. 2002;6:195-199.
29. Nielsen M.L., Davey D.D., Kline T.S. Specimen adequacy evaluation in gynecologic
cytopathology. Diagn Cytopathol. 1993;9:394-403.
30. Davey D.D., Woodhouse S., Styer P., et al. Atypical epithelial cells and specimen
adequacy. Arch Pathol Lab Med. 2000;124(2):203-211.
31. Davey D.D., Neal M.H., Wilbur D.C., et al. Bethesda 2001 Implementation and
reporting rates: 2003—practices of participants in the College of American Pathologists
Interlaboratory Comparison Program in cervicovaginal cytology. Arch Pathol Lab Med.
2004:;28(11):1224-1229.
32. Fidda N., Miron J., Rodgers W.H., et al. Impact of the new Bethesda system 2001 on
specimen adequacy of cervicovaginal smears. Diagn Cytopathol. 2004;30:235-239.
33. Quddus M.R., Sung C.J., Eklund C.M., et al. ASC:SIL ratio following implementation of
2001 Bethesda system. Diagn Cytopathol. 2004;30(4):240-242.
34. Prandi S., Beccati D., Aloysio G.D., et al. Applicability of the Bethesda system 2001 to a
public health setting. Cancer Cytopathol. 2006;108(5):271-276.
35. Colgan T.J., Woodhouse S.L., Styer P.E., et al. Reparative changes and the
falsepositive/false-negative Papanicolaou test. Arch Pathol Lab Med. 2001;125(1):134-140.
36. Snyder T.M., Renshaw A.A., Styer P.E., et alfor the Cytopathology Resource Committee.
Altered recognition of reparative changes in ThinPrep specimens in the College of
American Pathologists Gynecologic Cytology Program. Arch Pathol Lab Med.
2005;129(7):861-865.
37. Ponder T.B., Easley K.O., Davila R.M. Glandular cells in vaginal smears from
posthysterectomy patients. Acta Cytol. 1997;41:1701-1704.
38. Bwetra C. Columnar cells in posthysterectomy vaginal smears. Diagn Cytopathol.
1992;8(4):342-345.
39. Greenspan D.L., Cardillo M., Davey D.D., et al. Endometrial cells in cervical cytology:
review of cytologic features and clinical assessment. J Low Gen Dis.
2006;10(2):111122.
40. Browne T.J., Genest D.R., Cibas E.S. The clinical significance of benign-appearing
endometrial cells on a Papanicolaou test in women 40 years or older. Am J Clin Pathol.
2005;124(6):834-837.41. Interlaboratory Comparison Program in Cervicovaginal Cytology. 1993 PAP supplemental
questionnaire on Laboratory Practice: ASCUS. College of American Pathologists, 1994.
42. Ho G.Y.F., Bierman R., Beardsle L., et al. Natural history of cervicovaginal
papillomavirus infection in young women. N Engl J Med. 1998;338:423-428.
43. Raffle A.E., Alden B., Mackenzie E.F.D. Detection rates for abnormal cervical smears:
What are we screening for? Lancet. 1995;345:1469-1473.
44. Sidawy M.K., Tabbara S.O. Reactive change and atypical squamous cells of
undetermined significance in Papanicolaou smears: A cytohistologic correlation. Diagn
Cytopathol. 1993;9:423-429.
45. Sherman M.E., Schiffman M.H., Lorincz A.T., et al. Toward objective quality assurance
in cervical cytopathology. Am J Clin Pathol. 1994;102:182-187.
46. Stoler M.H., Schiffman M. Atypical Squamous Cells of Undetermined
Significance-Lowgrade Squamous Intraepithelial Lesion Triage Study (ALTS) Group. Interobserver
reproducibility of cervical cytologic and histologic interpretations: realistic estimates
from the ASCUS-LSIL Triage Study. JAMA. 2001;285(11):1500-1505.
47. The ALTS Group. Results of a randomized trial on the management of cytology
interpretations of atypical squamous cells of undetermined significance. Am J Obstet
Gynecol. 2003;188:1383-1392.
48. Stoler M.H. New Bethesda terminology and evidence based management guidelines for
cervical cytology findings. JAMA. 2002;287(16):2140-2141.
49. Ko V., Nanji S., Tambouret R., et al. Testing for HPV as an objective measure for quality
assurance in cervical cytology. Cancer Cytopath. 2007;111:67-73.
50. Jones B.A., Novis D.A. Follow-up of abnormal gynecologic cytology: a College of
American Pathologists Q-probes study of 16132 cases from 306 laboratories. Arch
Pathol Lab Med. 2000;124:665-671.
51. Sherman M.E., Solomon D., Schiffman M. Qualification of ASCUS: a comparison of
equivocal LSIL and Equivocal HSIL cervical cytology in the ASCUS LSIL Triage Study.
Am J Clin Pathol. 2001;116:386-394.
52. Sordon M., Dilworth H.P., Ronnett B.M. Atypical squamous cells, cannot exclude
highgrade squamous intraepithelial lesion. Diagnostic performance, human papillomavirus
testing, and follow-up results. Cancer Cyopathol. 2006;108:32-38.
53. Safaeian M., Solomon D., Wacholder S., et al. Risk of precancer and follow-up
management strategies for women with human papillomavirus-negative atypical
squamous cells of undetermined significance. Obstet Gynecol. 2007;109:1325-1331.
54. Cox J.T., Solomon D., Schiffman M. Prospective follow up suggests similar risk of
subsequent CIN 2 or 3 among women with CIN 1 or negative colposcopy and directed
biopsy. Am J Obstet Gynecol. 2003;188:1406-1412.
55. Nubia Muñoz N., Bosch F.X., Sanjosé S.D., et al. Epidemiologic classification of human
papillomavirus types associated with cervical cancer. N Engl J Med. 2003;348(6):518-527.
56. Lorincz A.T., Reid R., Jenson A.B., et al. Human papillomavirus infection of the cervix:
Relative risk associations of 15 common anogenital types. Obstet Gynecol.
1992;79:328-337.
57. Wright T.C., Schiffman M. Adding a test for human papillomavirus DNA to cervical
cancer screening. N Engl J Med. 2003;348(6):489-490.
58. Khan M.J., Castle P.E., Lorincz A.T., et al. The elevated 10-year risk of cervical
precancer and cancer in women with human papillomavirus (HPV) type 16 or 18 and
the possible utility of type-specific HPV testing in clinical practice. J Natl Cancer Inst.
2005;97:1072-1079.
59. Sherry L., Woodhouse S.L., Stastny J.F., et al. Interobserver variability in
subclassification of squamous intraepithelial lesions: Results of the College of American
Pathologists Interlaboratory Comparison Program in Cervicovaginal Cytology. Arch Pathol
Lab Med. 1999;123(11):1079-1084.
60. Renshaw A.A., Henry M.R., Birdsong G.G., et al. Cytologic features of squamous cell
carcinoma in conventional smears: Comparison of cases that performed poorly with
those that performed well in the College of American Pathologists Interlaboratory
Comparison Program in Cervicovaginal Cytology. Arch Pathol Lab Med.
2004;129(9):1097-1099.
61. Arbyn M., Sasieni P., Meijer C.J., et al. Clinical applications of HPV testing: A summary
of meta-analyses. Vaccine. 2006;24(Suppl 3):S78-S89.
62. Guido R., Solomon D., Schiffman M., Burke L. Comparison of management strategies for
women diagnosed as CIN 1 or less, postcolposcopic evaluation: Data from the ASCUS
and LSIL Triage Study (ALTS), a Multicenter Randomized Trial. J Low Genit Tract Dis.
2002;6:176.
63. Jones B.A., Davey D.D. Quality management in gynecologic cytology using
interlaboratory comparison. Arch Pathol Lab Med. 2000;124:672-681.
64. Evans M.F., Adamson C.S., Papillo J.L., et al. Distribution of human papillomavirus types
in ThinPrep Papanicolaou tests classified according to the Bethesda 2001 terminology
and correlations with patient age and biopsy outcomes. Cancer.
2006;106(5):10541064.
65. Renshaw A.A., Mody D.R., Lozano L.R., et al. Detection of adenocarcinoma in situ of
the cervix in Papanicolaou tests: comparison of diagnostic accuracy with other
highgrade lesions. Arch Pathol Lab Med. 2004;128:53-157.
66. Eddy G.L., Strumpf K.B., Wojtowycz M.A., et al. Biopsy findings in five hundred
thirtyone patients with atypical glandular cells of uncertain significance as defined by the
Bethesda System. Am J Obstet Gynecol. 1997;177:1188-1195.
67. Burja I.T., Thompson S.K., Sawyer W.L., et al. Atypical glandular cells of undetermined
significance on cervical smears. A study with cytohistologic correlation. Acta Cytol.
1999;43:351-356.68. Diaz-Montes T.P., Farinola M.A., Zahurak M.L., et al. Clinical utility of atypical
glandular cells (AGC) classification: cytohistologic comparison and relationship to HPV
results. Gynecol Oncol. 2007;14(2):366-371.
69. Selvaggi S.M. Cytologic features of squamous cell carcinoma in situ involving
endocervical glands in endocervical cytobrush specimens. Acta Cytol. 1994;38:687-692.
70. Novotny D.B., Maygarden S.J., Johnson D.E., et al. Tubal metaplasia. A frequent
potential pitfall in the cytologic diagnosis of endocervical glandular dysplasia. Acta
Cytol. 1992;36:1-10.
71. Lee K. Atypical glandular cells in cervical smears from women who have undergone cone
biopsy. Acta Cytol. 1993;37:705-709.
72. de Peralta-Venturino M.N., Purslow M.J., et al. Endometrial cells of the “lower uterine
segment” (LUS) in cervical smears obtained by endocervical brushings: A source of
potential diagnostic pitfall. Diagn Cytopathol. 1995;12:263-271.
73. Renshaw A.A., Schwartz M.R., Wang E., et al. Cytologic features of adenocarcinoma,
not otherwise specified, in conventional smears: comparison of cases that performed
poorly with those that performed well in the College of American Pathologists
Interlaboratory Comparison Program in Cervicovaginal Cytology. Arch Pathol Lab Med.
2006;130(1):23-26.
74. Austin R.M., Ramzy I. Increased detection of epithelial cell abnormalities by liquid-based
gynecologic cytology preparations. A review of accumulated data. Acta Cytol.
1998;42(1):178-184.
75. Ashfaq R., Gibbons D., Vela C., et al. ThinPrep Pap test. Accuracy for glandular disease.
Acta Cytol. 1999;43(1):81-85.
76. DeSimone C.P., Day M.E., Tovar M.M., et al. Rate of pathology from glandular cells
classified by the Bethesda system 2001 nomenclature. Obstet Gynecol.
2006;107:1285-1291.
77. Chhieng D.C., Gallaspy S., Yang H., et al. Women with atypical glandular cells: a long
term follow-up study in a high risk population. Am J Clin Pathol. 2004;122:575-579.
78. Arian S., Walts A.E., Thomas P., et al. The anal Pap smear: cytomorphology of
squamous intraepithelial lesions. Cytojournal. 2006:2-4.
79. Nayar R., Contreras N., Luzadeer R., et al. Screening for anal cancer: cytologic findings
and follow-up. Cancer Cytopathol. 2006;108:423. (abstract)
80. Saslow D., Runowicz C.D., Solomon D., et al. American Cancer Guideline for the early
detection of cervical neoplasia and cancer. CA Cancer J Clin. 2002;52:342.
81. Solomon D., Breen N., McNeel T. Cervical cancer screening rates in the United States
and the potential impact of implementation of screening guidelines. CA Cancer J Clin.
2007;57:105-111.!
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CHAPTER 7
Microbiology, Inflammation, and Viral infections
Prabodh K. Gupta, Cindy McGrath
Contents
Introduction
Vaginal Microbiology
General Features
Background Changes
Cellular Changes
Infections of the Female Genital Tract
Bacterial Infections
Viral Infections
Chlamydial Infection
Fungal Infections
Parasitic Infections
Concluding Remarks
Introduction
The lower female genital tract includes the vulva, the vagina, the cervix, and the uterine cavity. It is in
direct communication with the external environment, and prone to various noninfectious and infectious
in ammatory reactions. Although most of these infections remain con ned locally; they can progress and
the organisms may ascend to the fallopian tubes and the ovaries. Occasionally, microbial infections from
the lower genital tract can disseminate via the peritoneal space or the hematogenous or lymphatic routes.
It must be appreciated that while a large number of women harboring genital infections may remain
asymptomatic, vaginal infection can produce a number of clinical symptoms. Increased vaginal secretions,
along with sloughed vaginal epithelial cells, other infective organisms, bacteria, and in ammatory cells,
constitute acute vulvovaginitis. Inappropriate use of over-the-counter medications, personal hygiene, tight
clothing, impermeable panty hose (panty hose vulvitis), reaction to various laundry detergents, and washed
1clothes may contribute to the symptoms of vulvovaginitis. Often personal and social reasons delay medical
intervention.
Key features of vulvovaginitis
• Increased vaginal secretions;
• Sloughed vaginal epithelial cells;
• Bacteria;
• Infectious organisms; and
• Inflammatory cells.
Vaginitis accounts for nearly 6 million visits to the healthcare providers per year with an annual cost of
1over a billion dollars to society. Vulvovaginal irritation, itching, pain, ulceration with bleeding,
dyspareunia, and warty growths are some of the common presenting features of vaginal infections. Most!
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often, a woman may remain minimally symptomatic and may not seek medical help for various personal
and social reasons.
Vaginal Microbiology
Among healthy women, the vaginal milieu is polymicrobial and contains a large number and variety of
2aerobic as well as obligate and facultative anaerobic organisms. The most frequently recovered bacteria
include lactobacilli, Streptococcus viridans, and Staphylococcus epidermidis; none of which cause symptoms.
Bacteroides and Gardnerella vaginalis may be culturable from 20 and 30%, and 50% of the asymptomatic
women, respectively. Staphylococcus occurs infrequently in the healthy vaginal ora. Table 7.1 lists the
microorganisms that can be commonly recovered from vaginal specimens.
Table 7.1 Common microbial organisms in the vaginal flora
Lactobacilli Bacteroides species
Diphtheroids Peptococcus species
Staphylococcus species Peptostreptococcus species
Streptococcus species Fusobacterium species
Enterobacter (not group A) Clostridium species
Gardnerella vaginalis Bifidobacterium
Pregnancy, besides causing a growth of lactobacillary ora, does not appear to a8ect the microbial
composition of the vagina signi cantly. Estrogenic hormones and similar substances help in the epithelial
maturation of the vagina and support the growth of an extraordinary number of microbes. Transplacental
hormonal exchanges also in uence the vaginal epithelium of the newborn infant. Bacterial composition and
an adult type of microenvironment may occur in a newborn female infant.
Menarche and menopausal changes also a8ect the bacterial makeup of the vagina. Hormone or
hormonelike medications, contraceptives, intrauterine contraceptive devices (IUDs), barrier diaphragms, pessaries,
and other similar substances and contraception may directly or indirectly in uence the microbial balance
3of the lower genital tract. Common factors in uencing the vaginal microbial ora are presented in Table
7.2. It must be appreciated that the vaginal ora is in a dynamic state physiologically and in health. It
contains a large number of organisms that, under poorly understood conditions, may become pathogenic
and cause disease.
Table 7.2 Common factors influencing vaginal microbial flora
Physiologic Diseases and drugs Local factors
Parturition Hepatic disorders Infections
Pregnancy Hormonal imbalance IUD
Menstruation Metabolic diseases Pessary
Menopause Erosion and infections Diaphragm
Oral contraceptives Vaginal douche
Hormonal mimic drugs Surgery
Antibodies Trauma
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Sexual exposure
General Features
A number of general cytological features represent the various e8ects of infective processes. These include
the changes listed in Table 7.3. Speci c cytological changes frequently are associated with certain
infections and are described in their respective areas. Only some of these responses may occur under
specific inflammatory conditions.
Table 7.3 Cytologic features of vaginopancervical smears in infective processes
Cellular degenerative
General changes Cellular reactive
Background changes Nuclear Hyperplasia and
repair
Acute and chronic inflammation and cellular Cytoplasmic Degenerative
obscuring
Fresh and old blood Metaplasia
Cytolysis Parakeratosis
Cell distribution changes Hyperkeratosis
Pseudoparakeratosis
Multinucleation
Histiocytic
proliferation
Dysplasia
Background Changes
Inflammation and Cellular Obscuring
Overgrowth of microbes in the vaginal milieu may result in obscuring of morphologic details in the smear
(Fig. 7.1). In such smears, numerous polymorphonuclear leukocytes occur, often interspersed with a large
number of histiocytes. Excessive bacterial growth may also contribute to cellular obscuring. It must be
realized that no meaningful evaluation of cellular change may be possible on such smears. In all such cases,
it is almost mandatory that appropriate therapy be initiated and a repeat smear examined before an
opinion is rendered. Atrophic epithelium of the vagina is particularly prone to in ammatory changes (Fig.
7.2) that may mimic atypia. In such cases local hormonal application generally helps in proper
interpretation of cells. In specimens with overwhelming in ammatory exudates, it may not be possible to
di8erentiate vaginitis from cervicitis and endocervicitis. In ammatory exudates and vaginal microbial ora
4is considerably altered in the liquid-based gynecological slides (LBGS).
Postmenopausal atrophy and atypia:
• Important to treat with local estrogens;
• Repeat cytologic examination after 6 weeks; and
• Immediate colposcopy not recommended.#
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Fig. 7.1 Heavy, acute in/ ammatory exudate. (A) Vaginopancervical smear (Papanicolaou × LP). (B)
Note the reduced background inflammation in LBGS (Papanicolaou × LP)
Fig. 7.2 Atrophic smear with in/ ammatory background and some “atypical” cells (arrows). (A)
Vaginopancervical smear. (B) Same patient after topical estrogen application. Note the cellular maturation
and obvious “atypical” cells (arrow). LBGS (Papanicolaou × MP).
5Cellular specimens from speci c areas, e.g. the vaginal, cervical, and endocervical smear, or vulvar or
lateral vaginal wall scrapings, may reveal in ammatory response in one or more preparations that can help
localize the infective process within the lower genital tract. A speci c cervical in ammation with
predominant lymphohistiocytic reaction may occur in follicular cervicitis (discussed later). Granulomatous
reaction may occur in the presence of foreign bodies (e.g. suture material, surgical clips, IUD) or speci c
infections such as tuberculosis.
Bleeding
In infectious processes, both fresh and old blood may be observed. Postmenopausal women with atrophic,
thin vaginal mucosa may bleed more easily. Similar changes may occur in Trichomonas vaginalis infection,
which produces the typical “strawberry” cervical lesion. Whereas the fresh bleeding is recognizable without
much diE culty, old bleeding, observed as brin, should be di8erentiated from mucus. In direct smear
preparations, brin threads are uniformly thick and reveal nodal formations at the points of intersections of
interlacing threads. Sometimes, hemosiderin pigment may be observed within the macrophages and
extracellularly. Sometimes, old hemorrhage may contain hematoidin crystals that appear as “cockleburs,”
6as described by Hollander and Gupta (Fig. 7.3).
Fig. 7.3 Hematoidin crystals, also known as cockleburs, are seen associated with macrophage response.#
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(A) Vaginopancervical smear (Papanicolaou × MP). (B) Hematoidin crystals LBGS. Note the clean
background and lack of obvious macrophages (Papanicolaou × MP).
Cytolysis, the process of cellular degeneration due to bacterial overgrowth, commonly a8ects the
intermediate squamous epithelial cells. The process is believed to be glycogen dependent. Late menstrual
cycle and pregnancy as well as hormonal contraceptives often cause lactobacilli overgrowth. Pale staining,
vesicular nuclei with little or no cytoplasm of the intermediate cells predominate in such smears. Numerous
lactobacilli may occur interspersed with the remaining cellular remnants (Fig. 7.4).
Fig. 7.4 Lactobacilli. Vaginopancervical smear (Papanicolaou × HP).
Cellular Changes
In the cervicovaginal smears, intermediate and super cial cells occur predominantly among healthy and
normally menstruating women. Heavy in ammation frequently causes an exfoliation of parabasal cells in
the vaginal smear. Although often present in postmenopausal women and in the immediate postpartum
period, occurrence of these three types of cells in the premenopausal age group should be carefully
evaluated. Caution need to be exercised in rendering hormonal evaluation in smears with excessive
in ammation. The parabasal cells may exfoliate from ulceration of the squamous epithelium of the vagina
and the ectocervix.
Under the persistent e8ect of various microbial infections and in ammatory reactions, both squamous
and columnar epithelial cells may undergo degenerative changes. Almost all these changes are nonspeci c,
but their identi cation helps in the proper interpretation of more serious cellular alterations. This is critical
because most degenerative changes may be accompanied by concurrent regenerative, reactive, and
metaplastic changes. An extremely heterogenous group of cellular changes that includes reactive,
degenerative, metaplastic, and neoplastic features is termed atypical squamous cells (ASC). Morphological
features of these cells overlap and generally are nonspeci c, meaning that they cannot be precisely
separated between neoplastic and non-neoplastic changes. Additional studies are often necessary for further
characterization of these changes (discussed elsewhere).
Degeneration
In in ammatory states cytoplasm of the squamous and columnar cells may be completely or partially
disintegrated. However, the major changes are observed within the nuclei. These have been detailed by
7Frost. Brie y; the nuclei may become compact, dense, and pyknotic with loss of all chromatin details (Fig.
7.5). Such nuclei may have a distinct circumferential cytoplasmic clearing or hollow, causing a perinuclear
“halo” (Fig. 7.6), often seen in association with Trichomonas and other infections. Nuclei undergoing
degenerative changes frequently lose the sharp details of their nuclear envelope, the chromatin, and the
interphase. The nuclear chromatin may clump irregularly or appear beaded along the nuclear margins.
They may become blurry and opaque. Other changes include nuclear swelling, with partial or total
disintegration of the nuclear envelope, karyorrhexis, and karyopyknosis (Fig. 7.7).!
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Fig. 7.5 Nuclear degeneration. Vaginopancervical smear (Papanicolaou × HP).
Fig. 7.6 Perinuclear clearing or halos. Vaginopancervical smear (Papanicolaou × MP).
Fig. 7.7 Nuclear degeneration among endocervical cells infected with adenovirus infection.
Vaginopancervical smear (Papanicolaou × HP).
Degenerative, Regenerative (Repair), and Metaplastic Changes in Inflammation
The epithelial cells of the lower genital tract (ectocervix, endocervix, and transformation zone), under the
in uence of persistent irritation (infectious and non-infectious) and repair, undergo morphologic changes
commonly referred to as metaplasia. These essentially re ect a benign process of tissue repair. An
overestimation of reparative changes is a most common error in interpretive cytology. Geirsson and
co8workers referred to these as atypical reparative changes (ARC). The majority of these changes are believed
to be derived from the columnar and squamous epithelia, but reserve or pluripotential cells may be
involved in the genesis of the metaplasia. It must be appreciated that there is a continuum of changes
observed in the healing phase. Cytomorphologic changes, for convenience, are grouped under the term
metaplasia. Some of these morphologic changes can be indistinguishable from “early” intraepithelial
dysplastic alterations, observed within both the squamous and the glandular epithelia. These cellular
features are grouped as “atypical squamous or glandular cells of undetermined signi cance” (ASC, AGC)
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The earliest discernible changes are referred to as presquamous metaplasia: columnar di8erentiation
phase, or type I ARC of the cervical columnar epithelium. Under the e8ect of chronic irritation and repair
processes, the surface cells of the columnar epithelium continue to mature, and there is a proliferation of
the basal or reserve cells. These small, undi8erentiated cells commonly occur in small tissue fragments.
They have high nucleus-to-cytoplasmic (N/C) ratios and prominent nucleoli (Fig. 7.8). The nuclear
chromatin is ne and uniformly distributed, and the nuclear membrane is well delineated and thin. These
may have an in ammatory background. The cells of subluminal origin (progenitor cells) can often be
mistaken for undifferentiated neoplasia.
Fig. 7.8 Squamous metaplasia (atypical reactive cells, ARC I). LBGS (Papanicolaou × MP).
As the changes evolve, the subluminal cells di8erentiate from the germinal layers upward. These changes
re ect the immature squamous metaplastic epithelium and have been referred to as ARC II. The cells may
appear to be columnar and have excessive goblet cell proliferation and mucus production. Signet-ring forms
may be recognized. These cells can have numerous macronucleoli, coarse chromatin, and modest but pale
cytoplasm. If not carefully examined, the changes can be mistaken for a neoplastic lesion of the endocervix
(Fig. 7.9). Presence of heavy acute in ammation generally helps in the correct interpretation of these
changes. In the LBGS, in ammation is less obvious and these cells often occur as small tissue fragments and
may be reported as AGC.
Fig. 7.9 Squamous metaplastic changes with columnar cell hyperplasia (ARC II). LBGS
(Papanicolaou × MP).
As the changes progress from the subluminal, via the presquamous, to the keratinizing strati ed squamous
phase, the cells become oval or polygonal with sharp borders and dense cytoplasm. They lie in sheets and
reveal no obvious cilia and mucus. Intercellular bridges may be seen at times. These cells with their
metaplastic changes have been called ARC type III. The nuclear changes may be reactive or degenerative
with pyknosis. These cells may be mistaken for squamous cell carcinoma (Fig. 7.10).#
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Fig. 7.10 Metaplastic changes revealing keratinizing strati ed squamous metaplasia (ARC III).
LBGS (Papanicolaou × MP).
Squamous Epithelium
When stressed, as by a chronic irritation or an infective injury, squamous epithelium responds in a number
of ways. These changes essentially represent alteration of functional di8erentiation of the a8ected cells and
are mostly cytoplasmic in nature. Proper identi cation of these cytoplasmic features is necessary, as they
may mask a more serious underlying disease process. These changes—hyperkeratosis, parakeratosis, basal
cell hyperplasia, pseudoparakeratosis, and dyskeratosis—have been discussed elsewhere in this book. They
re ect abnormalities of maturation with normal keratin formation in cells that normally do not reveal these
changes.
Squamous epithelial cell changes
• Hyperkeratosis;
• Parakeratosis;
• Basal cell hyperplasia;
• Pseudoparakeratosis; and
• Dyskeratosis.
Dyskeratosis is mentioned here because of its relationship with viral infections and developing cancer.
This represents an abnormality of the squamous cells in which the cytoplasmic maturation is altered. The
a8ected cells reveal premature, hypermature, or atypical keratinization. It is a common occurrence in the
presence of chronic infections, such as those caused by human papillomavirus (HPV). The cytomorphologic
features are further detailed in the appropriate sections.
Endocervical Columnar Epithelium
In addition to the squamous metaplasia discussed earlier, endocervical cells may undergo other
morphologic changes including columnar cell hyperplasia and hyperplastic polyp formation.
Columnar Cell Hyperplasia
Endocervical cells frequently enlarge and produce excessive mucus. Such changes occur with chronic
8irritation of the endocervical canal, such as among women using IUDs or hormonal contraceptives, and
who are exposed to certain infections; these are discussed separately.
Hyperplastic Polyp
Hyperplastic endocervical columnar cells may proliferate to produce nger-like epithelial processes—
9 10polyps. As described by Ramzy and Frost, these polyps are three-dimensional structures with three
distinct planes—a oor or base composed of a sheet of polygonal cells, a middle plane that makes up the#
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sides of the polyp, and a top or surface layer that, like the base, is also a sheet of polygonal cells. In the
center of the polyp, a connective tissue core that contains broblasts, collagen, and capillary vessels, may
be recognizable.
Tubal Metaplasia
Generally, endocervical epithelium may contain ciliated columnar cells only in small numbers (5–10%).
Under conditions of chronic irritation, sheets of ciliated cells representing tubal metaplasia may occur in the
specimens collected from the transformation zone and the adjacent endocervix. These cells may show
11pseudostrati cation and atypia and can be a common source of misinterpretation as neoplastic cells.
INK 4A 12Tubal metaplastic cells may stain positively with p16 antibodies, which may lead to confusion
with neoplastic processes.
Columnar epithelial cell changes
• Hyperplasia
• Polyp formation
• Squamous metaplasia
• Tubal metaplasia
Endometrium
Heavy acute in ammation, with pronounced reactive, degenerative, and metaplastic changes may be
observed in these cells during the later half of menstrual bleeding, following instrumentation, in the
postpartum period, and in association with the usage of an IUD. Retained gestational products and foreign
bodies may result in extensive squamous metaplasia, multinucleated giant cell reaction, and calci cation.
Some of these changes are discussed later in this chapter.
Infections of the Female Genital Tract
Bacterial Infections
13Bacteria most commonly infect the female genital tract. Bibbo and Wied reported nonspeci c organisms
including mixed bacteria and coccobacilli in nearly 20% of patients. Among children these infections occur
commonly and may be hormonally dependent. Vaginal or vaginopancervical smears often reveal a number
14of bacilli and coccid organisms (mixed infections) as detailed by Wied and Bibbo. These organisms,
although di8usely scattered, may occur in clumps and as microcolonies. Appropriate microbiologic
isolation techniques are necessary for speci c species identi cation but is generally not considered
necessary for clinical management of the disease.
15Bacterial vaginitis (nonspeci c vaginitis) was rst described by Gardner and Dukes. They stated, “Any
woman whose ovarian activity is normal and who has a gray, homogenous, malodorous vaginal discharge
with a pH of 5.0 to 5.5 that yields no Trichomonads is likely to have Haemophilus vaginalis vaginitis.” It is
16also known as nonspeci c vaginosis/vaginitis so named by Blackwell and Barlow, or bacterial vaginosis
17(BV), a term used by the International Agency for Research on Cancer. This is the most common cause
for the clinical entity of bacterial vaginosis. Instead of making a speci c diagnosis of G. vaginalis infection,
reporting of a “shift in the bacterial ora” is the current term used to describe the organism variously
18named as Haemophilus vaginalis and Corynebacterium vaginalis. Gardner and Dukes rst described these
organisms. Regarding the etiology of BV, the statement by Fredricks and Marrazzo that “BV probably
results from infection with complex communities of bacteria that consist of metabolically interdependent
19(syntrophic) species” appears true.
Morphologically, the organisms are Gram-negative or Gram-variable, are 0.1–0.8 nm in diameter, and
appear bacillary or coccobacillary. The microbe, although it shares many characteristics with#
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20Corynebacterium, is catalase-negative and is now classi ed separately. Petersdorf and colleagues and
21Ledger and associates found that as many as 40–50% of women may have vaginal infection with G.
vaginalis and be asymptomatic. Among symptomatic women, leucorrhoea and pruritus with in amed
vaginal mucosa and occasional punctate hemorrhages are commonly observed. Increased growth and
17concentration of these organisms may not denote pathogenicity. It is believed that patients with pure G.
vaginalis infection are asymptomatic when the vaginal pH is less than 4.5. Secondary organisms interplay
with G. vaginalis and alter this synergistic relationship. A raised pH over 4.5 (5.0 to 6.5) and an interaction
with various bacteroides and peptococci may produce clinical disease. Recently, considerable interest has
been exhibited in the study of BV. Molecular identi cation of the associated bacteria has revealed the
presence of three bacteria in the Clostridiales order. These are named as bacterial vaginosis-associated
22bacterium (BVAB1, BVAB2, and BVAB3). BVAB are considered highly specific for BV infection.
Patients with a high pH of the vagina have a vaginal discharge with a distinct shy odor. When the pH is
23further raised by potassium hydroxide (KOH), this odor is manifest in the “whi8 test.” Such preparations
of vaginal reactions and KOH, when examined microscopically, have the diagnostic “clue cells” (Fig. 7.11).
These refer to normal polygonal squamous cells having thin, transparent cytoplasm covered by tiny
coccobacillary forms of G. vaginalis. Edges of the “infected” cells reveal the BV changes. The cell borders
may be indistinct and on a di8erent plane of focus. Similar clue cells are observed in the xed and stained
Papanicolaou preparations (Fig. 7.12). A variable amount of acute in ammation may be present in the
background. Mere complete or partial covering of the squamous epithelial cells by the organisms (Fig. 7.13)
or their sticking to the cellular margins (Fig. 7.14) per se should not be considered diagnostic for G.
vaginalis. To be diagnostic, clue cells should have bacterial organisms not only covering the surfaces of the
a8ected cells but also spreading beyond the margins of the squamous cells. In LBGS preparations,
organisms appear in a clean background (Fig. 7.15). Detection of BV is reported to be considerably less in
4LBGS-based preparations than in conventional slides. This may not be an entirely true observation. A high
degree of diagnostic accuracy exists in cytologic detection of clue cells and culture con rmation for G.
24vaginalis. Schnadig and co-workers cultured G. vaginalis in nearly 90% of the cases that contained clue
cells. This infection is believed to be sexually transmissible, and an accurate diagnosis is necessary.
Fig. 7.11 “Clue cells” phase contrast. Vaginopancervical smear (unstained × MP).#
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Fig. 7.12 Gardnerella vaginalis (BV) infection. Vaginopancervical smear (Papanicolaou × LP).
Fig. 7.13 Partial obliteration of the squamous cell by the coccobacillary organisms. Vaginopancervical
smear (Papanicolaou × MP).
Fig. 7.14 Organisms sticking to squamous cell. Vaginopancervical smear (Papanicolaou × MP).
Fig. 7.15 “Clue cells” in LBGS (Papanicolaou × LP).
Micrococcus Vaginitis (Toxic Shock)
This entity is now rarely observed in current practice. This group of microbes includes a large number of
Gram-positive coccoid organisms commonly observed in female genital tract smears, and Gram-negative
diplococci. Staphylococcus aureus may be recovered from the vagina in about 5% of normal women. These
organisms frequently cause vaginitis and vaginal discharge and may produce toxic shock syndrome. This
25association was documented by Shands and co-workers in 1980. These organisms characteristically occur
singly and can be seen within the polymorphonuclear leukocytes or other infected epithelial cells. In
vaginal smears, occasionally fragments of tampon bers may be observed (Fig. 7.16). However, the nding
of coccoid organisms or tampon bers in the vaginal smear does not have any correlation with the clinical
occurrence of toxic shock syndrome.>
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Fig. 7.16 Tampon bers. Occasionally, these may have a core center that may contain red blood cells.
Vaginopancervical smear (Papanicolaou × LP).
Lactobacillus Vaginitis (Cytolytic Vaginosis)
Lactobacilli are a heterogeneous group of organisms normally present in the vaginal ora. They occur in
abundance in the late luteal phase and in pregnancy, prefer an acid environment, and are common among
women using hormonal preparations (contraceptives and replacements) and in the premenarchal and
menopausal age groups. They are Gram-positive, immobile, non-spore-forming anaerobes or facultative
anaerobes. Certain species may be aerobic in their growth characteristics. In the presence of lactobacilli,
glycogen-rich intermediate cells are often lysed. Smears in such cases show cellular crowding, cytolysis with
cytoplasmic debris, and numerous bare nuclei occurring in a predominantly bacillary background. False
clue cells can be reported in these cases as the lactobacilli adhere to the edges of squamous cells.
Lactobacilli may be observed in up to 50% of healthy women depending on the day of the menstrual cycle.
In the symptomatic population, the observed gure may be lower, about 20%. It is debatable whether pure
lactobacilli (an unlikely occurrence) produce vaginitis, although vaginal discharge and leucorrhoea may
occur as a result of excessive cytolysis.
Gonococcus Vaginitis
These Gram-negative diplococci cause abundant, purulent vaginal exudates. The infection a8ects the
urethra and the perivaginal glands. On the surface of squamous cells, these organisms occur as bean-shaped
diplococci. The gonococci are better observed in the air-dried areas of the smears, such as the edges of the
smear. This is an uncommon occurrence in properly prepared LBGS. Within the air-dried distended
polymorphonuclear leukocytes, diplococci may be present in large numbers (Fig. 7.17). Gonococcus
vaginitis is a venereal infection with important social and medical implications. Although it is detectable
cytologically, we do not advise rendition of such a diagnosis on cytologic examination of Papanicolaou
stained smears alone; they may be indistinguishable from other cocci organisms, phagocytosed debris, or
Chlamydia organisms.
Fig. 7.17 Gonococcal organisms. These reveal diplococci within the polymorphonuclear leukocytes, and
on the surface. Vaginopancervical smear (Papanicolaou × OI).#
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Curved Anaerobic Bacterial Vaginitis
These motile, anaerobic, rod-shaped organisms resemble Wolinella and have been recognized as a cause of
2,26,27nonspeci c vaginitis by Hjelm and colleagues. In the Papanicolaou stained smears, these bacteria
cannot be easily diagnosed but are better detected in wet mount preparations. Clinically, the presentation is
of nonspecific vaginitis.
Vaginal Lactobacillosis
A recently recognized clinical picture has been reported among women who have used antifungal local
medications for genital Candida infection for a prolonged period of time, generally more than 20 months.
These result in the proliferation of giant lactobacilli accompanying often the yeast forms of Candida
organisms. A correct morphologic recognition of this condition is important for speci c treatment with
28,29appropriate antibiotics (Fig. 7.18).
Fig. 7.18 Vaginal lactobacillosis. This picture reveals numerous “giant” lactobacilli along with some
yeast forms of Candida (Papanicolaou × HP).
Foreign-Body Vaginitis
A forgotten tampon is the most common cause of this type of vaginitis, in which there is a secondary
overgrowth of anaerobic organisms. The tampons may irritate and ulcerate the vaginal wall and ectocervix.
Occasionally, fragments of tampons can be observed in vaginal smears. Their presence is not diagnostic of
vaginitis. Heavy acute inflammation, mucus, and foreign-body giant cells may be observed.
Allergic and Acute Vaginitis
Numerous eosinophils may occur in the cervical samples obtained from women with vaginal discharge.
Generally, the causes are noninfectious and associated with an allergic reaction to vaginal douche,
30contraceptives, or various items of clothing (Fig. 7.19).
Fig. 7.19 Allergic vaginitis. Note the numerous eosinophils in this preparation. Cervicovaginal smear
(Papanicolaou × MP).Desquamative Inflammatory Vaginitis (DIV)
This clinical condition is noninfectious in nature and may result from a number of blister-forming disorders
31including pemphigus vulgaris, lichen planus, and pemphigoid.
Pemphigus vulgaris may exfoliate parabasal size cells that have extremely prominent single or multiple
nucleoli, pale chromatin, and features of reactive cells. Mitosis may be observed. Nuclear and cytoplasmic
changes can simulate squamous cell carcinoma or atypical endocervical or metaplastic cells (Figs. 7.20,
327.21).
Fig. 7.20 Pemphigus vulgaris. In (B) cells show metaplastic changes. These features can be confused with
neoplastic as well as viral changes. Vaginopancervical smear (Papanicolaou × MP).
Fig. 7.21 Pemphigus vulgaris, cervix. Tissue biopsy reveals cellular changes similar to those seen in the
smear in Fig. 7.20. Cervical biopsy (H&E × MP).
Granuloma Inguinale
Gram-negative, encapsulated coccobacillary organisms called Calymmatobacterium granulomatis cause this
venereally transmitted infection. The infection produces large, ulcerated lesions that histologically reveal!
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in ammatory granulation tissue and numerous macrophages. These macrophages are easily identi able in
ethanol- xed Papanicolaou stained smears. They are plump and swollen and have a lobulated cytoplasm.
Within the cytoplasm, a large number of coccobacillary (1–2 m) structures (Donovan bodies) are seen.
These are safety-pin shaped with terminal or polar thickening of the cell walls (Fig. 7.22). The organisms
stain faintly with hematoxylin and eosin (H + E) dyes. They can be stained with Romanowsky or silver
stains. Varying degrees of acute in ammation are commonly observed in the smears. The infection is more
common in the tropics, and the incidence is high in India and New Guinea. Although reported, an
association of granuloma inguinale and squamous cell carcinoma is controversial. It is true that the
infection may cause extremely bizarre pseudoepitheliomatous hyperplasia of the squamous epithelium that
can mimic neoplasm.
Fig. 7.22 Donovan bodies. A single macrophage reveals the cytoplasmic lobules containing numerous
safety-pin-shaped bacillary structures. Vaginopancervical smear (Papanicolaou × OI).
Tuberculosis (Granulomatous Cervicitis)
This is a disease of the tropics and is usually secondary to extragenital, most often pulmonary, tubercular
infection. Involvement is more common in the fallopian tubes and the endometrium and is thus diE cult to
33adetect cytologically. Angrish and Verma reported a number of cases of cervical tuberculosis that were
detected cytologically. The cervical smears reveal large aggregates of epithelioid cells. These appear as pale,
cyanophilic cells in a syncytial formation with indistinct and arborizing borders and vesicular, oval nuclei
(Fig. 7.23). Intermixed with these one may occasionally observe Langhans-type multinucleated giant cells
(Fig. 7.24). These cells may contain as many as 20 to 30 peripherally arranged vesicular nuclei. A variable
number of lymphocytes may be present in the background. Secondary infection is common in these
ulcerated lesions, and heavy, acute in ammatory exudates may be present. A cytologic diagnosis of
granulomatous disease, probably tuberculosis, can be suggested under appropriate clinical and cytologic
settings.
Fig. 7.23 Epithelioid cells. Notice the syncytial formation of cells with ill-de ned margins.
Vaginopancervical smear (Papanicolaou × MP).!
Fig. 7.24 Cervical tuberculosis (H&E × LP).
Malacoplakia
Malacoplakia is a rare disorder that may a8ect the cervix. We have observed two cases occurring in
33postmenopausal women with atrophic smears and persistent vaginal discharge. Numerous macrophages
with the characteristic intracytoplasmic, laminated inclusions (Michaelis-Gutmann bodies) may be observed
(Fig. 7.25). They can be stained for calcium salts including calcium phosphates and carbonates by
34histochemical techniques such as Von Kossa's method.
Fig. 7.25 Michaelis-Gutmann bodies. Intracytoplasmic laminated structures from a case of malacoplakia
of the cervix. Vaginopancervical smear (Papanicolaou × HP).
Langerhans Cell Histiocytosis
This rare disease of unknown etiology may involve the lower female genital tract and the endometrium. It
has been included here because the lesions can both clinically and cytologically may be indistinguishable
from in ammatory or neoplastic processes. It can occur as “pure” genital tract or part of the generalized
systemic disease. Cervical cytology may contain atypical histiocytic cells (Fig. 7.26), numerous
macrophages with intranuclear grooves, eosinophils, and an occasional multinucleated giant cell (Fig.
7.27). This diagnosis may be considered in the presence of intranuclear grooves in the macrophages, and
eosinophils in the smear. The exact morphologic features vary from the stage of the disease.
Immunohistochemical stains S100 and CD1a as well as ultrastructural demonstration of Birbeck granules
35,36are helpful diagnostically.#
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Fig. 7.26 Langerhans cell histiocytosis. Note numerous histiocytic cells with intranuclear grooves
(arrowhead) and an occasional eosinophil (arrow). Vaginopancervical smear (Papanicolaou × HP).
Fig. 7.27 Langerhans cell histiocytosis. Note atypical histiocytic cells and numerous eosinophils. This
smear was obtained 14 months after that in Fig. 7.26. Vaginopancervical smear (Papanicolaou × HP).
Actinomyces
These organisms belong to the order of higher bacteria that also include Mycobacteriaceae and
Streptomycetaceae. There are three common species of Actinomyces—A. israelii, A. bovis, and A. naeslundii.
These bacteria are nonmotile, non-spore-forming, and anaerobic or facultative anaerobes. They are
Grampositive and occur in lamentous and diphtheroid forms. With the recent increase in IUD usage, genital
Actinomyces infection appears to also be increasing in prevalence. It is, however, true that the new device
37designs and the judicious usage make the clinical disease less likely.
Actinomyces occur commonly within the tonsillar crypts, tartar of teeth, and the alimentary tracts.
Actinomyces do not occur as commensals in the vaginal ora. In the female genital tract, ascending
infection is the most common mode of occurrence of clinical disease; however, rarely, hematogenous and
lymphatic spread, or dissemination of infection from the alimentary tract or other distant sources, may
occur. Ascending infection occurs in the presence of intrauterine or intravaginal contraceptives, IUDs of
various types being the most common. Vaginal pessaries, surgical clamps, and foreign bodies, including
forgotten tampons, all have been associated with vaginal Actinomyces. Among untreated women, clinical
disease may be manifest for as much as 12 months after the removal of the Actinomyces-associated IUD.
Gupta has reviewed the subject and the relationship of Actinomyces with clinical female genital tract
38disease. It is appropriate to say that nearly 10% of women using an IUD may develop vaginal
Actinomyces infection at some stage. If such users have symptoms of lower genital tract infection such as
pelvic pain, vaginal discharge, bleeding, fever, or lower abdominal tenderness, approximately one-quarter
of these women may have genital Actinomyces infection. Of the women using an IUD and being admitted to
the hospital for clinically suspected pelvic in ammatory disease, about 40% may harbor the organism in#
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39,40the lower genital tract. Dissemination of the infection to distant sites has been documented by de la
41 42Monte and co-workers, and by Hager and Majmudar.
Cytomorphology of Actinomyces
In close proximity to the calci ed and mineralized fragments of a disintegrating IUD, the Actinomyces
organism can be detected in Papanicolaou stained vaginal smears. Typically, the organisms appear as
spidery, amorphous clumps that are darker in the center (Fig. 7.28A). Morphologic features of the
Actinomyces colonies are more distinct in LBGS-based slides (Fig. 7.28B). These aggregates of Actinomyces in
42the cervicovaginal smears have been referred to as “Gupta bodies” by Hager and Majmudar. Upon
careful examination, numerous lamentous organisms with acute angle branching patterns are
recognizable in these clumps (Fig. 7.29). They can be uniformly thick and beaded. The laments generally
extend to the outer limits of the dark clumps. Only a few delicate, branching lamentous forms may occur
scattered randomly in the smear. In Papanicolaou stained smears, calci ed lamentous forms that may not
be stainable by antigen antibody techniques, club forms, or the Splendore Hoeppli phenomenon may be
identi ed. Typical sulfur granules may be observed in smears obtained from symptomatic patients (Fig.
7.30). These per se are not diagnostic of Actinomyces, and proper morphologic identi cation of the
lamentous forms is necessary in all cases. Gupta and co-workers have detailed various other morphologic
38,43forms.
Fig. 7.28 Actinomyces. (A) Vaginopancervical smear (Papanicolaou × LP). (B) LBGS (Papanicolaou ×
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Fig. 7.29 Colonies of Actinomyces. (A) Note the numerous lamentous structures radiating from the
center. Vaginopancervical smear (Papanicolaou × HP). (B) Higher magni cation of the colonies of
Actinomyces (Papanicolaou × HP).
Fig. 7.30 Sulfur granule. In the center are radiating lamentous structures of Actinomyces organisms.
Vaginopancervical smear (Papanicolaou × MP).
Actinomyces organisms can be stained with modi ed Gram, periodic acid-Schi8 (PAS), and silver stains.
A de nitive species diagnosis requires speci c antigen antibody reaction using an immunoenzymatic or
44immunofluorescence or bacterial culture procedures.
A number of organisms, including Candida, dermatophytes, and Nocardia, along with bacterial
aggregates, and foreign substances such as sulfa drug crystals and contraceptive creams, may resemble
6Actinomyces organisms. Hematoidin crystals described by Hollander and Gupta have a resemblance to
sulfur granules. The di8erential diagnosis of Actinomyces as seen in the vaginopancervical smear is
presented in Table 7.4.
Table 7.4 Differential diagnosis of Actinomyces in vaginal smearsOther Candida, Aspergillus, Nocardia, Penicillium, Trichophyton, Leptotrichia, lactobacilli
organisms
Miscellaneous Filamentous structures: Fibrin, mucus, sulfa crystals, cotton and synthetic fibers
structures
Nonfilamentous structures: Contraceptive cream, bacterial clumps, hematoxylin pigment,
spermatozoa, hematoidin, foreign material (spores, pollen, douche ingredients)
We believe that genital Actinomyces is an exogenous infection. Orogenital contact may be an important
mode of acquiring the genital Actinomyces infection. The “tail” of the IUD most likely acts as a carrier for
the ascent of the organisms. The tissue damage produced by the body and edges of the IUD causes a change
in the oxygen reduction potential and alteration in the microbial milieu of the lower genital tract. The
changed environment is conducive to the growth of these organisms. Actinomyces has been observed with
all types of IUDs, including currently marketed models. Infection is more common with devices with
polyfilamentous thread and with angular forms.
Key features of genital Actinomyces
• Always associated with an IUD or a foreign body;
• May cause no symptoms;
• Occur as dark, woolly clumps (Gupta bodies);
• Parallel filaments, branching at acute angle;
• Difficult to culture; and
• May be confirmed by special stains.
Occasionally, Actinomyces may occur in association with “black yeast,” a fungus Aureobasidium pullulans,
commonly found in areas with poor hygienic conditions. It has large, dark-fruiting bodies (Fig. 7.31). As
reported by de Moraes-Ruehsen and associates, Entamoeba gingivalis, a protozoan of the oral cavity, may be
45found in association with Actinomyces in vaginal specimens (Fig. 7.32). An orogenital route of this
Actinomyces infection is a distinct possibility. These nonpathogenic protozoa should be distinguished from
Entamoeba histolytica that occur in the alimentary tract and which may also cause lower genital tract
infection.
Fig. 7.31 Aureobasidium pullulans. These black yeast organisms can vary in color from light yellow,
goldbrown, to black. Vaginopancervical smear (Papanicolaou × HP).
Reproduced with permission from Gupta PK: Intrauterine contraceptive device: Vaginal cytology, Pathologic changes,
and their clinical implications. Acta Cytol 1982;26:571-613.!
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Fig. 7.32 Entamoeba gingivalis. (A) Vaginopancervical smear (Papanicolaou × HP). (B) LBGS
(Papanicolaou × HP).
IUD-Associated Cellular Changes
In addition to the alterations in the microbial environment and Actinomyces infection, usage of the IUD is
associated with cellular changes occurring in the various genital tract epithelia, as early as 10–12 weeks
after an IUD insertion. These result from chronic irritation by the IUD tail and the body a8ecting the
adjacent tissues within the endocervix and the uterine cavity. It is important to recognize these morphologic
features as they can mimic and be confused with dysplastic and neoplastic cellular changes of the
squamous, metaplastic, endocervical, and endometrial epithelia. These changes appear more pronounced in
LBGS and interpretation can be problematic especially in the paucity of an in ammatory background.
There is no de nite evidence for the association of squamous dysplastic changes and IUD usage. Squamous
cell changes are essentially reactive and reparative in nature. These occur in about 40% of women using
IUDs. DNA analysis of IUD-associated cellular changes does not reveal any aneuploidy.
The morphological picture is further complicated by interplay among the reactive-proplastic and
degenerative-retroplasticchanges occurring over a prolonged period and a8ected by polymicrobial and
physiologic factors.
Endocervical columnar cells may become hyperplastic with large papillary tissue fragment formations.
46 47Bibbo and co-workers and Gupta and colleagues have systematically reviewed these changes.
Columnar cell hyperplastic changes should be distinguished from adenocarcinoma (Fig. 7.33). They may
mimic papillary tumors of ovarian or endometrial origin. Single cells can be extremely bizarre and resemble
neoplasia. Cells may show large cytoplasmic vacuoles referred to as “bubble gum” cells. The presence of
heavy in ammation and degenerative changes helps diagnostically. The salient features of these cellular
changes are summarized in Table 7.5. The presence of psammoma or calci ed bodies among IUD users is
not an indication of neoplasm.
Fig. 7.33 “Bubble-gum” cells occurring in a patient with an IUD. (A) Vaginopancervical smear
(Papanicolaou × HP). (B) IUD-associated glandular cells LBGS (Papanicolaou × MP).
Table 7.5 Comparison of IUD-associated columnar-type cells and adenocarcinoma cells
Feature IUD columnar cells Tumor cells
Tumor diathesis Absent Present
Distribution Endocervical Random#
Inflammation Present Variable
Cellular degeneration Present Absent|
“Bubble gum” cytoplasm Present Absent
Bare nuclei Absent Present
Cellular preservation Poor Good
Atypical histiocytic cells Absent Present
Another cell type, best described as indeterminate cell changes or “IUD cells,” probably arises from the
47endometrial surface. Such conclusions are supported by the work of Gupta and co-workers. These cells
with a high nucleus-to-cytoplasmic ratio should be distinguished from the third type of cell described by
48Graham and from in situ carcinoma (HSIL, CIN III) cells. Nuclear degeneration, the presence of nucleoli,
and a hiatus between normal and abnormal cells help di8erentiate these cells from true neoplastic cells
(Fig. 7.34) . Table 7.6 summarizes the salient features of these cells. Occasionally, the endometrial-type
reactive cells and the IUD cells may occur together.
Fig. 7.34 “IUD cells.” These high nucleocytoplasmic (N/C) ratio cells appear to be of endometrial origin.
They frequently show multinucleation, nuclear degeneration, and nucleoli. If not carefully examined, these
can be easily mistaken for cervical intraepithelial neoplasm. (A) Vaginopancervical smear (Papanicolaou ×
HP). (B) IUD cells in LBGS (Papanicolaou × OI).
Table 7.6 Comparison of IUD cells and cervical intraepithelial neoplasia (CIN/HSIL) cells
Feature IUD cell CIN/HSIL cell
Distribution Endocervical Endocervical
Tissue fragments Rare Common (LBGS)
Inflammation Present Absent
Cellular degeneration Present Absent
Preservation Poor Good
Cellular hiatus Present Absent
Nucleoli Present Absent
Multinucleation Present Absent
IUD columnar cells Present Absent
Binucleated and multinucleated giant forms and psammoma body formation are other ndings that may
be observed in the presence of the IUD and Actinomyces. These develop from endometrial surface changes.
Extensive squamous metaplasia of the endometrial surface may occur in some cases as the result of
prolonged endometritis accompanying the IUD.
Key features of IUD-associated cellular changes#
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• Bubble gum cells;
• IUD cells;
• Metaplastic cells;
• Mesenchymal proliferation;
• Multinucleation; and
• Psammoma body formation.
Leptotrichia buccalis
These microbes, also known as just Leptotrichia or Leptothrix, are Gram-negative, non-spore-forming
anaerobic organisms. They occur in the oral and vaginal cavities as very thin, segmented, large, filamentous
structures. Occasionally, branching may be observed (Fig. 7.35). Morphologically they may be
indistinguishable from certain forms of Doederlein's bacillus. Most frequently (75–80%), cases of
Leptotrichia have concomitant T. vaginalis infection. Numerous other infective organisms, including Candida
and G. vaginalis, may occur in the presence of L. buccalis infection.
Fig. 7.35 Leptotrichia buccalis. Vaginopancervical smear (Papanicolaou × HP).
13Bibbo and Wied made an investigative study on the prevalence of Leptotrichia in cervicovaginal
smears. They observed Leptotrichia organisms in 75% cases with trichomonads, 1.5% with Doederlein's
bacillus, and about 1% among patients with fungal or BV infection. Nearly half (47%) of the 1,000 patients
studied were oral contraceptive users. Pregnancy and menopause were other physiologic features, followed
by the postpartum state, that were often associated with the presence of L. buccalis in cervical smears.
Sometimes acute inflammatory changes may be observed in the presence of Leptotrichia.
Mycoplasma
These are the smallest known organisms capable of growing in cell-free media. Jones and Davson
61documented a correlation between the occurrence of a “dirty” smear and mycoplasma, and Mardh and
co-workers con rmed these ndings and reported the occurrence of coccoid organisms both on the surface
49and in between the squamous epithelial cells in dirty smears in cases of Mycoplasma. Such features
48a,49appear to have limited practical value.
Follicular Cervicitis
Also referred to as lymphocytic cervicitis, this is a speci c type of cervical, and sometimes vaginal, lesion in
which the predominant feature is the occurrence of lymphoid follicles in the subepithelial areas. When
examined cytologically, numerous mature and reactive lymphoid cells and germinal macrophages (tingible
50bodies) are seen (Fig. 7.36A). Cellular changes are uncommonly recognized in LBGS (Fig. 7.36B) and are
often more problematic to interpret. Small lymphoid cells appear in aggregates especially in LBGS and do51not clearly reveal tingible bodies. Lack of mucus and small size of the lymphocytes appear to contribute
to these morphologic changes. At times, a capillary from the germinal center of the lymphoid follicle may
be scraped and observed in the smear (Fig. 7.37). There is evidence that nearly 50% of the cases of
52,53follicular cervicitis are associated with Chlamydia infection. Follicular cervicitis is not uncommonly
seen in postmenopausal atrophic smears. The precise pathogenesis of this condition in this age group is not
well understood. These cells should be distinguished for malignant lymphomas, histocytes, endometrial
cells, and metastatic tumor cells.
Key features of follicular cervicitis
• Must identify tingible-body macrophage;
• Difficult to diagnose in LBGS due to lymphocytic dispersion; and
• Cells should be distinguished from:
– Lymphoma;
– Metastatic tumor cells;
– Endometrial cells; and
– Histiocytes.
Fig. 7.36 ( A ) Follicular cervicitis. Vaginopancervical smear (Papanicolaou × OI). (B) Follicular
cervicitis, LBGS. Diagnosis is diE cult to make. Tingible macrophages (arrow) and lymphocytes may be
observed (Papanicolaou × HP).
Fig. 7.37 Follicular cervicitis. Note the occurrence of a germinal follicle and a capillary in this picture.
Vaginopancervical smear (Papanicolaou × LP).
Viral Infections
Diseases caused by these intracellular organisms are among the most common in the human body and
include a most heterogeneous group of clinical conditions. Although some of the viral infections have been
a8ecting humanity for thousands of years, changes in society, social habits, medical practice, and advances
in diagnostic capabilities have resulted in a great many new viral diseases. Even though smallpox has been
eradicated from the world, with antibiotics and immunosuppressive therapies, numerous dormant viralinfections have become manifest.
Being intracellular by nature, viruses co-opt cellular metabolic processes in their replication cycles. In
addition to the nature of the a8ected tissues, the virus, general and local immune responses, and particular
enzymatic derangements are important in determining the cytomorphologic changes and the nature of the
tissue injury or injuries. In some common viral infections, these cellular changes may be quite typical and
considered of diagnostic significance.
General Features of Viral Infection
Inclusion Formation
Inclusions are discrete, dense, homogeneous, round, or oval intracellular structures consisting of viral
particles in a matrix, and generally represent a stage in the replication of the virus. They do not occur in all
viral infections, their formation depending upon a particular agent and on the a8ected tissue. Certain
inclusions are typical and diagnostic. Inclusions may be observed within the nuclei, the cytoplasm, or both.
Hydropic or Ballooning Degeneration
This particular cellular change is often an e8ect of organelle membrane damage caused by the virus.
Certain degenerative changes precede or accompany the development of inclusion bodies and are often
used in the diagnostic evaluation of cellular changes (Fig. 7.38).
Fig. 7.38 Hydropic degeneration. Vaginopancervical smear (Papanicolaou × HP).
Necrosis
Viruses may cause coagulative necrosis and characteristic cytoplasmic changes. Most often, the cytoplasm
becomes opaque and thickened and loses its transparency and crispness. Nuclear degenerative changes with
karyolysis and karyorrhexis may occur (Fig. 7.39). Only ghost forms of the infected cells may remain.
Fig. 7.39 Nuclear degeneration. LBGS (Papanicolaou × HP).#
#
Giant Cell Formation
Alterations in the membrane composition of the infected cells contribute to the fusion of cells to produce
syncytial and giant forms. Sometimes nuclear inclusions may occur within the multinucleated forms.
Cellular Proliferation
Transient cellular proliferation is commonly seen in viral infections. These changes may be extreme and
mimic dyskaryosis and neoplastic forms (Fig. 7.40).
Fig. 7.40 ( A) Atypical cellular proliferation, LBGS (Papanicolaou × MP). (B) “Atypical” cellular
changes in cervical herpes, LBGS (Papanicolaou × HP).
Cellular Cohesion
In certain viral infections, the initial step is attachment to the host cell; viral proteins (antireceptors) adsorb
to the cell surface furnished with appropriate receptors. The interaction may alter not only the surface of
the infected cell but also the structure of the virion. Although not fully understood, viral detachment and
readsorption perhaps contribute to cell clumps or plaque formation.
Cytoskeleton Changes
Cytoplasmic and nuclear changes frequently occur not as a result of the damage caused by the virus, but
rather as a result of speci c reorganization of the cellular or skeletal elements necessary for its growth.
Alteration in intermediate keratin laments and microtubules, and cellular metabolism contribute to the
formation of ciliocytophthoria (CCP) seen in certain viral infections. It should be distinguished from
detached ciliary tufts (DCT) described by Hollander and Gupta that may be observed in lower genital tract
6,54smears in the absence of a viral infection. In vivo hemadsorption observed occasionally may be a
related phenomenon (Fig. 7.41).#
#
!
!
Fig. 7.41 Hemadsorption. This patient had herpes infection at the time when these and many other
similar cells were seen. Vaginopancervical smear (Papanicolaou × HP).
Oncogenesis
Both in vitro and in vivo neoplastic transformation of viral-infected cells may occur. Numerous DNA viruses
and a group of retroviruses are capable of neoplastic transformation. These commonly manifest as
dyskaryosis and atypical nuclear alterations.
Quite often, in the presence of orid viral infection, no discernible morphologic changes may occur in the
infected cells and tissues.
The previously mentioned cellular manifestations may or may not be re ected in all cytologic
preparations and in the presence of all viral infections.
Specific Infections
Speci c viral infections commonly observed in the female genital tract include herpes infection: Herpes is a
Greek word meaning “to creep.” It is believed that this word was used in relation to certain clinical features
of an infection that eventually was found to be related to the particular DNA virus. There are at least six
different viruses in this group causing disease in humans. These are:
• Herpes simplex virus, type 1 and type 2 (HSV 1, HSV 2);
• Cytomegalovirus (CMV);
• Varicella-zoster virus;
• Epstein-Barr (EB) virus; and
• Lymphoma-associated viruses.
In the cytologic preparations from the female genital tract herpes, CMV and varicella may be detected.
Herpes Simplex Virus
55Distinction between HSV 1 and HSV 2 was made based on serologic studies by Schneweis. Most people
acquire antibodies to HSV 1 during the rst 2 years of their life. Herpetic vulvovaginitis or stomatitis due to
HSV 1 may occur at the time of initial infection, generally in infancy or early adolescence. Infection is
mostly asymptomatic, or it may be accompanied by upper respiratory tract or ocular symptoms.
Morphologically, HSV 1 and HSV 2 cellular changes appear identical.
Although congenital or neonatal transmission may occur, HSV 2 generally occurs after puberty and the
onset of sexual activity. Cutaneous lesions, commonly vesicles, tend to occur in the same area repeatedly;
the interval between successive eruptions varies considerably even in the same individual. Stress, menses,
and other unrelated ailments may precipitate an eruption in an otherwise healthy person. Following the
initial infection, the virus remains dormant in the sacral (S2 through S4) dorsal root ganglia in the spinal
56cord. McDougall has documented its presence in the spinal cord.#
!
!
#
#
!
#
!
Recently, there has been an increase in the occurrence of HSV 2 cases; it is generally attributed to
changed sexual and social habits. Using seroepidemiologic data, an association of HSV 2 and cervical
55,57-63cancer has been reported by Kessler and others. The precise role of HSV 2 in the development of
human cervical cancer is far from resolved; evidence, however, accepts HPV as the most important
causative infection.
Herpes Simplex Genitalis Virus, Type 2
For nearly 2500 years, people have used the word herpes in medical literature. Corey has brie y discussed
64the history of genital herpes. John Astruc rst described genital herpes in 1736 in the French literature.
More than 100 cases of “herpes progenitalis” were reported in the late nineteenth century. Lipschutz
65established experimental transmission of herpes in human beings in 1921. He concluded that there were
differences between oral (HSV 1) and genital herpes (HSV 2) infections.
HSV 2 infection is one of the most common sexually transmitted genital infections; more than 300,000
new cases are recorded in the United States annually. The prevalence of infection varies depending on the
group studied. Although in general populations the incidence of infection is not well established, genital
HSV infection was diagnosed among 4.2% of those attending the Sexually Transmitted Disease Clinic in
Seattle, Washington, in 1980. Women presenting at student health services have been found to have HSV 2
infection about seven to ten times more commonly than gonorrhea. Data from the Centers for Disease
Control and Prevention (CDC) suggest that the prevalence of HSV 2 is increasing and that the infection is
occurring in social groups that previously did not have the disease.
Primary infection may be asymptomatic or accompanied by severe constitutional symptoms. Commonly,
fever, headache, and myalgia occur before the appearance of mucocutaneous lesions. Visible lesions appear
between 2 and 7 days following exposure to the virus. Local pain and itching, dysuria, vaginal discharge,
and inguinal lymphadenopathy may be present. The lesions are painful and often multiple. Large
ulcerations that start as papules or vesicles spread rapidly. They form pustules that coalesce and break
down. Unless complicated by secondary infection, these ulcers heal in 5 to 10 days with reepithelialization.
Residual scarring is uncommon. Systemic symptoms and inguinal lymphadenopathy occur mainly in
primary HSV 2 infection.
The cytologic diagnosis of HSV infection is important and must be made on well-preserved cells that have
typical diagnostic features and have not been altered by air-drying, xation, or in ammation. Such a
diagnosis may determine proper management of patients, especially pregnant women with genital
ulcerations. An HSV diagnosis, with its social and medical implications, should be rendered only when
unequivocal evidence is present.
In addition to diagnosis in the standard Pap test, direct sampling of visible lesions can be performed.
Such smears should be prepared from the edge and bed of the ulceration, not from the contents of the
vesiculae. The latter generally contain serosanguineous material with acute in ammatory cells, eosinophils,
and some macrophages. Although use of air-dried smears and their examination after Romanowsky stain
66(Tzanck preparation) have been advocated, we do not recommend this for genital lesion diagnoses.
Heavy in ammation, cellular obscuring, and degeneration often make interpretation diE cult and may
severely compromise the diagnostic value of air-dried smears. Cellular samples obtained from the cleared
ulcer beds should be immediately fixed in 95% ethanol and examined after Papanicolaou staining.
The virus may infect the immature squamous, metaplastic, and endocervical columnar cells. Initially, the
changes are proplastic and somewhat nonspeci c. The infected cells can occur singly, in groups, and in
tissue fragments. There is cytomegaly and karyomegaly, and the nucleocytoplasmic ratio is not much
altered. These cells demonstrate a combination of reactive (proplastic) and degenerative (retroplastic)
changes. The nuclei of the infected cells show changes in the chromatin structure consisting of hydropic or
ballooning degeneration. The chromatin material becomes extremely nely divided and is uniformly
dispersed in the nuclear sap. The chromatin–parachromatin interphase is obliterated, and nuclei assume a
faintly hematoxylinophilic, homogenized appearance. Some chromatin material may be matted against the
inner leaf of the nuclear envelope, which may appear uniformly thick and conspicuous. The altered nuclear#
#
morphology is commonly referred to as ground glass, bland, gelatinous, glassy, or opaque. In some cases
the redistribution of chromatin may result in a beaded appearance of the nuclear margins. Nucleoli may be
present and conspicuous, may have associated chromatin, and may not appear typically bright acidophilic.
Although the nucleoli generally remain round or oval, sometimes irregular shapes may be observed.
In the later stages of HSV infection, the cells undergo the e8ects of viral replication and DNA integration.
The cells may assume multinucleation, which is observed in nearly 80% of the smears from cases of genital
HSV infection. The infected nuclei may have the same homogeneous chromatin pattern described
previously. The nuclei appear tightly packed within the cells and reveal distinct internuclear molding (Fig.
7.42). At times they may be overlapping and not molding. Large and single intranuclear inclusions appear
within these nuclei. The nuclear inclusions are generally round or oval. They can be angulated and sharp
(Fig. 7.43). They lack structure and are densely eosinophilic. Depending on the staining procedure
employed, they may appear cherry red. A clear zone, or halo, which separates it from the nuclear
membrane, surrounds the intranuclear inclusion. Most often the halo is as clear as the background of the
slide. Sometimes it may retain delicate, homogeneous, di8use hematoxylinophilia. Small, inconspicuous
chromatin granules can occur in the peri-inclusion halo. Inclusions may occur in infected single cells
observed in nearly one-third of the cases of HSV 2 infection. Intranuclear inclusions may not be present in
all of the nuclei within the multinucleated giant cells.
Fig. 7.42 “Early” herpes genitalis infection. Vaginopancervical smear (Papanicolaou × HP).
Fig. 7.43 Herpes genitalis infection. Vaginopancervical smear (Papanicolaou × HP).
The cytoplasm in the infected cells at this early stage of HSV infection is dense. It may lose its transparent
appearance and become opaque. Often it stains bright cyanophilic.
HSV-infected cells can become atypical; the enlarged cells may assume bizarre shapes (Fig. 7.44). They
may be hyperchromatic or degenerated and may be misinterpreted as tumor cells. The cytoplasm may show
changes of the cytoskeletal structure and become dense or opaque uniformly or focally. The latter may
represent keratohyaline material. Degenerative vacuoles, the ectoplasmic–endoplasmic di8erentiation with
67spiral brils of Erbeth, as described by Patten, may be present between the two zones. The brillary
apparatus of Herxheimer appears as delicate, uniformly thin spirals that originate at the nucleus, travel