Handbook of Cervical Cytology: Special Emphasis on Liquid-Based Cytology [2 ed.] 9789390020218, 9789352702657

Citation preview

Handbook of

Cervical Cytology

Special Emphasis on Liquid-based Cytology

Handbook of

Cervical Cytology

Special Emphasis on Liquid-based Cytology

Pranab Dey MBBS MD MIAC FRCPath

Professor Department of Cytology and Gynecologic Pathology Postgraduate Institute of Medical Education and Research Chandigarh, India

The Health Sciences Publisher New Delhi | London | Panama

Jaypee Brothers Medical Publishers (P) Ltd

Headquarters Jaypee Brothers Medical Publishers (P) Ltd 4838/24, Ansari Road, Daryaganj New Delhi 110 002, India Phone: +91-11-43574357 Fax: +91-11-43574314 Email: [email protected]

Overseas Offices J.P. Medical Ltd 83 Victoria Street, London SW1H 0HW (UK) Phone: +44 20 3170 8910 Fax: +44 (0)20 3008 6180 Email: [email protected]

Jaypee-Highlights Medical Publishers Inc City of Knowledge, Bld. 235, 2nd Floor, Clayton Panama City, Panama Phone: +1 507-301-0496 Fax: +1 507-301-0499 Email: [email protected]

Jaypee Brothers Medical Publishers (P) Ltd 17/1-B Babar Road, Block-B, Shaymali Mohammadpur, Dhaka-1207 Bangladesh Mobile: +08801912003485 Email: [email protected]

Jaypee Brothers Medical Publishers (P) Ltd Bhotahity, Kathmandu, Nepal Phone: +977-9741283608 Email: [email protected]

Website: www.jaypeebrothers.com Website: www.jaypeedigital.com © 2018, Jaypee Brothers Medical Publishers The views and opinions expressed in this book are solely those of the original contributor(s)/author(s) and do not necessarily represent those of editor(s) of the book. All rights reserved. No part of this publication may be reproduced, stored or transmitted in any form or by any means, electronic, mechanical, photocopying, recording or otherwise, without the prior permission in writing of the publishers. All brand names and product names used in this book are trade names, service marks, trademarks or registered trademarks of their respective owners. The publisher is not associated with any product or vendor mentioned in this book. Medical knowledge and practice change constantly. This book is designed to provide accurate, authoritative information about the subject matter in question. However, readers are advised to check the most current information available on procedures included and check information from the manufacturer of each product to be administered, to verify the recommended dose, formula, method and duration of administration, adverse effects and contraindications. It is the responsibility of the practitioner to take all appropriate safety precautions. Neither the publisher nor the author(s)/editor(s) assume any liability for any injury and/or damage to persons or property arising from or related to use of material in this book. This book is sold on the understanding that the publisher is not engaged in providing professional medical services. If such advice or services are required, the services of a competent medical professional should be sought. Every effort has been made where necessary to contact holders of copyright to obtain permission to reproduce copyright material. If any have been inadvertently overlooked, the publisher will be pleased to make the necessary arrangements at the first opportunity. Inquiries for bulk sales may be solicited at: [email protected] Handbook of Cervical Cytology: Special Emphasis on Liquid-based Cytology First Edition: 2018 ISBN: 978-93-5270-265-7

Dedicated to Shree Shree Satyananda Giri, Rini and Madhumanti

Preface

This is a short handbook on cervical cytology for the practising cytologists, students and cytoscreeners. Here I have given stress on the interpretation of the cervical smears with large numbers of microphotographs. The differential diagnosis and management of the lesions also have been highlighted. Special emphasis is given on liquid-based cytology preparation, and the various practical view points of the interpretation of liquid-based cytology are also discussed. I hope that this book will be helpful in learning cervical cytology. Pranab Dey

Acknowledgments

First and foremost, I would like to express my gratitude to God Almighty for His incessant blessing. I am extremely thankful to Shri Jitendar P Vij (Group Chairman), Mr Ankit Vij (Group President) and Ms Ritu Sharma (Director–Content Strategy) of M/s Jaypee Brothers Medical Publishers (P) Ltd, New Delhi, India. They provided me full support in publishing this book. I wish to express my gratitude to Late Professor Subhash Kumari Gupta who taught me cervical cytology. My special thanks to Professor Arvind Rajwanshi for his support in daily routine activities in the department which is very much necessary for any creative work. I am grateful to Dr Suvradeep Mitra who gave me his suggestions and active help in the various stages of this work. Lastly, I would like to thank my wife Rini and daughter Madhumanti for their constant encouragement, without their help it was not possible to write this book.

Content

1. Anatomy, Normal Cytology of the Female Genital Tract and Approach of Reporting

1

Normal anatomy and histology of the female genital tract  1 Cytology 5 Cells of adjacent organ  16

2. Sample Collection, Liquid-based Cytology and Automated Screening

Cervical smear sampling  23 Procedure of sample collection  24 Liquid-based cytology  24 Automated screening devices  31

3. The Bethesda System of Reporting Cervical Cytology

56

Atypical squamous cells of undetermined significance  57 Liquid-based cytology  59 Atypical squamous cell, cannot exclude high grade   squamous intraepithelial lesion  63

6. Squamous Intraepithelial Lesions and Invasive Squamous Cell Carcinoma

41

Reactive cellular changes  41 Organisms 48 Atrophic changes  54 Glandular cell status post-hysterectomy  55

5. Atypical Squamous Cells

33

Introduction to different classification  33 Bethesda system, 2014  34 Explanations 34 Epithelial cell abnormalities  39

4. Negative for Intraepithelial Lesion or Malignancy

23

Low grade intraepithelial lesions  70 High grade intraepithelial lesions  79 Keratinizing high grade squamous intraepithelial lesion  89 Squamous cell carcinoma  91

69

xii Handbook of Cervical Cytology: Special Emphasis on Liquid-based Cytology 7. Abnormality of Glandular Cells

8. The Presence of Endometrial Cells

133

Cervical malignancies  133

10. Screening, Quality Control and Management of Cervical Lesion

126

Endometrial cells  126

9. Other Malignant Tumors

101

Atypical endocervical cells  101 Atypical glandular cell, endocervical origin, favors neoplasia  104 Atypical endometrial cells  104 Endocervical adenocarcinoma in situ  108 Endocervical adenocarcinoma  111 Endometrial adenocarcinoma  116

138

Screening strategy  138 Different screening methods for cervical pre-cancer  139 Quality control of cervical cytology  144 Indicators of good screening program  145 Management 146

11. Sample Cases with Answers

150

Index 167

Abbreviation

AGC Atypical glandular cell Atypical glandular cell of undetermined significance AGCUS Adenocarcinoma in situ AIS Atypical squamous cells, cannot exclude HSIL ASC-H American Society for Colposcopy and Cervical Pathology ASCCP Atypical squamous cell of undetermined significance ASCUS British Society of Clinical Cytology BSCC Cervical intraepithelial neoplasia CIN Conventional preparation CP Food and Drug Administration FDA Fluorescence resonance energy transfer FRET Hybrid capture HC Hyperchromatic crowded groups HCG Human papillomavirus HPV High grade squamous intraepithelial lesion HSIL Herpes simplex virus HSV Intrauterine contraceptive device IUCD Liquid-based cytology LBC Low grade squamous intraepithelial lesion LSIL Lower uterine segment LUS Nucleo-cytoplasmic ratio N/C ratio NILM Negative for intraepithelial lesion or malignancy PAP Papanicolaou’s Polymerase chain reaction PCR Positive predictive value PPV Squamous cell carcinoma SCC Squamous intraepithelial lesion SIL SP SurePath TBS The Bethesda system Transcription mediated amplification TMA

xiv Handbook of Cervical Cytology: Special Emphasis on Liquid-based Cytology TP ThinPrep TV Trichomonas vaginalis TZ Transformation zone VIA Visual inspection with acetic acid WHO World Health Organization

1

Chapter

Anatomy, Normal Cytology of the Female Genital Tract and Approach of Reporting

Normal anatomy and histology of the female genital tract Female genital tract is composed of: •• Vulva •• Vagina •• Cervix •• Uterus •• Fallopian tube •• Ovaries. Figure 1.1 shows the different parts of the female genital tract.

Vulva This is the external female genital part which is composed of mons pubis, labia majora, labia minora, clitoris, prepuce and vestibule.

Figure 1.1: Schematic diagram of the female genital tract

2 Handbook of Cervical Cytology: Special Emphasis on Liquid-based Cytology Mons pubis: The soft fibrofatty tissue over the pubic bone and symphysis pubis is known as mons pubis. Labia majora: These are laterally situated two soft folds of skin that extend from the mons pubis to the perineum. Labia minora: They are situated medial to labia majora. Anteriorly the two ends of labia minora encircle the clitoris. Clitoris: This is the soft erectile tissue that represents male counterpart of penis.

Vagina This is 8 to 9 cm fibromuscular tube that joins the vulva with the uterus. The upper part of the vagina merges with ectocervix. There is a free space between the cervix and the posterior vaginal wall which is known as posterior vaginal fornix. In this potential space the exfoliated cells accumulate. Histology It has three layers: 1. The inner mucosal layer is composed of nonkeratinizing stratified squamous epithelium. 2. The middle muscular layer. 3. Outer connective tissue layer. The squamous epithelial layers of the vagina is responsive to estrogen and contains glycogen. This glycogen provides nutrient to the bacterial flora of the lower genital tract. The lactic acid produced by the bacterial flora maintains acidic pH and protects from pathogenic invasion.

Uterus The uterus consists of three parts: fundus, body, and cervix. Fundus: This is the uppermost part of the uterus and it is situated above the origin of the fallopian tube. Body: This is the central wide part of uterus. Histology of Body and Fundus Uterus consists of (1) inner endometrium, (2) middle myometrium, and (3) outer serosa. The mucosal lining of endometrium is composed of columnar cells. The endometrium undergoes cyclical changes and shredded out in regular interval if pregnancy does not occur. There are two layers of endometrium: 1. Outer functionalis layer: This is superficial layer and is regularly shredded out during menstruation. 2. Inner basalis layer: This is the deeper layer and helps in regeneration of the endometrium after menstrual shedding.

Anatomy, Normal Cytology of the Female Genital Tract and Approach of Reporting

Cervix The cervix is a cylindrical tube of 3 cm in length in nulliparous woman. This is the lower portion of the uterus and is connected with the body of uterus by isthmus. The upper part of the cervical canal is connected with uterus by internal os and the lower part of it opens into the vagina through an orifice known as external os. The part of the cervix that remains within the vagina is known as ectocervix (Figure 1.2). The endocervical canal is the passage in between the external and internal os of the cervix. Histology of Cervix Ectocervix: It is lined by non- keratinizing stratified squamous epithelium. It consists of three layers (Figure 1.3): 1) Basal or parabasal zone is composed of basal and parabasal cells, 2) Intermediate zone is made of intermediate

Figure 1.2: Schematic diagram of cervix

Figure 1.3: Histology of squamous lining epithelium (Hematoxylin and eosin stain)

3

4 Handbook of Cervical Cytology: Special Emphasis on Liquid-based Cytology

Figure 1.4A: The section shows tall columnar lining and also subepithelial endocervical glands (Hematoxylin and eosin stain)

Figure 1.4B: Endocervical columnar lining epithelial cells in higher magnification (Hematoxylin and eosin stain)

squamous cells, and 3) Superficial zone is formed by flattened superficial squamous cells. Endocervix: It is made of single layer of mucus secreting tall columnar cells with basally placed nuclei (Figure 1.4 a, b). The endocervical lining often gives picket fence appearance. The same columnar cell also makes the lining of the endocervical glands. Transformation Zone (TZ) This is also called as squamocolumnar junction. It is the meeting point between the stratified squamous epithelium of the ectocervix and mucin secreting columnar epithelium of the endocervix (Figure 1.5). The TZ is not a fixed point and its position varies with age. The cervical pre-neoplastic

Anatomy, Normal Cytology of the Female Genital Tract and Approach of Reporting

Figure 1.5: Squamocolumnar junction: This is the meeting point between squamous epithelium and mucin secreting columnar cells (Hematoxylin and eosin stain)

lesion originates from the TZ and therefore this area is the target of the cervical screening.

Ovaries These two oval structures are 3–5 cm diameter each and are situated lateral to fallopian tubes and uterus. The ovary secretes female sex hormones estrogen and progesterone. The other important function of the ovary is production of mature ovum. Each ovary contains overall 400000 ova. The ovum comes out from the ovary at the time of ovulation and passes to the fallopian tube. If fertilization does not occur then this ovum is expelled from the uterus. The fertilized ovum is embedded in the endometrium.

Fallopian Tubes Fallopian tubes are 9–11 cm long tubular structure that extend from the cornual ends of uterus laterally up to each ovary. Both the tubes directly open to the peritoneal cavity to receive the ovum from ovary.

Cytology The normal cellular components of cervical smear are: • Squamous cells • Columnar cells • Metaplastic squamous cell • Endometrial cells • Inflammatory cells • Others These cellular constituents are important to identify for the proper interpretation of the cervical smear.

5

6 Handbook of Cervical Cytology: Special Emphasis on Liquid-based Cytology Squamous Cells

Superficial Squamous Cells The superficial squamous cells are the predominant constituents of the cervical smear. These cells are scrapped from the outer most layer of the cervical squamous epithelium. These cells are large and polygonal in shape with abundant orangeophilic to green cytoplasm (Figure 1.6). The diameter of the cell is 30–45 micron and nucleus is about 5–7 micron. The cytoplasm of the cells often shows kerato-hyaline granules which are small dark brown in color in Papanicolaou’s (PAP) stain (Figure 1.7). At times the air may be trapped in between the smear and cover slip and brownish granules like artifact may be created. This cell is labelled as “corn flakes” cell (Figure 1.8). The characteristics of superficial cells are: •• Large polygonal cells •• Abundant thin pink to green cytoplasm

Figure 1.6: Superficial cells and intermediate cells: Superficial cells show orangeophilic cytoplasm and central pyknotic nuclei. The intermediate cells have abundant cytoplasm with central vesicular nuclei

Figure 1.7: Keratohyaline granules in superficial squamous cells (see arrow)

Anatomy, Normal Cytology of the Female Genital Tract and Approach of Reporting

Figure 1.8: Corn flakes cells: Brownish granules like structure formed due to air trapping between the slide and cover slip (see arrow)

•• Cytoplasmic orangeophilia is often seen which indicates intracellular keratin. •• Centrally placed small pyknotic nuclei. Intermediate Cells These cells are almost of same size and shape as that of superficial cells. They contain abundant greenish cytoplasm. The basic difference of superficial cells and intermediate cells lies in the nucleus. The nuclei are relatively large and vesicular in the intermediate cells compared to pyknotic nuclei in the superficial cells (Figure 1.6). The characteristic features of intermediate cells include: •• Large polyhedral cells •• Abundant pale green cytoplasm •• Centrally placed vesicular nucleus. Note: Intermediate cells with folded margins look like boat shaped. These cells are labelled as navicular cells. Navicular cells are seen in pregnant patients due to the progesterone effect on cervical epithelium.

Parabasal and Basal Cells The parabasal and basal cells are smaller in size (Figure 1.9). The diameter of parabasal cell is 10–25 micron and nucleus is 6–8 micron. The nuclei of the cells are relatively large with homogenous bland chromatin. The number of parabasal cell increases as age increases. The cytological features of parabasal cells are: •• Small round to oval cells •• Dense cytoplasm •• Relatively large round nucleus with condensed chromatin

7

8 Handbook of Cervical Cytology: Special Emphasis on Liquid-based Cytology

Figure 1.9: Parabasal cells: Small cell with thick cytoplasm and enlarged round nuclei (see arrow)

Note: Basal and parabasal cells are frequently seen in atrophic smear of postmenopausal patient. The parabasal cells may also be increased in inflammatory smear. The cells may be mistaken as dysplastic cells as they have high nucleo-cytoplasmic (N/C) ratio. However, the nuclear chromatin is homogenous in the parabasal cells.

Changes of Squamous Epithelium The following changes may occur in the squamous epithelium: Hyperkeratosis The term hyperkeratosis is characterized by the presence of thick keratin layer over the squamous lining of the cervix. In cervical cytology smear hyperkeratosis is represented by single or clusters of anucleated squamous cells (Figure 1.10). The nuclei of the cells may be only seen as clear central zone. 1 Hyperkeratosis is a non-specific finding and may be seen in inflammation, post cryotherapy or uterine prolapse. However, hyperkeratosis may be an associated finding in Human Papilloma virus (HPV) infection. 2 Parakeratosis The term parakeratosis means the presence of nucleated squamous cells in keratin layer of the hyperkeratosis. On cytology smear the parakeratotic cell is characterized by superficial squamous cells with small, pyknotic or hyperchromatic nuclei2, 3 (Figure 1.11). The presence of parakeratosis is also a non-specific finding. Probably the female with the presence of parakeratosis in the cervical smear has higher chance of HPV infection. Pseudoparakeratosis The term “pseudoparakeratosis” indicates the glandular cells with deep orangeophilic cytoplasm simulating parakeratotic cells. These type of cells are seen in patients who receive oral contraceptive. The pseudoparakeratotic cells may also be seen as degenerated parabasal cells in atrophic background.

Anatomy, Normal Cytology of the Female Genital Tract and Approach of Reporting

Figure 1.10: Hyperkeratosis: Clusters of anucleated squamous cells (see arrow)

Figure 1.11: Parakeratotic cells: The cells with orangeophilic cytoplasm and dense pyknotic nuclei (see arrow)

Dysplastic cells Discussed later.

Endocervical Cells The endocervical cells are round to columnar in shape (Figure 1.12, 1.13). The cells may be present in small clusters with honey comb appearance (Figure 1.14) or they may be discrete or in small strips. The cells contain moderate amount of cytoplasm which may be vacuolated. The diameter of endocervical cell is about 18–20 micron and nucleus is 8–10 micron. The nuclei of the cell are round with finely granular chromatin having occasional tiny nucleoli. Occasionally the endocervical cells may be multinucleated.

9

10 Handbook of Cervical Cytology: Special Emphasis on Liquid-based Cytology

Figure 1.12: Endocervical cell: loose cluster and discrete cells (see arrow)

Figure 1.13: Endocervical cells: Higher magnification showing columnar cells with basally placed nuclei

Figure 1.14: Endocervical cells: Cells showing honeycomb appearance

Anatomy, Normal Cytology of the Female Genital Tract and Approach of Reporting

Figure 1.15: Tubal metaplasia: Columnar endocervical cells with cilia (see arrow)

Figure 1.16: Tubal metaplasia: Columnar cell with basal plate and cilia (see arrow)

The characteristic features of endocervical cells are: •• Small round to columnar cell •• Vacuolated cytoplasm and basally placed nuclei •• Round nuclei with small nucleoli and fine granular chromatin •• Honeycomb like arrangement is often seen •• Picket fence appearance in side view Note: The relative frequency of endocervical cells are more in liquid-based cytology (LBC) due to use of the brush that samples cells from the endocervical canal. Endocervical cells with tuft of cilia from terminal plate are seen in tubal metaplasia (Figures 1.15 and 1.16). The cells should not be mistaken as dysplastic cells.

11

12 Handbook of Cervical Cytology: Special Emphasis on Liquid-based Cytology Metaplastic Squamous Cells The endocervical cells may undergo squamous metaplasia. This is the commonest change in the endocervical epithelium. The immature parabasal cells are the main constituents of the metaplastic cells. The cells are geometrical shaped with projected cytoplasmic margin and are arranged in interlocking manner that gives a mosaic like pattern (Figures 1.17 and 1.18). The squamous metaplastic cells are relatively small with scanty cytoplasm. The nuclei are enlarged with slightly irregular contour and mildly hyperchromatic. The cytological features of squamous metaplastic cells include: •• Relatively small with scanty dense cytoplasm resembling parabasal cells •• Cells have projected margin that resembles spider-leg •• Round nuclei with fine chromatin and small nucleoli •• Mosaic like pattern in clusters

Figure 1.17: Metaplastic endocervical cells: Cells with polyhedral appearance, small in size with cytoplasmic projections (see arrow)

Figure 1.18: Metaplastic endocervical cells: Higher magnification of the cells (see arrow)

Anatomy, Normal Cytology of the Female Genital Tract and Approach of Reporting Note: Metaplastic squamous cells should not be mistaken as dysplastic cells. The following features are helpful in distinguishing metaplastic cells from the dysplastic cells: •• Relatively small nucleus •• Regular nuclear margin •• Fine chromatin •• Low N/C ratio.

Whereas, the cells of SIL have higher N/C ratio, irregular nuclear margin and hyperchromatic nuclei.

Endometrial Cells The endometrial cells can be normally found in women with reproductive age period. The presence of endometrial cells in post-menopausal patient is abnormal. The endometrial cells are usually present in small tight cohesive clusters (Figures 1.19 and 1.20). The cells show scanty cytoplasm and round dark nuclei. Nuclei may compress each other causing moulding. Single cell death or apoptotic cells may be seen in cluster of endometrial cells. The salient cytological features of endometrial cells include: •• The cells are usually arranged in small tight spherical clusters •• Small round cells with scanty cytoplasm •• Round dark nuclei •• Nuclear molding •• No nucleoli. Note: Benign endometrial cells may be seen in endometrial polyp, endometritis, endometriosis and abortion. It may also be present during menstrual period. The endometrial cells may be mistaken as: cells of high grade squamous intraepithelial lesion (HSIL), poorly differentiated squamous cell carcinoma and well differentiated adenocarcinoma.

Figure 1.19: Endometrial cells: Tight cohesive cells with scanty cytoplasm and round nuclei with condensed chromatin (see arrow)

13

14 Handbook of Cervical Cytology: Special Emphasis on Liquid-based Cytology

Figure 1.20: Endometrial cells: Tight clusters of round cells with scanty cytoplasm (see arrow)

Inflammatory Cells Polymorphonuclear leukocytes and lymphocytes are frequently seen in cervical smear. Mere presence of inflammatory cells does not indicate inflammation. Histiocytes These cells are also normally found in the cervical smear. The cells are: •• Large cells •• Moderate cytoplasm •• Central kidney shaped nuclei Note: These cells may be seen in various conditions such as pregnancy, foreign body reactions, and endometrial cancer.

Others

Spermatozoa The cells can be mistaken as Candidal spores. Long tail of spermatozoa are easily identified in the smear (Figure 1.21). Lactobacilli Lactobacilli are gram positive rod shaped bacilli and are normally present in cervical smear (Figure 1.22). Ova of Parasites Rarely ovum of parasites are contaminated in cervical smears. Giant Cells The multinucleated giant cells are not uncommon in cervical smear (Figures 1.23 and 1.24). They are mostly nonspecific in nature. The differential diagnosis of multinucleated giant cells in the cervical smear include: •• Foreign body giant cells,

Anatomy, Normal Cytology of the Female Genital Tract and Approach of Reporting

Figure 1.21: Sperms: Sperm with tail (see arrow)

Figure 1.22: Lactobacilli: Rod like structures. Normal commensal of cervical flora (see arrow)

Figure 1.23: Giant cell: Multinucleated giant cell in cervical smear (see arrow)

15

16 Handbook of Cervical Cytology: Special Emphasis on Liquid-based Cytology

Figure 1.24: Multinucleated giant cell in viral infection (HSV) (see arrow)

•• •• •• •• •• ••

Viral infection, Tuberculosis Syncytio and cytotrophoblasts Radiation exposure Squamous cell carcinoma and Choriocarcinoma.

Cells of adjacent organ Urothelial Cells

•• Columnar cells with round regular nuclei •• Difficult to identify.

Colonic Cells

•• Aggregates of columnar cells in rows. Acellular Material •• Contaminants (Figure 1.25): Dust of surgical gloves and lubricant jellies etc.

Essential Information Needed for Interpretation of Cervical Cytology Some information is mandatory before the interpretation of any cervical cytology smear, such as (Box 1.1): a. Patient’s age: Mentioning the age of the patient in the clinical request form is mandatory. The diagnostic interpretation may vary according to age such as presence of endometrial cells above 45 years of age should be considered as abnormal. b. Date of last menstruation and the status of menopause: Menstrual history particularly the date of last menstruation may help to interpret

Anatomy, Normal Cytology of the Female Genital Tract and Approach of Reporting

Figure 1.25: Acellular material in the smear (see arrow)

c.

d. e.

f.

the endometrial cell clusters around Box 1.1: Essential information the menstrual period. Similarly •• Patient’s age menopause may affect the pattern •• Date of last menstruation and of cervical smear. Parabasal and the status of menopause basal cells are usually more in post•• Intrauterine contraceptive device menopausal smear and may show •• Hormone status reactive nuclear enlargement that •• Previous history of SIL or histopacan be mistaken as malignancy. thology Intrauterine contraceptive device •• History of radiation or chemo(IUCD): The history of IUCD may therapy. help to recognize the “bubble gum” cells and IUD cells. These cells can be mistaken as the cells of adenocarcinoma. Hormone status: The history of hormonal replacement is essential to recognize the changes due to hormone. Previous history of squamous intraepithelial lesion (SIL) or histopathology: The previous history of SIL helps to search and recognize the dysplastic cells. It also helps to take decision to have further colposcopic or histopathological examination. Previous history of histopathology or cone biopsy is also necessary. The large number of ciliated endocervical cells are often seen after sampling from a cone biopsy. History of radiation or chemotherapy: Radiation or chemotherapy may cause cytomegaly having nuclear enlargement. It is easier to interpret such cells with proper history.

How to Recognize a Dysplastic Cell In every smear a thorough search is needed to identify the dysplastic cells. The area of LBC smear is small and it is much easier to find out the abnormal cells. Moreover, relatively clean background free of mucus, necrotic tissue

17

18 Handbook of Cervical Cytology: Special Emphasis on Liquid-based Cytology and inflammatory cells make the search Box 1.2: Recognition of dysplastic cells process easier. One should always screen •• Altered arrangement of cell clusthe smear thoroughly either horizontally ters: Nuclear overcrowding or vertically in a zig-zag way. The essen- •• Altered chromatin pattern: tial characteristics of dysplastic cells are Speckled chromatin, fine chro(Box 1.2): matin, coarse granular chromatin •• Hyperchromasia

Arrangement •• High nucleocytoplasmic ratio The dysplastic cells are predominantly •• Irregular nuclear contour. arranged singly (Figure 1.26). Occasionally the dysplastic cells may be arranged as tight cohesive cluster known as hyperchromatic crowded group of cells. The hyperchromatic crowded group (HCG) may be noted in both benign and dysplastic cells (Figure 1.27). Irregular margin of the cluster and the

Figure 1.26: Discrete dysplastic cells (see arrow)

Figure 1.27: Hyperchromatic crowded group (HCG) of dysplastic cells

Anatomy, Normal Cytology of the Female Genital Tract and Approach of Reporting

loss of polarity of the peripheral cells are the features of HCG produced by dysplastic cells. Moreover, one should note the individual cells of the HCG to recognize the dysplastic cells. Chromatin Pattern Alteration Alteration of the chromatin pattern of the cell is the most important characteristic to recognize a dysplastic cell. Normal cells show homogenous bland chromatin. The dysplastic cells show: speckled chromatin, fine granular, even or irregular coarsely granular chromatin (Figures 1.28, 1.29 and 1.30). The type of change of chromatin pattern has little effect on grade of dysplasia. Hyperchromasia The majority of the dysplastic cells show hyperchromatic nuclei. The hyperchromatic nuclei are dark colored and should always be differentiated

Figure 1.28: Dysplastic cell with coarse granular chromatin (see arrow)

Figure 1.29: Dysplastic cell with granular chromatin (see arrow)

19

20 Handbook of Cervical Cytology: Special Emphasis on Liquid-based Cytology

Figure 1.30: Dysplastic cell with speckled chromatin (see arrow)

Figure 1.31: Dysplastic cell with hypochromatic and pale nucleus (see arrow)

from pyknotic nuclei or nuclei with smudged chromatin. Rarely dysplastic cells are hypochromatic and pale in color (Figure 1.31). The pale dyskaryotic cells show abnormal chromatin pattern. Nuclear Enlargement and N/C Ratio Simple enlargement of nucleus along with excess cytoplasmic area may not be significant. Nuclei are relatively large in parabasal and endocervical cells. However, increased nucleo-cytoplasmic (N/C) ratio is pathognomonic of dysplastic cells (Figure 1.32). Nuclear Contour Nuclear margin irregularity is also one of the characteristic features of dysplasia. There may be minor irregularity of the nuclear margin in the cells

Anatomy, Normal Cytology of the Female Genital Tract and Approach of Reporting

Figure 1.32: High nucleocytoplasmic ratio and irregular nuclear membrane in dysplastic cells (see arrow)

Figure 1.33: Dysplastic cell with thick nuclear membrane (see arrow)

of low grade squamous intraepithelial lesion (LSIL) and grossly irregular nuclear contour in HSIL (Figure 1.32). The alteration of nuclear margin is usually more evident in LBC preparation as the cells are better fixed in liquid-based cytology and the air drying is completely avoided. However, the nuclear margin may be completely regular in dysplastic cells. Thickened Nuclear Membrane Nuclear membrane is often thickened in dysplastic cells (Figure 1.33). This is also an important feature to recognize dysplastic cells. Thickened nuclear membrane is more frequent in HSIL and is more evident in LBC.

21

22 Handbook of Cervical Cytology: Special Emphasis on Liquid-based Cytology

References 1. Izadi-Mood N, Sarmadi S, Alijani S, Sanii S. The significance of hyperkeratosis in pap smears with squamous intraepithelial lesion. Acta Cytol. 2012;56(4):379-82. 2. Zahn CM, Askew AW, Hall KL, Barth WH Jr: The significance of hyperkeratosis/parakeratosis on otherwise normal Papanicolaou smears. Am J Obstet Gynecol 2002; 187:997-1001. 3. Kern SB. Significance of anucleated squames in Papanicolaoustained cervicovaginal smears. Acta Cytol. 1991;35:89-93.

2

Chapter

Sample Collection, Liquid-based Cytology and Automated Screening

Cervical smear sampling Optimal collection of cervical cytology specimen is the first important step for proper interpretation. The following things are needed for cervical cytology smear collection: •• Instrument –– Devices to collect the cervical sample: Ayres spatula/cervix brush/ endocervical brush (Figure 2.1) –– Speculum: To examine the cervix and to take smear •• Fixative –– Coplin jar with 95% ethanol –– Spray fixative, or –– Fixative supplied by the company for liquid based-cytology (LBC) •• Glass slide for conventional preparation (CP) •• Laboratory form: The laboratory form with full clinical details.

Figure 2.1: Different devices to collect the cervical smear: (1) Cervix brush, (2) Ayer’s spatula, and (3) Endocervical brush

24 Handbook of Cervical Cytology: Special Emphasis on Liquid-based Cytology Optimum Clinical Condition and Required Information a. It is preferable that the patient should be in the mid cycle and by any chance she should not be in the menstruation. b. The patient should be restrained from sexual intercourse 2 days prior to the test. c. Any sorts of vaginal cream, lubricant jelly or tampon should also be avoided. d. Name of the patient, age, date of last menstrual period, any symptoms, and previous screening history.

Procedure of sample collection The patient should be in the dorsolithotomy position as in this position the cervix is better visualized. The speculum is introduced through vagina without using any lubricant jelly. Water can be used to moisturize the speculum and rarely water soluble lubricant is used. Usually broom stick like device is used for LBC preparation. The broom is introduced so that the central part enters into the endocervical canal and the shorter bristles of the broom are touched to the ectocervix. The broom is rotated four to five times in a clockwise direction. Then the broom is withdrawn and is dipped in the fixative solution provided by the company. The broom should be repeatedly swirled to shake the material into liquid. Finally the broom should be discarded and the vials should be labelled properly and sent to the laboratory. In case of conventional preparation either broom or spatula is used. The collection of the cervical sampling method is almost same. The smear is prepared on the glass slide and immediately fixed in the 95% ethyl alcohol for 30 minutes. Alternatively, spray fixative can be used. Spray fixative contains alcohol base and carbowax. Before Papanicolaou’s staining the carbowax must be removed by dipping the slide in alcohol.

Liquid-based cytology Presently two companies are available for LBC: (1) ThinPrep (TP) (Cytyc Corp, Marlborough, MA) and (2) SurePath (SP) (TriPath Imaging Inc., Burlington, NC). Both these companies are approved by food and drug administration (FDA). Unlike smear preparation and fixation in the conventional preparation, these techniques provide the methods of immediate fixation of the cells in liquid fixative.

Thin Prep Preparation Technique (Figures 2.2A and B) The Thin Prep 5000 processor is now presently available in the market. The processor can automatically prepare up to 20 samples in 45 minutes time period. The microscopic slides are always provided by the ThinPrep (Cytic, UK) company.

Sample Collection, Liquid-based Cytology and Automated Screening

A

B

Figures 2.2A and B: Schematic diagram showing basic principle of ThinPrep

Dispersion The sample is collected in 20 ml of PreservCyt solution for fixation. A cylindrical tube with attached filter in one end is dipped within the vial containing the specimen in PreservCyt solution. The cylinder is rotated within the vial rapidly and this rotatory movement disperses the cells singly. Cell Collection A gentle negative suction pressure is used through the cylinder and the fluid from the vial is drained out whereas the cells are struck on the external surface of the filter. The collection of the cell is controlled by adjusting the negative pressure with the help of inbuilt software in the TP processor. Cell Transfer Now the TP filter is inverted and the cells attached on the surface of the filter are imprinted automatically on the circular area of the glass slides. The cells are adhered on the glass slide by positive air pressure. The slide is immediately immersed within a fixative.

25

26 Handbook of Cervical Cytology: Special Emphasis on Liquid-based Cytology SurePath Preparation Technique (Figures 2.3A and B) In SP technique at first the cells are collected in the SUREPATH® Preservative fluid in a vial. The vial is transferred to the laboratory for further processing. Vortexing The sample is vortexed to disperse the cells. A total of 48 sample can be vortexed each time (Figure 2.4). Cell Enrichment The sample is then mixed with PREPSTAIN® Density Reagent and is centrifuged for 2 minutes in 200 g (Figures 2.5 and 2.6). The unwanted

A

B

Figures 2.3A and B: Schematic diagram showing basic principle of SurePath

Sample Collection, Liquid-based Cytology and Automated Screening

Figure 2.4: Vortexing: The specimen is vortexed for 15 second

Figure 2.5: The sample is mixed with Prepstain density reagent

elements such as blood, mucus and necrotic debris are separated by the centrifugation. The supernatant fluid is decanted by using vacuum suction. The reaming fluid is again centrifuged by 800 g for 10 minutes (Figure 2.6). A cell rich pellet is formed. Resuspension The cell pellet is revortexed for 15–20 seconds to resuspend the cellular material within the fluid and then transferred into the PrepStain slide processor.

27

28 Handbook of Cervical Cytology: Special Emphasis on Liquid-based Cytology

Figure 2.6: The tubes are centrifuged and then supernatant fluid is discarded

Figure 2.7: Specimen finally ran into BD PrepStain slide processor

Density Gradient Sedimentation (Figure 2.7) In the PREPSTAIN® settling chamber, the cells are deposited with the help of gravity on a precoated slide (modified poly-L-lysine). Simultaneously automatically Papanicolaou’s stain is done with the help of program software. Table 2.1 shows comparison of ThinPrep and SurePath technique. 1

Sample Collection, Liquid-based Cytology and Automated Screening Table 2.1: Comparison of ThinPrep versus SurePath Features

ThinPrep

SurePath

Fixative

PreservCyt fluid is methanol SurePath preservative fluid is based fixative ethanol based

Sample preparation

Easy

Cell dissociation

By rotatory motion of the vial By vortexing

Cell separation

By negative suction

Density gradient

Monolayer

Monolayer preparation

Multilayer

Time consuming

Hyperchromatic crowded Infrequent group of cells

Frequent

Cell concentration

Relatively less

More

Inadequacy rate

More

Less

Advantages and Disadvantages of LBC Preparation

Advantages LBC preparation of cervical smear has following advantages: •• Better cellular preservation and no air drying artefact •• Overall cellularity is much more than conventional preparation •• Relatively clean background •• Lesser screening time as the cells are limited in small area •• Monolayer cell preparation in ThinPrep •• Multiple smears can be prepared from a single sample •• HPV testing can be done from the residual sample. Disadvantages of LBC Preparation •• Costly instrument •• Training is required for interpretation.

Significant Differences of the Cytology in LBC Compared to CP There are few important features present in the LBC: Background The background of the cytology smear is always clean and free from any blood, mucus, and necrotic debris (Figure 2.8). Cell Size The cells are immediately dipped in fluid and due to wet fixation overall cell shape is more rounded and cell size is also smaller compared to CP.

29

30 Handbook of Cervical Cytology: Special Emphasis on Liquid-based Cytology

Figure 2.8: Clean and small smear free from blood and mucus in liquid-based cytology compared to conventional smear.

Cell Crowding Cellular dispersion is overall more in TP than SP. Hyperchromatic crowded group of cells are frequently seen in SP. Hyperchromasia The nuclei of the cells are relatively less hyperchromatic compared to CP of cells. Cytoplasmic and Nuclear Detail As the cells are immediately fixed in liquid fixative so the nuclear details are excellent in LBC.

Specific Changes in LBC Following specific changes can are seen in LBC compared to CP: •• Atypical squamous cell of undetermined significance (ASCUS) is relatively easily recognized due to better cellular preservation •• ASC-H: Here the cells look much smaller •• LSIL: The nuclear hyperchromasia is minimal •• HSIL: The cells are usually more dispersed than in clusters. The nuclear hyperchromasia may be lost in many cells •• Squamous cell carcinoma: Necrotic tissue debris are mostly removed in case of carcinoma and necrotic material is adhered in the periphery of the cell cluster giving the appearance of “clinging diathesis”. Overall cellularity of the tumor cells may be low. At times the cells are present in small round pattern and may simulate glands of adenocarcinoma •• Feathering pattern of adenocarcinoma is better seen •• Endometrial cell: The cells are mostly present as three dimensional clusters. Single cells are also present. The nuclear morphology is better preserved with nucleoli and details chromatin pattern

Sample Collection, Liquid-based Cytology and Automated Screening

•• Follicular cervicitis: The lymphoid cells are usually present in clusters instead of discretely •• Bacterial vaginosis: Clue cells are seen in a clean background •• Trichomonas vaginalis: The typical background is lost and the TVs are smaller in size and round in shape •• Candida: Epithelial cells often show Shish Kebab like appearance •• Atrophic smear: The granular background may be mistaken as granular debris of squamous cell carcinoma. The nuclei of the cells are small in size and the bare nuclei are less in number •• Hyperchromatic crowded group of cells: This type of cellular crowding is frequently seen in SP.

Automated screening devices Presently two automated cervical smear screening systems are available.

BD FocalPoint GS Imaging System (FPGS) BD FPGS is FDA approved automated device for primary screening of SurePath and conventional smear. This system has two components: 1. BD FocalPoint TM Slide Profiler 2. BD FocalPoint GS Review Station BD FocalPoint TM Slide Profiler The cervical cytology slide is labelled with barcode and then loaded in the slide tray of BD FocalPoint™ Slide Profiler. There are total 36 slide trays and each tray can contain 8 slides. The device reads the bar code and screens the entire smear both in low and high power. The system studies several cytological features such as nuclear size, nucleo-cytoplasmic ratio etc and each slide is given a “device score” according to its level of abnormality. The slide is categorized as no further review and review group. The whole data regarding the examined slides are transferred to the review station. BD FocalPoint GS Review Station This is the accessory system of BD FocalPointTM Slide Profiler. Here, the cytologist rapidly relocate field of view of the so called abnormal area and decide whether the slide needs full rescreening or it should be labelled as no abnormality.

HOLOGIC ThinPrep Imaging System This system includes: 1. ThinPrep imaging system, and 2. ThinPrep review system.

31

32 Handbook of Cervical Cytology: Special Emphasis on Liquid-based Cytology ThinPrep Imaging System This is FDA approved system to screen cervical smears. Here, the system captures images of the slide and analyse the images and stores the result. The image processor screens the slide and selects 22 microscopic fields for reviewing the potential abnormal cells. Review Scope It is an automated microscope with a motorized stage. At first the bar code of the already imaged slide is scanned. This guides the cytotechnologist to review the 22 microscopic field of interest. The cytotechnologist reviews the 22 microscopic field manually. If no abnormal cells are detected then the slides are passed as normal. However, if any abnormal cell is suspected then the whole slide is screened thoroughly and is referred to the cytopathologists. Main goal of automated screening system: 1. Reduces false negative cases. 2. Reduces the cost of the screening in comparison to manual screening. Unfortunately, the presently available automated screening systems are very costly and economically not feasible. Until the overall price is reduced significantly the automated screening will not be successful.

References 1. Zhao FH, Hu SY, Bian JJ, Liu B, Peck RB, Bao YP, Pan QJ, Frappart L, Sellors J, Qiao YL. Comparison of ThinPrep and SurePath liquid-based cytology and subsequent human papillomavirus DNA testing in China . Cancer Cytopathol. 2011;119(6):387-94.

3

Chapter

The Bethesda System of Reporting Cervical Cytology

Introduction to different classification Earlier in 1969, Reagan et al. 1 classified cervical preneoplastic lesion as mild dysplasia, moderate dysplasia, severe dysplasia and carcinoma in situ. However, there are overlapping cytomorphological features of severe dysplasia and carcinoma in situ. Therefore, Richart RM2 presented the concept of cervical intraepithelial neoplasia (CIN) and classified the preneoplastic lesions of cervix as CIN 1, CIN 2, and CIN 3. According to Richart RM2 mild dysplasia and moderate dysplasia represents as CIN 1 and CIN 2 respectively. He clubbed severe dysplasia and carcinoma in situ as CIN 3. The Bethesda system (TBS) of classification clubbed CIN 2 and CIN 3 as high grade squamous intraepithelial lesion (HSIL).3 The CIN 1 represented in TBS as low grade squamous intraepithelial lesion (LSIL). Figure 3.1 has shown the relationship between the different classifications. Similar to TBS, British Society of Clinical Cytology (BSCC) classified the preneoplastic lesions of cervix as low grade dyskaryosis and high grade dyskaryosis that represented LSIL and HSIL in TBS.4

Figure 3.1: Schematic diagram showing the comparison of different classification used in cervical smear reporting

34 Handbook of Cervical Cytology: Special Emphasis on Liquid-based Cytology Bethesda System, 2014 The Bethesda system (TBS) 2001 of reporting cervical cytology smear provides uniform guidelines for reporting cervical cytology. 3 This classification is mildly modified in 2014 Bethesda system.5 Table 3.1 enumerates TBS, 2014 as: a) specimen adequacy, b) general categorization and c) terminologies. The predominant changes in Bethesda 2014 classification •• No major changes in this classification from 2001 Bethesda system •• In the category Other: The age of reporting benign-appearing endometrial cells is raised from 40 years to more than 45 years.

Explanations In any cervical smear at first overall Box 3.1: Overall approach of reporting cervical smear assessment should be done regarding the clinical history, staining quality, and •• Clinical history and proper labelling of the slide adequacy of the specimen (Box 3.1). After the initial assessment of adequacy the •• Staining quality smear should be broadly classified as •• Assessment of adequacy of the specimen negative for intraepithelial lesions and • • General categorization epithelial cell abnormality. This general • • Proper terminology categorization should be followed by •• Further comment, if any. proper terminology of the smear.

Specimen Adequacy TBS 2014 system has discarded the category “satisfactory but limited” and considered only two categories regarding the adequacy of the sample: 1. Satisfactory for evaluation. 2. Unsatisfactory for evaluation.

Minimum Number of Cells Required for Reporting 1. Conventional smear: According to TBS 2014, there should be at least 8000 to 12000 squamous cells. 2. Liquid-based cytology: The number of squamous cell in LBC is much less and only 5000 squamous cells are considered as minimum number of cells.

Which Cells Should be Considered (Figures 3.2 and 3.3) The well visualized and well preserved squamous cells should be taken into consideration. Only the squamous cells or metaplastic squamous cells should be considered for cellularity. Endocervical cells, endometrial cells, and inflammatory cells should not be considered for cellular adequacy.

The Bethesda System of Reporting Cervical Cytology Table 3.1: The Bethesda System 20145 The Bethesda System 2014 Specimen type •• Conventional or liquid-based cytology •• Specimen adequacy Satisfactory for evaluation •• Adequate number of squamous cells: –– Conventional smear: 8000 to 12000 squamous cells –– Liquid-based cytology: 5000 squamous cells •• Mention the presence or absence of endocervical cells or transformation zone components: at least 10 endocervical or metaplastic cells should be present, no need for cell clusters. •• Any other quality elements such as drying artifact, obscured by blood or inflammation etc. Partially obscured: 50 to 75% epithelial cells cannot be visualized, Unsatisfactory: when more than 75% epithelial cells cannot be visualized. Unsatisfactory for evaluation •• Specimen rejected: Specimen rejected due to broken slide or mislabelled specimen •• Fully evaluated unsatisfactory specimen: Specimen processed but unsatisfactory because: –– Insufficient squamous cells –– More than 75% of smear is obscured by blood or inflammatory cells. General Categorization (Optional) •• Negative for intraepithelial lesion or malignancy •• Epithelial cell abnormality •• Interpretation/Result •• Negative for intraepithelial lesion or malignancy –– Non-Neoplastic Findings (optional to report) ◆◆ Non-neoplastic cellular variations ◊ Squamous metaplasia ◊ Keratotic changes ◊ Tubal metaplasia ◊ Atrophy ◊ Pregnancy-associated changes. ◆◆ Reactive cellular changes associated with: ◊ Inflammation (includes typical repair) ◊ Lymphocytic (follicular) cervicitis ◊ Radiation ◊ Intrauterine contraceptive device (IUD) ◆◆ Glandular cells status post-hysterectomy. 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 consistent with herpes simplex virus •• Cellular changes consistent with cytomegalovirus Other: Endometrial cells (in a woman aged more than 45 years) (Also specify if “negative for squamous intraepithelial lesion”) Contd...

35

36 Handbook of Cervical Cytology: Special Emphasis on Liquid-based Cytology Contd... Epithelial Cell Abnormality Squamous cell •• Atypical squamous cell (ASC) –– Of undetermined significance (ASC-US) –– Cannot exclude HSIL (ASC-H) •• Low-grade squamous intraepithelial lesion (LSIL) •• High- grade squamous intraepithelial lesion (HSIL) •• Squamous cell carcinoma Glandular cell •• Atypical –– Endocervical in origin –– Endometrial in origin –– Glandular cell, not otherwise specified •• Atypical –– Endocervical, favour neoplastic –– Glandular, not otherwise specified •• Endocervical adenocarcinoma in situ •• Adenocarcinoma –– Endocervical –– Endometrial –– Extra uterine –– Not otherwise specified •• Other malignant neoplasm Adjunctive testing Provide a brief description of the test method(s) and report the result so that it is easily understood by the clinician Computer-assisted interpretation of cervical cytology If case examined by an automated device, specify the device and result Educational notes and comments appended to cytology reports (optional) Suggestions should be concise and consistent with clinical follow-up guidelines published by professional organizations (references to relevant publications may be included)

Cell Counting Only, the approximate number of cells should be estimated and the cytologists are not expected to count the individual cells. A minimum number of 10 microscopic filed at 40 X should be assessed for the cellularity. Mention the Presence or Absence of Endocervical Cells or Transformation Zone Components As endocervical cells represent the transformation zone so the presence of either endocervical cells or metaplastic endocervical cells should be mentioned. At least 10 endocervical or metaplastic cells should be present in the smear and there is no need for cell clusters.

The Bethesda System of Reporting Cervical Cytology

Figure 3.2: Adequate cervical smear: well stained squamous cells in adequate number

Figure 3.3: Scanty cellularity in cervical smear

Partially Obscured In partially obscured smear almost 50–75% epithelial cells cannot be visualized due to blood or inflammatory cells. If more than 75% of the epithelial cells are obscured then the smear should be labelled as unsatisfactory for reporting.

Unsatisfactory for Evaluation

•• Specimen rejected: Specimen should be rejected if the slides are broken or specimen is mislabelled. •• Fully evaluated unsatisfactory specimen: Specimen may be unsatisfactory due to: –– Inadequate number of squamous cells –– More than 75% of smear is obscured by blood or inflammatory exudate. (Figure 3.4)

37

38 Handbook of Cervical Cytology: Special Emphasis on Liquid-based Cytology

Figure 3.4: Inflammatory exudate covering more than 75% of the smear

It is advisable to have a good look of the unsatisfactory slides as these smears may also show the pathology such as the presence of endometrial cells in post-menopausal women or organisms. How Strict are the Criteria The criteria of adequacy of the specimen are not strict as: •• The individual laboratory can use their own judgement in borderline cellularity. •• The criteria of cellularity can be relaxed in case of significant clinical history, post-menopausal patient and atrophic cervix.

General Categorization It is not necessary to strictly follow “General categorization” as it is an optional component in TBS 2014. This is used for rapid triaging of the report by the clinicians. Negative for Intraepithelial Lesion or Malignancy The two components “within normal limit” and “benign cellular changes” of TBS 1991 have been combined together to make a single category as “negative for intraepithelial lesion or malignancy” in TBS 2001/2014. Others The presence of benign looking endometrial cells in woman over 45 years of age should be labelled as “others”.

Epithelial Cell Abnormality These categories are mutually exclusive and non-overlapping. However, the smears may show overlapping features. In this circumstances, the cytologists should categorize the clinically most significant lesion.

The Bethesda System of Reporting Cervical Cytology

Interpretation and Result As the cervical smear is primarily a screening test so the terminology “diagnosis” is replaced by “interpretation and result”. The diagnosis depends on the clinical features and other laboratory investigations.

Negative for Intraepithelial Lesion or Malignancy (NILM) Cytology smear that does not show any abnormality of the epithelial cell is reported as NILM. TBS 2001/2014 has kept optional choice to report other non-neoplastic findings, e.g. reparative processes, radiation, atrophy, and intrauterine contraceptive devices. Mere presence of an organism may not indicate infection. Therefore, the term “infection” has been replaced by “organism” in TBS 2001/2014.

Epithelial cell abnormalities Atypical Squamous Cells In 2001/2014 TBS, “atypical squamous cell” is labelled as: •• “Atypical squamous cells of undetermined significance” (ASC-US) •• “Atypical squamous cells, cannot exclude HSIL” (ASC-H). The category ASC favours reactive atypia is discarded and the cytopathologists therefore should downgrade this terminology to NILM. Atypical squamous cells, cannot exclude HSIL (ASC-H) indicates that cytological features are almost similar to HSIL but not confirmatory to the final interpretation of HSIL. The term ASC-H represents HSIL and its mimickers. The reproducibility of ASC-H is variable and no specific treatment should be done on the basis of this interpretation. ASC-H comprises of only 5 to 10% of ASC cases.6

Squamous Intraepithelial Lesion (SIL) The SIL is divided as LSIL and HSIL. This two tier division is kept as the clinical management of these two groups are completely different. Moreover, the interpretation of LSIL/HSIL is reproducible than three tier division of cervical intraepithelial lesion (1, 2, and 3).

Squamous Cell Carcinoma The term “squamous cell carcinoma” represents the invasive carcinoma. TBS, 2001/TBS 2014 does not encourage to differentiate keratinizing from non-keratinizing squamous cell carcinoma in cervical smear.

Atypical Glandular Lesion The atypical glandular cell has been categorized as: •• Atypical glandular cells: Endocervical, endometrial or not otherwise specified •• Atypical glandular cells favour neoplastic: Endocervical, or not otherwise specified.

39

40 Handbook of Cervical Cytology: Special Emphasis on Liquid-based Cytology In 2001/2014 TBS, the cytopathologists are supposed to find out the primary site of the atypical glandular cells such as endocervical or endometrial and should specify in report. In case of difficulty one may just write “atypical glandular cell, not otherwise specified”.

Endocervical Adenocarcinoma in Situ (AIS) This has been kept as a separate entity. There may be some amount of overlapping between AIS with invasive carcinoma in one hand and AIS with atypical glandular cells favour neoplastic in other hand.

Automated Review and Ancillary Testing It is essential to mention about: •• The name of automated scanning system •• The type of HPV screening test.

References 1. Reagan JW, Ng ABP, Wentz WB. Concepts of genesis and development in early cervical neoplasia. Obstet Gynecol Survey. 1969;24:860-74. 2. Richart RM. Cervical intraepithelial neoplasia: a review. Pathol Ann. 1973;8:301-28. 3. Solomon D, Davey D, Kurman R, et al. for the Forum Group Members and the Bethesda 2001 Workshop. The 2001Bethesda System—terminology for reporting results of cervical cytology. JAMA. 2002;287:2114-9. 4. Denton KJ, Herbert A, Turnbull LS, Waddell C, Desai MS, Rana DN, Dudding N, Smith JH; British Society of Clinical Cytology. The revised BSCC terminology for abnormal cervical cytology. Cytopathology. 2008;19(3):137-57. 5. Nayar R, Wilbur DC. The Pap test and Bethesda 2014. Cancer Cytopathol. 2015;123(5): 271-81. 6. Sherman ME, Solomon D, Schiffman M; ASCUS LSIL Triage Study Group. 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(3):386-94.

4

Chapter

Negative for Intraepithelial Lesion or Malignancy

Negative for intraepithelial lesion or malignancy (NILM) means absence of any evidence of intraepithelial lesion or malignancy and it represents: •• Reactive cellular changes associated with –– Inflammation –– Intra uterine contraceptive device –– Radiation. •• Organism –– Trichomonas –– Bacterial vaginosis –– Fungal morphology consistent with Candida –– Bacterial morphology consistent with actinomycosis –– Cellular morphology consistent with herpes simplex virus. •• Atrophic smear •• Glandular cell status post-hysterectomy.

Reactive cellular changes Inflammation (Figures 4.1–4.6) The cervical smears of acute inflammation shows inflammatory exudates. The inflammatory cells often stick with the epithelial cells. The epithelial cells show degenerative changes such as pyknosis, karyorrhexis, and karyolysis. The cytoplasm shows perinuclear vacuolation. In addition, there may be increased number of parabasal cells. The endocervical cells show squamous metaplastic changes. The smear may show reactive endocervical cells with enlarged mildly pleomorphic nuclei with prominent nucleoli. The following features are characteristic of inflammation: •• Excess neutrophils in the smear •• Occasional necrosis •• Inflammatory cells adhered to the epithelial cells

42 Handbook of Cervical Cytology: Special Emphasis on Liquid-based Cytology

Figure 4.1: Inflammatory smear: Inflammatory exudates sticking with the cells (see arrow)

Figure 4.2: Inflammatory smear: The cells how karyorrhexis and karyolysis (see arrow)

Figure 4.3: Inflammatory smear: Para nuclear vacuoles (see arrow)

Negative for Intraepithelial Lesion or Malignancy

Figure 4.4: Reactive endocervical cells: cluster of endocervical cells showing nuclear enlargement

Figure 4.5: Reactive endocervical cells: Endocervical cells with enlarged nuclei having prominent nucleoli. Note the absence of nuclear moulding

Figure 4.6: Reactive endocervical cells: Endocervical cells with nuclear enlargement and pleomorphism

43

44 Handbook of Cervical Cytology: Special Emphasis on Liquid-based Cytology •• Excess parabasal cells •• Eosinophilia of the cell cytoplasm and vacuolation •• Pyknosis, karyorrhexis, karyolysis of nucleus. Note: The mere presence of inflammatory cells does not indicate inflammation. Excess number of inflammatory cells may be present in premenstrual and menstrual smear. The cytoplasmic and nuclear degenerative changes are necessary to label the smear as inflammatory.

In liquid-based cytology (LBC), the necrotic background may not be evident and the amount of polymorphs may also be less. Multiple clusters of endocervical cells are often seen in LBC due to endocervical brush sampling. At times the reactive endocervical cells Box 4.1: may be mistaken as atypical glandular cells because of nuclear enlargement Reactive endocervical cells may and pleomorphism (Box 4.1). However, be mistaken as atypical glandular the nuclear chromatin of these cells are cells. However, they lack nuclear bland and the cells do not show nuclear overlapping and chromatin is bland. overlapping.

Chronic Inflammation Acute inflammatory process may subside in course of time and may show features of chronic inflammation. Chronic inflammation is characterized by: •• Abundant lymphocytes •• Histiocytes •• Occasionally epithelioid cell granulomas in granulomatous inflammation. Follicular Cervicitis This is a type of chronic inflammatory conditions characterized by: •• Abundant lymphoid cells with polymorphic population •• Aggregates of lymphoid cells •• Many tangible body macrophages •• Polymorphs •• Dirty necrotic background. Note: Abundant lymphoid cells in the cervical smear may be falsely diagnosed as lymphoma.

Intrauterine Contraceptive Device (Figures 4.7–4.10) In case of use of intrauterine contraceptive device (IUCD) the cervical cytology smears show small isolated clusters of glandular cells. The cells contain multiple vacuoles or large vacuoles that pushes the nucleus to the periphery of the cell.1,2 The large vacuolated cells are known as “bubble gum cell”. The so called “IUD” cells are probably of endometrial origin. These cells are small with round hyperchromatic nuclei having high nucleocytoplasmic ratio. The smears of IUCD device are often associated with actinomycosis infection. 3

Negative for Intraepithelial Lesion or Malignancy

Figure 4.7: IUCD changes: Endocervical cells with large vacuoles (see arrow)

Figure 4.8: IUCD changes: Cell with high N/C ratio (see arrow)

Figure 4.9: IUCD changes: Careful observation shows degenerated nuclei (see arrow)

45

46 Handbook of Cervical Cytology: Special Emphasis on Liquid-based Cytology

Figure 4.10: IUCD changes: Multinucleated cells (see arrow)

In case of IUCD devices the cervical smear shows following changes: •• Columnar endocervical cells with vacuolated cytoplasm •• These cells have enlarged nucleus with prominent nucleoli •• At times these cells show large vacuolated cytoplasm labelled as “bubble gum cell”. •• IUD cells: –– Small round cells with high N/C ratio –– Hyperchromatic nuclei –– They are probably originated from endometrial cells. •• Occasionally multinucleated giant cells, psammoma bodies or calcification may be seen •• One fourth of IUCD cases are also associated with actinomycosis infection. Notes: The vacuolated endocervical cells and “bubble gum cells” along with the presence of psammoma bodies in the smear may be mistaken as adenocarcinoma. However, the following features are helpful to distinguish it from adenocarcinoma (Box 4.2):

Box 4.2: IUCD changes are the potential source of misdiagnosis (adenocarcinoma) Please note: •• Characteristic history •• Absent of tumor diathesis •• Degenerative changes in nuclei

1. The absence of “tumor diathesis” 2. The presence of inflammation 3. The presence of degenerative changes in the nuclei 4. Poor cellular preservation 5. History of IUCD use Smaller IUD cells may simulate HSIL. However, the regular nuclear margin and bland chromatin pattern help to distinguish IUD cells from HSIL.

Reactive Cellular Changes Associated with Radiation (Figures 4.11–4.14) The radiation induced changes are characterized by:

Negative for Intraepithelial Lesion or Malignancy

Figure 4.11: Radiation changes: Multiple multinucleated giant cells (see arrow)

Figure 4.12: Radiation changes: Both nuclear and cytoplasmic enlargement in radiation. There is no abnormality of nuclear chromatin (see arrow)

Figure 4.13: Radiation with squamous cell carcinoma: Smear shows may discrete squamous cells with marked increase of N/C ratio. Nuclear chromatin is coarsely clumped (see arrow)

47

48 Handbook of Cervical Cytology: Special Emphasis on Liquid-based Cytology

Figure 4.14: Radiation with squamous cell carcinoma: Multinucleated giant cells and malignant cells both are present (see arrow)

•• Markedly enlarged cells, i.e. cytomegaly present •• Both nucleus and the cell are enlarged so nucleo-cytoplasmic ratio is not altered •• Abundant and vacuolated cytoplasm •• Nuclear chromatin is smudged with single to multiple prominent nucleoli •• Multinucleated giant cells are frequently seen. In case of chronic effect the cells are mildly enlarged and clusters of squamous cell with spindle shaped elongated nuclei are seen. The cells resemble smooth muscle cells. Note: The cells with radiation changes may simulate malignancy. However, preserved N/C ratio of the cells, many multinucleated giant cells and significant degenerative changes of the nuclei are also helpful to confirm radiation induced changes. Moreover, history of radiation will always be present.

Organisms Trichomonas vaginalis (TV) (Figures 4.15 and 4.16)

•• •• •• ••

Round-to-oval “pear-shaped” organisms, 15–30 micron long Red cytoplasmic granules Round nuclei with pale vesicular chromatin Flagella may be seen in LBC preparation, which are absent in conventional preparation (CP) •• Squamous cells show perinuclear halo and increased eosinophilia •• The typical dirty background of CP is absent in LBC •• The collection of neutrophils around the surface of the squamous cells known as “cannon ball” or “pus ball” may not be evident in LBC. Note: Cytoplasmic fragments of the cells and mucus globules may often be mistaken as TV. However, the presence of nucleus helps to identify TV. Careful observation is needed to identify TV in LBC as the organisms are slightly smaller in size and the typical dirty background of the smear is absent (Box 4.3). At times the squamous

Negative for Intraepithelial Lesion or Malignancy

Figure 4.15: Trichomonas vaginalis: Round shaped organism with relatively clean background

Figure 4.16: Trichomonas vaginalis: Round to oval organism with nuclei (see arrow)

cells may show considerable nuclear atypia in case of TV infection. Leptothrix is often associated with TV infection. Infection by TV often shows mild degree nuclear enlargement and pleomorphism. The reactive atypia due to TV infection should not be labelled as ASCUS.

Box 4.3: Trichomonas vaginalis infection often shows nuclear atypia (ASCUS). One should be careful in this matter. In LBC, the identification of TV may be difficult due to clear background

Bacterial Vaginosis (Shift in Bacterial Flora) (Figure 4.17 and 4.18) Bacterial vaginosis is characterized by: •• Clue cells: Squamous cells are filled with small coccobacilli. These cells are known as “clue cells” •• The background of the smear is flimsy •• Numerous small coccobacilli and curved bacilli are seen •• Lactobacilli are absent. Note: The LBC smear shows much clear background than CP. However, clue cells are readily identified.

49

50 Handbook of Cervical Cytology: Special Emphasis on Liquid-based Cytology

Figure 4.17: Bacterial vaginosis: Clue cells present. Note the relatively clean background.

Figure 4.18: Clue cells: Superficial and intermediate cells with cytoplasmic black dots (see arrow)

Candida (Figures 4.19 and 4.20)

•• Yeast form: Pink colored, oval 3 to 7 micron diameter encapsulated regular structure •• Pseudohyphae form: Long slender non-septate structure with constriction •• In addition: –– Peculiar herringbone or fern like arrangement of the epithelial cells are often seen. This is also known as “shish kebab” like arrangement of the cells. •• Squamous cells often show nuclear enlargement, perinuclear haloes, and increased eosinophilia. •• Many fragmented neutrophils are seen in the background.

Negative for Intraepithelial Lesion or Malignancy

Figure 4.19: Candida: Long pseudohyphae of candida

Figure 4.20: Candida with Shish kebab appearance (see arrow)

Note: •• Herringbone pattern is more common in LBC •• Thin mucous strands may simulate candidal pseudohyphae. However, mucous strands are pale blue in color •• Perinuclear halo in the squamous cells may often be mistaken as koilocytosis •• Nuclear atypia of the squamous cell may simulate atypical squamous cell.

Leptothrix (Figure 4.21)

•• A gram negative, non-spore forming anaerobic organism •• Thin, segmented, and long filamentous organism. Note: Leptothrix are commonly associated with trichomonas infection.

51

52 Handbook of Cervical Cytology: Special Emphasis on Liquid-based Cytology

Figure 4.21: Leptothrix: Long thin organism (see arrow)

Figure 4.22: Actinomycosis: Lower magnification shows clump of actinomycosis (see arrow)

Actinomycosis (Figures 4.22 and 4.23)

•• Clumped of greyish blue filamentous bacteria radiate from a dense centre •• The parallel filaments radiating from the central mass gives “Cotton ball” or “Wooly body” appearance •• Background shows many polymorphonuclear leucocytes, macrophages and occasional multinucleated giant cells. Note: Actinomycosis is almost always associated with IUD or foreign body material.

Herpes Simplex (HSV) Cytomorphologically there are no specific features to distinguish HSV1 and HSV2 virus. The salient cytological features of HSV include (Figure 4.24 and 4.25): •• Large squamous epithelial cells

Negative for Intraepithelial Lesion or Malignancy

Figure 4.23: Actinomycosis: Filamentous organisms giving sunray appearance (see arrow)

Figure 4.24: Herpes simplex: Many uninucleated and binucleated cells (see arrow)

Figure 4.25: Herpes simplex: Multinucleated cells with faceted nuclei and ground glass appearance (see arrow)

53

54 Handbook of Cervical Cytology: Special Emphasis on Liquid-based Cytology •• Multinucleated cell with molding of the nuclei. The nuclei compress each other •• Nuclear chromatin is homogenous and gives ground glass appearance •• Large eosinophilic intranuclear inclusion •• Bizarre nucleus is often seen. Notes: Multinucleated giant cells are also seen in foreign body reaction, trophoblastic cells and as a non-specific reaction. The typical ground glass nuclei and intranuclear large inclusions are helpful in diagnosis.

Atrophic changes (Figures 4.26 and 4.27) The atrophic smear is characterized by: •• Abundant flat monolayer sheets of parabasal and basal cell population •• In liquid-based cytology (LBC) preparation the parabasal cells may be discrete

Figure 4.26: Atrophic smear: Smear shows abundant parabasal cells

Figure 4.27: Atrophic smear: Both parabasal cells and metaplastic squamous cells are present

Negative for Intraepithelial Lesion or Malignancy

•• The parabasal cells show enlarged nuclei which may be 3–5 times of the area of the intermediate cells. This nuclear enlargement is generalized •• Nuclei may show mild hyperchromasia and uniformly distributed chromatin. No chromatin clumping is seen •• There may be degenerated orangeophilic or eosinophilic parabasal cells and histiocytes in the smear •• Elongated spindle shaped cells may be confused with malignancy •• Typical dark blue round shaped amorphous material known as “blue blobs” are usually absent in LBC •• In conventional smear, inflammatory exudates and bluish granular exudates are present that may simulate tumor diathesis. However, in LBC smear the inflammatory exudates are minimum. Notes: The presence of parabasal cells with enlarged nuclei, mild hyperchromasia and background basophilic material with inflammatory exudates may be mistaken as squamous cell carcinoma. However, careful examination of nuclear character of the parabasal cell immediately eliminate this possibility.

Glandular cell status post-hysterectomy In case of post hysterectomy cases, the cervical smear may show benign looking columnar endocervical cells. The cells are present in small groups. The cytoplasm shows pale mucinous material. In addition, inflammatory cells, atrophic pattern and occasional atypical cells may also be seen. 4 Note: The above mentioned changes are benign and possibly due to radiation or chemotherapy effect. These changes do not need any aggressive workup.

References 1. Fornari ML. Cellular changes in the glandular epithelium of patients using IUCD—a source of cytologic error. Acta Cytol. 1974;18(4):341-3. 2. Risse EK, Beerthuizen RJ, Vooijs GP. Cytologic and histologic findings in women using an IUD. Obstet Gynecol. 1981;58(5):569-73. 3. Pillay B, Gregory AR, Subbiah M. Cytopathologic changes associated with intrauterine contraceptive devices. A review of cervico-vaginal smears in 350 women. Med J Malaysia. 1994;49(1):74-7. 4. Tambouret R, Pitman MB, Bell DA. Benign glandular cells in posthysterectomy vaginal smears. Acta Cytol. 1998;42(6):1403-8.

55

5

Chapter

Atypical Squamous Cells

Atypical squamous cells (ASC) in TBS, 2001/2014 is classified as: •• Atypical squamous cells of undetermined significance (ASC-US) •• Atypical squamous cell, cannot exclude high grade high grade squamous intraepithelial lesion (ASC-H). ASC is the most common interpretation of the cervical cytology. The cervical smear is interpreted as ASC when the cytological features are equivocal and insufficient for the diagnosis of SIL. Therefore, ASC is a diagnosis of exclusion. The reproducibility of ASC is poor and complete consensus is reached only in 1/3rd cases of ASC.1,2 This poor reproducibility of ASC is due to variable diagnostic threshold and diagnostic accuracy. The prevalence of ASC is indirectly proportional with the reporting of SIL. The rate of ASC-US diagnosis should be less than 5% of total cervical smear examined in the laboratory. It is preferable that the overall ASC-US reporting rate should not be more than two to three times of the SIL. ASC is a heterogeneous entity and it comprises of both SIL and benign lesions that mimic SIL. In 2001 TBS, ASC is classified into ASC-US and ASC-H. There is no provision to report ASC-US favor reactive changes. Out of all ASC lesions about 80–90% cases are ASC-US and only 10% cases are ASC-H. It has been shown that 10 to 20% of ASC-US cases show either LSIL or HSIL3 and about 30 to 40% ASC-H cases show HSIL on histopathology. 3 Box 5.1: Atypical squamous cells •• •• •• •• ••

ASC is classified into ASC-US and ASC-H Poor reproducibility, complete consensus in 33% cases ASC-US reporting rate should not be more than two to three times of the SIL ASC-US represents 80–90% cases and only 10% cases are ASC-H On histopathology –– Only 10–20% of ASC-US cases show either LSIL or HSIL –– About 30–40% ASC-H show HSIL.

Atypical Squamous Cells

Atypical squamous cells of undetermined significance (ASC-US) The ASC-US is defined as the cytological features supersede that of reactive changes but not so much to give a definitive diagnosis of SIL. Truly speaking ASC-US is a lesion of exclusion. The criteria of ASC-US include (Figure 5.1–5.5): •• The nuclear enlargement of the squamous cell is 2.5 to 3 times more than the nucleus of the normal intermediate cell •• Superficial and intermediate group of cells show atypia •• The cells show mildly increased nucleo-cytoplasmic ratio •• Regular nuclear margin •• Mild nuclear hyperchromasia with fine granular chromatin •• Cytoplasm of the cell is dense orangeophilic.

Figure 5.1: ASC: The intermediate squamous cells with three times enlarged nuclei compared to normal intermediate cells

Figure 5.2: ASC: Enlarged nuclei with bland nuclear chromatin (see arrow)

57

58 Handbook of Cervical Cytology: Special Emphasis on Liquid-based Cytology

.

Figure 5.3: Single multinucleated cell in ASC

Figure 5.4: ASC: Nucleus of the cell more than 3 times than that of intermediate cell. Nuclear margin is regular

Figure 5.5: Bi and multinucleated cells in ASC

Atypical Squamous Cells

Liquid-based cytology •• Both CP and LBC show similar type of atypical cells in smear •• The cells of LBC may be slightly smaller than CP. Notes: In case of ASC-US the superficial and intermediate cells show atypia and basal and parabasal cells do not display any such change. The cytoplasm of the cells often show orangeophilia indicating squamous differentiation. The cells may show changes that are suggestive of HPV infection, however, classical koilocytotic changes eliminate the diagnosis of ASC-US. These cases should always be labelled as LSIL. ASC-US group may include atypical metaplastic squamous cell, atypical squamous cells in atrophy, and atypical parakeratotic squamous cells. The different pattern of smears in ASC-US include:

•• Atypical squamous cell with mature intermediate type cytoplasm: This is the most frequent pattern of ASC-US. Here, predominantly superficial and intermediate cells are involved. The nuclei of the cells are enlarged and usually 2.5–3 times larger than the intermediate cell nuclei. The margin of the nuclei may be irregular. Chromatin is finely granular. •• Atypical metaplastic squamous cells (Figures 5.6–5.8): The smear shows metaplastic squamous cells with dense cytoplasm, high N/C ratio, hyperchromatic nuclei and mildly irregular nuclear membrane. The nuclear chromatin is fine granular. These type of smears should be distinguished from HSIL, reparative changes and carcinoma. •• Atypical squamous cell in the setting of atrophy: The smears show cells with reactive and atypical changes in the background of atrophy. The individual cells show nuclear enlargement, pleomorphism, hyperchromasia, irregular nuclear contour. The cytoplasm may be orangeophilic. 4 •• Atypical parakeratotic squamous cells (Figure 5.9): The smears show singly or in small three dimensional clusters of atypical cells. The cells are small polygonal with dense orangeophilic cytoplasm having small

Figure 5.6: Atypical metaplastic squamous cells with enlarged nuclei

59

60 Handbook of Cervical Cytology: Special Emphasis on Liquid-based Cytology

Figure 5.7: Loose clusters of metaplastic cells showing ASCUS changes

Figure 5.8: Metaplastic cells showing ASCUS changes: Discrete metaplastic cells with enlarged nuclei

Figure 5.9: Atypical parakeratotic cells with nuclear enlargement

Atypical Squamous Cells

dark nuclei. Nuclei show irregular and angulated contour with irregularly distributed chromatin.5 •• ASC-US with equivocal changes of HPV (Figure 5.10): In this type of smear there is perinuclear halo suggestive of koilocytosis. The koilocytotic cells may or may not have nuclear enlargement. In addition to this doubtful koilocytotic cells the smear also shows occasional bi or multinucleated cells with dark hyperchromatic nuclei. The ASC-US cases should always be distinguished from: 1. Improperly fixed smear: It is totally avoided in LBC preparation 2. Inflammatory atypia (Figure 5.11A and B): Inflammatory changes in the cytoplasm and nuclei such as cytoplasmic vacuolation, nuclear pyknosis, karyorrhexis, karyolysis, excess parabasal cells and inflammatory exudates help to distinguish inflammatory atypia from true atypia. 3. Artifact producing changes: Various staining artefact, nuclear overlapping etc. may create problem. 4. Therapy induced atypia (Figure 5.12): Radiation and chemotherapy induced atypia should not be included in ASC-US. The nuclear chromatin is smudgy and overall nuclear preservation is poor in therapy induced atypia. N/C ratio is not altered in such cases. 5. Naked large nuclei with stripped out cytoplasm: Truly speaking these naked nuclei should not be considered during reporting of ASC-US. 6. Other large atypical cells such as decidual cells, trophoblastic cells etc. 7. Folic acid deficiency (Figure 5.13): Folic acid deficiency may produce diffuse nuclear enlargement in most of the cells. The nuclei are Normochromatic.

Figure 5.10: ASC: Enlarged nuclei with equivocal changes of koilocytosis (case taken from case based approach in exfoliative cytology book)

61

62 Handbook of Cervical Cytology: Special Emphasis on Liquid-based Cytology

Figure 5.11A: Inflammation causing atypia

Figure 5.11B: Atypical looking endocervical cells in an inflammatory lesion

Figure 5.12: Radiation induced atypia: Atypical cell with enlarged nuclei in a case of radiation induced changes in cervical smear. The smear should not be classified as ASCUS (see arrow)

Atypical Squamous Cells

Figure 5.13: Folic acid deficiency causing enlarged nuclei and may simulate ASC

Atypical squamous cell, cannot exclude high grade squamous intraepithelial lesion (ASC-H) ASC-H cases are defined as “suggestive of HSIL but fall short of cytological criteria for a definitive interpretation of HSIL”. The criteria of ASC-H are (Figures 5.14–5.17): •• Single and loose cohesive group of cells •• Hyperchromatic crowded groups are present •• Immature small squamous cells •• Nucleus: Mild to moderate enlargement, hyperchromasia and pleomorphism. Nucleoli is usually absent •• Nuclear size is 1.5–2.5 times larger than normal metaplastic cells. •• Increased nucleocytoplasmic ratio.

Liquid-based Cytology

•• The overall size of ASC-H cells are much smaller in LBC preparation •• The nuclei may not be much hyperchromatic in LBC and therefore much attention should be given to high nucleocytoplasmic ratio and nuclear margin irregularity. The nuclei of the adjacent metaplastic cells in the smear is helpful for comparison to the ASC-H cells. Notes: Smears of ASC-H show only occasional atypical cells compared to large number of abnormal cells in HSIL. The nuclear changes in ASC-H are always less marked than HSIL. However, the difference of ASC-H and HSIL are too some extent subjective. The differential diagnosis of ASC-H always includes the mimickers of HSIL. The pattern of ASC-H include:

•• Small cell pattern or atypical immature metaplasia (Figures 5.18 and 5.19): The atypical are small in size with increased N/C ratio. The cells simulate immature metaplastic cells. The following nuclear features help to differentiate immature metaplastic cells from ASC-H:

63

64 Handbook of Cervical Cytology: Special Emphasis on Liquid-based Cytology

Figure 5.14: ASC H: Smaller cells with large hyperchromatic nuclei

Figure 5.15: ASC H: Higher magnification showing enlarged hyperchromatic nuclei

Figure 5.16: ASC H: Occasional discrete small cells with enlarged hyperchromatic nuclei

Atypical Squamous Cells

Figure 5.17: ASC H: Discrete intermediate cells with enlarged nuclei

Figure 5.18: ASC-H: Metaplastic cells with enlarged hyperchromatic nuclei

Figure 5.19: ASC H: Group of metaplastic cells with hyperchromatic enlarged nuclei

65

66 Handbook of Cervical Cytology: Special Emphasis on Liquid-based Cytology

Figure 5.20: ASC-H: Hyperchromatic crowded group of atypical cells with overlapping nuclei

Figure 5.21: ASCH: Higher magnification showing atypical cells with enlarged hyperchromatic nuclei

a. Nuclear enlargement : Double nuclear area b. Nuclear margin irregularity c. Hyperchromasia d. Irregular clumped chromatin. •• Atrophic pattern: The occasional atypical cells are noted in an atrophic background. The cells are small in size with high N/C ratio. Atrophic smear or radiation induced atypia are the common mimickers of these type of ASC-H. •• Hyperchromatic crowded group (Figures 5.20 and 5.21): The cells are aggregated as tight clusters and at times mimic glandular cells. However, the cells of hyperchromatic crowded group are polygonal in shape with definite cell border having dense orangeophilic cytoplasm. The nuclei

Atypical Squamous Cells

Figure 5.22: Cluster of endometrial cells simulating ASC-H

Figure 5.23: Atypical glandular cells simulating ASC-H

are hyperchromatic having high N/C ratio. Prominent nucleoli is not a feature of ASC-H. The presence of prominent nucleoli indicate glandular involvement.

Mimickers of ASC-H •• Endometrial cells (Figure 5.22): Clusters of endometrial cells with dark nuclei may simulate ASC-H. Endometrial cells are better visualized in LBC and can be distinguished from ASC-H by the nuclear morphology. •• Histiocytes: The histiocytes are always present singly and nuclei are pale. •• Atypical glandular cells (Figure 5.23): Atypical glandular cells in small clusters may simulate ASC-H. The cells with vacuolated cytoplasm and vesicular chromatin are helpful differentiating features.

67

68 Handbook of Cervical Cytology: Special Emphasis on Liquid-based Cytology •• HSIL involving glands •• Atypia in IUD, or radiation: atypical cells may be seen in IUCD or radiation and may simulate ASC-H. In case of radiation there is no increased N/C ratio and the nuclear chromatin is usually not well preserved.

References 1. Sherman ME, Schiffman MH, Lorincz AT, et al. Toward objective quality assurance in cervical cytopathology: correlation of cytopathologic diagnoses with detection of highrisk human papillomavirus types. Am J Clin Pathol. 1994;102:182-7. 2. Smith AE, Sherman ME, Scott DR, et al. Review of the Bethesda System atlas does not improve reproducibility or accuracy in the classification of atypical squamous cells of undetermined significance smears. Cancer. 2000;90:201-6. 3. 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;183:1383-92. 4. Solomon D, Frable WJ, Vooijs GP, et al. ASC-US and AGUS criteria: International Academy of Cytology Task Force summary: Diagnostic Cytology Towards the 21st Century: an International Expert Conference and Tutorial. Acta Cytol. 1998;42:16-24. 5. Voytek TM, Kannan V, Kline TS. Atypical parakeratosis: a marker of dysplasia? Diagn Cytopathol. 1996;15:288-91.

6

CHAPTER

Squamous Intraepithelial Lesions and Invasive Squamous Cell Carcinoma

INTRODUCTION TBS 2001/2014, classified SIL as: • Low-grade squamous intraepithelial lesion (LSIL) • High-grade squamous intraepithelial lesion (HSIL) TBS 2001 has classified squamous intraepithelial lesions in two tier system. The logic behind the two tier grading system are: 1. Most of the LSIL does not progress to higher grade and either persist or regress. 2. It is expected that two tier grading is more reproducible than three tier grading (CIN 1, 2, and 3). LSIL encompasses both HPV induced changes and cervical intraepithelial lesions grade 1 (CIN 1), whereas HSIL includes cervical intraepithelial lesions grade 2 (CIN 2) and cervical intraepithelial lesions grade 3 (CIN 3). The majority of LSIL is caused by low risk HPV 6 and 11. Almost 60% LSIL cases regress within 2 year period, 30% persist and 10% progress to HSIL. Very few cases of LSIL (less than Box 6.1: Essential information on SIL 1%) ultimately progress to invasion (Box 6.1). There is no morphological marker • LSIL is predominantly caused by HPV 6 and 11 to predict whether a particular LSIL case • 60% LSIL cases regress within 2 will progress to HSIL or not. Therefore, it year period is necessary to have periodic screening • 30% LSIL cases persist in such cases. High risk HPVs (HPV 16, • 10% LSIL cases progress to HSIL 18) are found in 97% of HSIL cases. Only • Less than 1% LSIL ultimately progress to invasion 0.5% of all cervical smears show HSIL. Near about 40% CIN 2 and 33% CIN 3 • High risk HPV 16 and 18 are seen in 97% of HSIL cases regress and 40% of CIN 2 and 56% of • Only 20% cases of HSIL progress CIN 3 persist. Only 20% cases of CIN2 to invasive carcinoma. progress to invasive carcinoma.

70 Handbook of Cervical Cytology: Special Emphasis on Liquid-based Cytology LOW GRADE INTRAEPITHELIAL LESIONS (LSIL) In case of LSIL the lower 1/3rd of the cervical squamous epithelium shows dysplastic cells. These cells show mild nuclear enlargement and pleomorphism (Figure 6.1). The amount of abnormal or dysplastic cells is related with the extent of the disease. More severe the abnormality more amount of dysplastic cells are noted in the cervical smear. The dysplastic cells of LSIL closely resemble superficial and intermediate cells. The cells show well defined cytoplasmic margin with enlarged nuclei occupying more than 1/3 of the cytoplasmic area. Nuclear margin is mildly irregular. Nuclei are mildly hyperchromatic with inconspicuous nucleoli.

Cytological Features of LSIL Include (Figures 6.2–6.6)

• • • • • • •

•

Predominantly single or sheets of superficial and intermediate cells The cells are large with moderate to abundant cytoplasm The atypical cells have well defined cytoplasmic margin The nuclei of the cells are enlarged and nuclear area is more than three times of the normal intermediate cell nuclei Nucleocytoplasmic (N/C) ratio is Box 6.2: Koilocytosis mildly increased • The presence of koilocytosis alNuclei show mild hyperchromasia ways indicates at least LSIL with mildly irregular nuclear margin • Distinct perinuclear cytoplasmic Nuclei show evenly distributed vacuoles with sharp condensed granular chromatin with absent or thick cytoplasmic margin and enlarged nuclei are characteristic inconspicuous nucleoli of koilocytosis Koilocytosis (Figures 6.7– 6.9) (Box • Differential diagnosis: Glycoge6.2): Koilocytosis is characterized nated cells, inflammatory smear by distinct perinuclear cytoplasmic particularly TV infection. vacuole with sharp condensed thick

Figure 6.1: LSIL (CIN1): Only lower third of epithelium shows atypical cells

Squamous Intraepithelial Lesions and Invasive Squamous Cell Carcinoma

Figure 6.2: LSIL: Cell shows enlarged mildly hyperchromatic nucleus. Nucleus is three times enlarged than that of normal intermediate cell (see arrow)

Figure 6.3: LSIL: Loose cluster of cells with enlarged mildly hyperchromatic nuclei

Figure 6.4: LSIL: Cells with enlarged nuclei and mildly irregular nuclear margin

71

72 Handbook of Cervical Cytology: Special Emphasis on Liquid-based Cytology

Figure 6.5: LSIL: Loose cluster of cells with enlarged nuclei. Nuclei have regular outer margin

Figure 6.6: LSIL: Cells show enlarged nuclei with mildly irregular nuclear margin. Nuclear hyperchromasia is not prominent

Figure 6.7A: Histopathology of koilocytosis: Koilocytotic cells with enlarged nuclei and perinuclear haloes

Squamous Intraepithelial Lesions and Invasive Squamous Cell Carcinoma

Figure 6.7B: Histopathology of koilocytosis: Higher magnification of koilocytotic cells

Figure 6.8: LSIL: Koilocytotic cells with nuclear atypia (see arrow)

Figure 6.9: LSIL: Smear shows koilocytotic cell with enlarged nuclei and perinuclear halo. Note the thick cytoplasmic margin (see arrow)

73

74 Handbook of Cervical Cytology: Special Emphasis on Liquid-based Cytology cytoplasmic margin. Nuclei are enlarged. The perinuclear halo along with the nuclear abnormalities are essential for the qualification of koilocytosis. The presence of koilocytosis is the characteristic feature of LSIL but it is not an essential feature to interpret LSIL. • Frequent bi and multinucleation is also seen.

Liquid-based Cytology In LBC preparation the atypical cells may Box 6.3: LSIL in LBC not show significant hyperchromasia. However, the single discrete atypical cells • Minimal hyperchromasia are easily identifiable in LBC preparation • Clusters of atypical cells • Nuclear enlargement and pleoby observing the nuclear enlargement morphism maintained and nuclear margin irregularity (Box 6.3). • Easily identifiable koilocytotic cells.

Cytological Features of HPV Effect (Box 6.4) • Koilocytosis (Figures 6.10 and 6.11): Box 6.4: Cytological features of HPV Koilocytosis is the characteristic fea- infection ture of HPV infection. It is important • Koilocytosis to identify true koilocytes from the • Bi and multinucleation other cells that simulate koilocyto- • Parakeratosis sis. The following cells may simulate • Frayed edges of the cells koilocytes: • Dysplastic cells. – Trichomonas vaginalis (TV) infection: Trichomonas infection may cause perinuclear haloes simulating koilocytes. However, nuclei of the cells are not enlarged. The presence of TV along with mild nuclear atypia practically eliminates the possibility of koilocytosis. – Artefacts: Artifactual perinuclear haloes in the cells may pose problem. – Occasional enlarged endocervical cells: At times the endocervical cells may show nuclear enlargement with perinuclear clearing. These cells closely simulate koilocytes. However, the accompanying endocervical cells helps in the elimination of any possibility of koilocytosis due to HPV infection. • Variably enlarged nuclei (Figure 6.12): The nuclei of the cells are variably enlarged. • Bi- and multinucleation (Figures 6.13 and 6.14): Frequent bi- and multinucleated cells are seen. • Parakeratosis (Figures 6.15 and 6.16): The parakeratotic cells are often seen in HPV infection. The cells are smaller with abnormal keratinization in the cytoplasm. • Abnormal shaped cells: Cells with tad pole shaped, fiber cells and frayed edges are also noted in HPV infection. Careful evaluation is needed to avoid mistake of these features as squamous cell carcinoma.

Squamous Intraepithelial Lesions and Invasive Squamous Cell Carcinoma

Figure 6.10: True koilocytosis: Truly koilocytotic cells with perinuclear halo and enlarged nuclei (see arrow)

Figure 6.11: False koilocytosis: Glycogenated cells with perinuclear halo (see arrow)

Figure 6.12: HPV changes: Nuclear enlargement of the cell (see arrow)

75

76 Handbook of Cervical Cytology: Special Emphasis on Liquid-based Cytology

Figure 6.13: HPV changes: Bi-nucleated cell (see arrow)

Figure 6.14: HPV changes: Multi-nucleated cell (see arrow)

Figure 6.15: HPV changes: increased number of parakeratotic cells

Squamous Intraepithelial Lesions and Invasive Squamous Cell Carcinoma

Figure 6.16: LSIL: Parakeratotic cells showing nuclear atypia

Diagnostic Difficulties in LSIL LSIL must be distinguished from its Box 6.5: Mimickers of LSIL mimickers as described below (Box 6. 5): • Reactive inflammatory changes 1. Reactive inflammatory changes in in squamous epithelial cells squamous epithelial cells: Inflam- • Air drying artifact matory smears may show nuclear • Non-specific perinuclear halos enlargement in squamous cells. Howmimicking koilocytosis ever, these reactive cells show regular • Reactive changes in endocervical cells nuclear margin with homogenous • ASC-US bland chromatin. 2. Air drying artifact: Air drying artifact • Folic acid deficiency in CP shows nuclear enlargement and • Radiation effect causing nuclear enlargement. this may be mistaken as LSIL. 3. Non-specific perinuclear halos (Figure 6.17): Nonspecific perinuclear halos may be seen in trichomonas infection. No nuclear atypia is seen in these conditions. 4. Reactive changes in endocervical cells (Figure 6.18): As mentioned before, reactive endocervical cells may have enlarged nucleus and polygonal shape which may simulate LSIL. The cells are often recognized by the accompanying typical endocervical cells. 5. ASCUS (Figure 6.19): The difference between ASCUS and LSIL is too some extent subjective. Nuclear changes in ASCUS is quantitatively and qualitatively less than that of LSIL. 6. Folic acid deficiency (Figure 6.20): In case of folic acid deficiency the majority of the cells show nuclear enlargement. Other than nuclear enlargement no other nuclear abnormalities are seen. 7. Radiation effect causing nuclear enlargement: Radiation injury may cause nuclear enlargement. However, in such condition cytomegaly

77

78 Handbook of Cervical Cytology: Special Emphasis on Liquid-based Cytology

Figure 6.17: False koilocytosis: Inflammatory smear showing false koilocytotic cells

Figure 6.18: Reactive endocervical cells: Reactive endocervical cells with nuclear enlargement simulating LSIL

Figure 6.19: ASCUS: Smear from ASCUS showing nuclear enlargement and simulate LSIL. Note relatively bland chromatin of the cells

Squamous Intraepithelial Lesions and Invasive Squamous Cell Carcinoma

Figure 6.20: Folic acid deficiency showing nuclear enlargement

occurs and the nucleocytoplasmic ratio of the cell is not altered. The patient always give history of radiation exposure in radiation induced changes.

HIGH GRADE INTRAEPITHELIAL LESIONS (HSIL) In case of HSIL the histopathology section of the cervix shows involvement by dysplastic cells by more than basal 1/3rd of the epithelium. The cells show moderately enlarged and pleomorphic nuclei (Figure 6.21).

The Cytological Features of HSIL Include (Figures 6.22–6.29) The cells are arranged discretely, sheets or as syncytial aggregates. • Overall the cells are smaller in size and less mature in HSIL. The mostly parabasal sized cells are affected • Cytoplasm is dense or lacy and orangeophilic in keratinized variant • The cells show moderate nuclear pleomorphism. The degree of nuclear shape and size variation are usually more in HSIL compared to LSIL • The nuclei of the cells show following changes: – Marked enlargement – Markedly increased N/C ratio – Coarsely granular chromatin – Marked irregular nuclear contour with frequent indentation – Nucleoli is commonly absent. However, the dysplastic cells may show prominent nucleoli if the endocervical glands are involved by the dysplastic cells.

Liquid-based Cytology The number of abnormal cells may be less in the smear in LBC preparation. The cells are usually in dispersed population. In comparison to CP,

79

80 Handbook of Cervical Cytology: Special Emphasis on Liquid-based Cytology

Figure 6.21: HSIL: Histopathology of HSIL showing full thickness involvement

Figure 6.22: HSIL: Cluster of cells with round, moderately hyperchromatic nuclei

Figure 6.23: HSIL: The cells are small with enlarged hyperchromatic nuclei

Squamous Intraepithelial Lesions and Invasive Squamous Cell Carcinoma

Figure 6.24: HSIL: Discrete cells with nucleus occupying whole of the cytoplasm

Figure 6.25: HSIL: Nuclei of the cells are hypochromatic

Figure 6.26: HSIL: Small cells with high N/C ratio. Nuclei show coarse granular chromatin

81

82 Handbook of Cervical Cytology: Special Emphasis on Liquid-based Cytology

Figure 6.27: HSIL: Metaplastic cells showing nuclear enlargement and pleomorphism. Nuclear chromatin is coarse

Figure 6.28: HSIL: Occasionally only a few discrete abnormal cells may be seen

Figure 6.29: Both metaplastic cells and HSIL: Both metaplastic cells and HSIL have been highlighted

Squamous Intraepithelial Lesions and Invasive Squamous Cell Carcinoma

the cells in LBC are smaller in size (Box 6.6). These abnormal cells may not show significant hyperchromasia in LBC. However nuclear margin irregularity, nuclear enlargement, and pleomorphism help to identify such dysplastic cells. Hyperchromatic crowded group of cells may be noted which should be interpreted carefully.1

Box 6.6: HSIL in LBC • Abnormal cells are less in the smear • Dispersed single cells • Smaller in size • Less hyperchromatic • Other nuclear abnormalities such as nuclear enlargement, pleomorphism and margin irregularities are maintained • Hyperchromatic crowded group of cells may be noted.

Notes: The cells of HSIL are relatively smaller in size with enlarged nuclei. Nuclear enlargement of cells of HSIL is almost same but the N/C ratio is markedly increased. The nuclei show marked hyperchromasia, membrane irregularity and coarse granular chromatin. The severity of the nuclear changes in HSIL help to differentiate the lesion from LSIL (Table 6.1). At times the HSIL cells are much smaller with no cytoplasmic differentiation. These cells simulate endocervical glandular cells and difficult to be recognized.

The differential diagnosis of HSIL include Box 6.7: Mimickers of HSIL (Box 6.7): • Squamous metaplastic cells • Squamous metaplastic cells (Figure • Atrophic smear 6.30): Metaplastic squamous cells • Endometrial cells have relatively large nucleus and • ASC-H dense chromatin and may mimic • Overstained cells HSIL. However, metaplastic cells have • LSIL regular chromatin pattern compared • Squamous cell carcinoma. to coarse granular chromatin in HSIL. • Atrophic smear (Figure 6.31): In atrophic smears the small parabasal cells with high N/C ratio may mimic HSIL. Attention should be given to other nuclear features such as coarse granular nuclear chromatin and irregular nuclear margin that help to distinguish it from atrophic smear. • Histiocytes: Mononuclear histiocytes with enlarged nuclei may also simulate HSIL. The fine chromatin texture, normochromasia and relatively

Table 6.1: Distinguishing cytological features of LSIL versus HSIL LSIL

HSIL

Superficial and intermediate cells are Parabasal, basal and metaplastic are predominant cell population predominant cell population Cell cytoplasm is relatively more

Cell cytoplasm is relatively less

Nucleus is enlarged with relatively low N/C Nucleus is moderately enlarged with ratio relatively high N/C ratio Nuclear margin is mildly irregular

Nuclear margin is moderately irregular

Fine nuclear chromatin

Coarse granular nuclear chromatin

83

84 Handbook of Cervical Cytology: Special Emphasis on Liquid-based Cytology

Figure 6.30: Metaplastic squamous cells: Metaplastic squamous cells with enlarged nuclei simulating HSIL

Figure 6. 31: Atrophic smear: Large number of parabasal cells present. These cells often simulate HSIL

•

•

• •

regular nuclear contour are helpful features to distinguish histiocytes from HSIL. Endometrial cells (Figure 6.32): Small groups of endometrial cells with dark nuclei and high N/C ratio may simulate HSIL. However, endometrial cells are always present in tight cohesive well circumscribed clusters and individual cells are smaller than that of HSIL. ASC-H: ASC-H is a close mimicker of HSIL. The difference is too some extent subjective. The qualitative and quantitative changes in ASC-H are less compared to HSIL. Overstained cells: Overstained nuclei may show false hyperchromasia and mimic HSIL. Squamous cell carcinoma: In absence of tumor diathesis, fibre cells and tadpole cells squamous cell carcinoma may mimic HSIL. This is a

Squamous Intraepithelial Lesions and Invasive Squamous Cell Carcinoma

Figure 6.32: Endometrial cells: Cohesive cluster of endometrial cells may also simulate HSIL

problem in LBC preparation as tumor Box 6.8: Potential pitfalls of HSIL diathesis is mostly removed in such A. Pale cell dyskaryosis preparation. B. Sparse population of dyskaryotic • Several potential pitfalls in the cell diagnosis of HSIL may be encountered C. Small cell severe dyskaryosis (Box 6.8) that include: D. Bland dyskaryosis A. Pale cell dyskaryosis2 (Box 6.9): All E. Hyperchromatic crowded group types of squamous cells may show of cells. pale cell dyskaryosis (Figures 6.33 and 6.34). These cells may be seen Box 6.9: Pale cell dyskaryosis in all grades of dysplasia and even • Small group of cells squamous cell carcinoma. It is still • Hypochromatic pale nuclei not clear whether these cells are • Regular nuclear margin staining artifact or true feature of the • Speckled chromatin cells. These cells are present usually • Minimal chromatin abnormality in small groups or singly. The cells are Interpretation error: Inflammatory small with pale hypochromatic nuclei cells. having regular nuclear contour. Fortunately pale cells are seen in addition to classical hyperchromatic dysplastic cells. Predominant population of pale cells may create diagnostic confusion. Commonly these cells are mistaken as inflammatory cells. B. Sparse dyskaryotic cell3: At times dysplastic cells may be present only in sparse population (Figure 6.35 and 6.36). Scattered single scanty dysplastic cells may be mistaken as false negative smear. The total 13 to 14 severely dysplastic cells may be noted in such smear. C. Small cell severe dyskaryosis (Box 6.10): Small severe dyskaryotic cell are present singly or in small clusters. The cells are small and of same size as that of neutrophils. The cells have enlarged nuclei occupying most of the cytoplasm with high N/C ratio. Nuclear membrane is regular. These

85

86 Handbook of Cervical Cytology: Special Emphasis on Liquid-based Cytology

Figure 6.33: Pale cell to miss: Cell with enlarged pale nucleus (see arrow)

Figure 6.34: Pale cell to miss: Cell with enlarged pale nucleus (see arrow)

cells are identified by their speckled Box 6.10: Small cell severe dyskaryosis chromatin. At times this chromatin • Predominantly single and occapattern may be the only clue to sional clusters recognize such cells. The common • Small size as that of neutrophil mimickers of the small dysplastic • High N/C ratio cells are histiocytes, endometrial • Regular nuclear margin cells, lymphocytes, and mature • Speckled or irregularly clumped metaplastic cells. Endometrial cells chromatin are usually in tight clusters with • Inconspicuous nucleoli. small homogenously dark stained Differential diagnosis: Histiocytes, nuclei. Histiocytes have pale grey lymphocytes, endometrial cells, cytoplasm with kidney shaped metaplastic squamous cells.

Squamous Intraepithelial Lesions and Invasive Squamous Cell Carcinoma

Figure 6.35: Sparse cell to miss: Occasional dysplastic cell (see arrow)

Figure 6.36: Sparse cell to miss: Occasional dysplastic cell in higher magnification (see arrow)

nuclei having bland chromatin. Degenerated histiocytes may show eosinophilic cytoplasm and dark pyknotic nuclei. These cells may mimic small dyskaryotic cells. Therefore, it is always recommended to diagnose small dyskaryotic cell only in presence of classical dysplastic cells. D. Bland dyskaryosis: The cells are usually present in loose clusters with disorganized pattern (Box 6.11). The nuclei of the cells are hyperchromatic with regular nuclear margin. At low power magnification the chromatin of the nuclei look bland. However, careful observation in higher magnification reveals spackled chromatin. These cells may be mistaken as immature metaplastic cells or endocervical cells.4 E. Hyperchromatic crowded groups (Figures 6.37 and 6.38): The cells of HSIL may be present as thick crowded group with overlapping nuclei.

87

88 Handbook of Cervical Cytology: Special Emphasis on Liquid-based Cytology This crowded cells are known as Box 6.11: Bland dyskaryosis hyperchromatic crowded group • Clusters of cells with disordered (HCG). This type of HCG may easily architecture be mistaken as endometrial cells or • High N/C ratio endocervical cells. HCG composed • Hyperchromatic nuclei of dysplastic cells are recognized by • Regular nuclear margin the following criteria: • Speckled chromatin. a. Irregular margin of the HCG Differential diagnosis: immature b. The loss of polarity of the periph- metaplastic cells or endocervical cells. eral cells of the cluster c. Hyperchromatic nuclei, and d. The presence of occasional mitosis.

Figure 6. 37: Hyperchromatic crowded group: Thick crowded group with overlapping nuclei. Note the irregular margin of the group.

Figure 6.38: Hyperchromatic crowded group: Higher magnification showing large pleomorphic cells

Squamous Intraepithelial Lesions and Invasive Squamous Cell Carcinoma

KERATINIZING HIGH GRADE SQUAMOUS INTRAEPITHELIAL LESION (FIGURE 6.39 TO 6.44) Keratinizing HSIL shows single and clusters of dysplastic cells (Box 6.12). The cells have abundant dense orangeophilic cytoplasm with central enlarged nuclei. These abnormally keratinized cells show hyperchromatic enlarged pleomorphic nuclei with irregular nuclear membrane and high nucleo cytoplasmic ratio. Often the nuclei of the cells are spindle shaped or tadpole like resembling keratinized squamous cell carcinoma. Mitosis may also be seen.

Box 6.12: Keratinizing HSIL • Single and clusters of dysplastic cells • Cell with dense orangeophilic cytoplasm • Enlarged hyperchromatic nuclei • High N/C ratio • Irregular nuclear membrane • Spindle shaped cells and tadpole cells present • Mitosis present • Absent tumor diathesis Differential diagnosis: keratinizing squamous cell carcinoma, atrophic vaginitis may show pseudo keratinized cells

Differential Diagnosis The keratinizing HSIL should always be distinguished from keratinizing squamous cell carcinoma. Absent of tumor diathesis is an important distinguishing feature. Occasionally atrophic vaginitis may also show pseudo keratinized cells.

HSIL with Glandular Involvement (Figures 6.45 and 6.46) When HSIL involves the endocervical glands the smear shows many hyperchromatic crowded groups of cells. The margin of the crowded group is irregular and the cells show loss of polarity. The cells also exhibit nuclear atypia, occasional cytoplasmic vacuoles and prominent nucleoli. The careful

Figure 6.39: Keratinizing HSIL: Many parakeratotic cells are seen

89

90 Handbook of Cervical Cytology: Special Emphasis on Liquid-based Cytology

Figure 6.40: Keratinizing HSIL: The cells with enlarged nuclei along with spindle cells

Figure 6.41: Keratinizing HSIL: Clusters of parakeratotic cells

Figure 6.42: Keratinizing HSIL: Discrete parakeratotic cells with enlarged nuclei

Squamous Intraepithelial Lesions and Invasive Squamous Cell Carcinoma

Figure 6.43: Keratinizing HSIL: Cells with enlarged nuclei having dense orangeophilic cytoplasm

Figure 6.44: Keratinizing HSIL: Occasionally elongated fibre like cells may be present

examination of the peripheral cells of the Box 6.13: HSIL with glandular involvecluster suggests the squamous origin of ment the cells (Box 6.13).

SQUAMOUS CELL CARCINOMA Squamous cell carcinoma (SCC) is the commonest carcinoma of the cervix. It occurs in the elderly patients between 40 to 55 years of age. World Health Organization (WHO) recommends two tiered system of classification of squamous cell carcinoma of cervix on histology (Figure 6.47):

• Many hyperchromatic crowded groups of cells • Irregular outer margin of the cell clusters • Loss of polarity of the peripheral cells • Cells look more of squamous than endocervical • Cytoplasmic vacuoles and prominent nucleoli.

91

92 Handbook of Cervical Cytology: Special Emphasis on Liquid-based Cytology

Figure 6.45: HSIL with gland involvement: The loose cluster of cells with enlarged hyperchromatic nuclei and cytoplasmic vacuolation

Figure 6.46: HSIL with gland involvement: Same case shows simultaneous clusters of cells with orangeophilic cytoplasm and moderately hyperchromatic nuclei

• Keratinizing squamous cell carcinoma • Non-keratinizing squamous cell carcinoma. However, TBS, 2001/2014 does not promote to sub classify squamous cell carcinoma into any further category. The smears of SCC show all the cytological features that are seen in HSIL. In addition, the smears show evidence of tumor diathesis, fiber cells and tadpole cells. The overall cellularity of squamous cell carcinoma is much more than HSIL. The cells may be arranged in syncytial clusters and discretely. The Box 6.14: Squamous cells carcinoma cells may be polyhedral or oval shaped with orangeophilic cytoplasm. The nuclei • All the cytological features of HSIL of the cells show moderate to markedly • In addition: tumor diathesis, fiber pleomorphism with irregular clumped cells, tadpole cells. chromatin and often large prominent nucleoli (Box 6.14).

Squamous Intraepithelial Lesions and Invasive Squamous Cell Carcinoma

Figure 6.47: Histopathology of squamous cell carcinoma

Figure 6.48: Squamous cell carcinoma: In absence of tumor diathesis the cells of squamous cell carcinoma may simulate HSIL

The cytological features of squamous cells carcinoma include (Figure 6.48–6.58): • Large cells with moderate to marked pleomorphic nuclei • The cells show enlarged nuclei with high N/C ratio • Moderate to marked hyperchromatic nuclei • Irregular contour of nuclei • Irregular coarse chromatin • Nucleoli are small to large and occasionally macronucleoli may be seen • Cytoplasm is dense orangeophilic indicating intracellular keratinization • Tadpole cells: These are elongated cells with enlarged hyperchromatic nuclei in one corner • Fibre cell: Spindle shaped hyperchromatic nuclei • Tumor diathesis: Dirty background with necrotic tissue fragments and RBCs.

93

94 Handbook of Cervical Cytology: Special Emphasis on Liquid-based Cytology

Figure 6.49: Squamous cell carcinoma: Discrete and loose clusters of malignant cells

Figure 6.50: Squamous cell carcinoma: Markedly enlarged nuclei with hyperchromatic nuclei having clumped chromatin

Figure 6.51: Squamous cell carcinoma: Squamous pearl along with malignant cells

Squamous Intraepithelial Lesions and Invasive Squamous Cell Carcinoma

Figure 6.52: Squamous cell carcinoma: Markedly pleomorphic nuclei with coarsely clumped chromatin

Figure 6.53: Squamous cell carcinoma: : Coarse granular chromatin

Figure 6.54: Squamous cell carcinoma: Discrete tumor cells with very high N/C ratio

95

96 Handbook of Cervical Cytology: Special Emphasis on Liquid-based Cytology

Figure 6.55: Squamous cell carcinoma: Fibre cells with elongated nuclei

Figure 6.56: Squamous cell carcinoma: Tadpole cell with one sided nuclei and elongated cytoplasmic process

Figure 6.57: Squamous cell carcinoma: Lower magnification showing tumor diathesis (see arrow)

Squamous Intraepithelial Lesions and Invasive Squamous Cell Carcinoma

Figure 6.58: Squamous cell carcinoma with tumor diathesis: Tumor cells clinging with necrotic tissue

LBC Features of Squamous Cell Carcinoma Tumor diathesis is not always obvious in case of LBC (Box 6.15). The other important features of SCC in LBC have been highlighted in Box 6.15. In case of non-keratinizing SCC the characteristic cytological features are: • Relatively smaller cells and resemble the cells of HSIL • Hyperchromatic nuclei with high N/C ratio • Cytoplasm is scanty and orangeophilia may not be evident • Clinging tumor diathesis is more often seen.

Box 6.15: LBC features of squamous cell carcinoma • Tumor diathesis shows RBCs, inflammatory cells and necrotic debris with attached tumor cells. • Necrotic cells typically attaches with the surface of the cells giving rise to “clinging diathesis” • The cells are usually smaller in size • The cytoplasm of the tumor cells are more condensed • Usually the cells show more prominent nucleoli • The chromatin of the cells may be evenly distributed and the cells may not look malignant • The nuclear enlargement, pleomorphism and nuclear margin irregularity are seen.

Notes: The demonstration of tumor diathesis is the single most important feature in case of diagnosis of invasive SCC. In LBC preparation this tumor diathesis may not be evident as the necrotic fragments are largely removed during sample preparation. The tumor diathesis may be present as granular amorphous debris of cells and RBCs in the background (Box 6.16). This type of necrotic background may also be seen in atrophic smear and menstrual smear. Clinging tumor diathesis is more reliable for invasion. Here, the necrotic tissue is attached with the tumor cells. This type of clinging tumor diathesis is more prominent in case of non-keratinizing squamous cell carcinoma.

Fibre cells are slender elongated cells with hyperchromatic nuclei. These cells are more frequent in keratinizing squamous cell carcinoma and are rarely seen in HSIL (Figures 6.59A and B). One should be careful in identifying

97

98 Handbook of Cervical Cytology: Special Emphasis on Liquid-based Cytology

Figure 6.59A: Fiber cell in squamous cell carcinoma

Figure 6.59B: Spindle cells in atrophic smear may simulate fiber cells

the fiber cells, as various other cells may Box 6.16: Tumor diathesis in LBC mimic fiber cells such as: • Tumor diathesis is largely rea. Endometrial stromal cells, moved in LBC b. Fibroblasts, • Only clinging tumor diathesis: Tumor cells cling with the nec. Smooth muscle cells, and crotic tissue d. Stromal cells in atrophic smear. • Beware of atrophic smear and The smear of SCC should always be menstrual smear that simulate distinguished from the following cells tumor diathesis. (Box 6.17): • HSIL (Figure 6.60 and 6.61): HSIL is the closest mimicker of SCC. The nuclear abnormalities are more severe in SCC than HSIL. However, this is subjective and the presence of tumor diathesis is the most important differentiating feature between HSIL and invasive carcinoma. • Atypia of repair: Marked reparative changes may mimic invasive carcinoma. In both SCC and reparative smear the cells show moderate nuclear

Squamous Intraepithelial Lesions and Invasive Squamous Cell Carcinoma

Figure 6.60: HSIL: Discrete and clusters of dysplastic cells in HSIL may be misdiagnosed as squamous cell carcinoma

Figure 6.61: Higher magnification of HSIL simulating carcinoma

enlargement, pleomorphism with Box 6.17: Mimickers of squamous cell large prominent nucleoli. However, carcinoma coarse irregular clumped chromatin • HSIL in carcinoma in comparison to fine • Atypia of repair and granular nuclear chromatin in • Atrophic smear reparative cells helps in the diagnosis. • Endometrial cells. • Atrophic smear (Figures 6.62 and 6.63): Atrophic smear may mimic SCC due to dirty background, “blue blobs” material and cells with moderately enlarged hyperchromatic nuclei. Cells of the atrophic smear show smudgy chromatin pattern compared to irregular coarse chromatin in SCC. • Endometrial cells: The clusters of endometrial cells in a background of granular debris and RBCs in a menstrual background may simulate non-keratinizing squamous cell carcinoma. Nuclei are regular and homogenously dark and bland in endometrial cells.

99

100 Handbook of Cervical Cytology: Special Emphasis on Liquid-based Cytology

Figure 6.62: Atrophic smear: Enlarged parabasal cells in a dirty background in atrophic smear may simulate squamous cell carcinoma. However, in LBC this type of dirty background is not seen

Figure 6.63: Atrophic smear in higher magnification: Careful observation show bland nuclear chromatin of the cells

REFERENCES 1. Wilbur DC, Dubeshter B, Angel C, Atkison KM. Use of thin-layer preparations for gynecologic smears with emphasis on the cytomorphology of high-grade intraepithelial lesions and carcinomas. Diagn Cytopathol. 1996;14(3):201-11. 2. Smith PA, Turnbull LS. Small cell and pale dyskaryosis. Cytopathology. 1997;8:3-8. 3. Gupta N, John D, Dudding N, Crossley J, Smith JH. Factors contributing to false-negative and potential false-negative cytology reports in SurePath™ liquid-based cervical cytology. Cytopathology. 2013;24(1):39-43. 4. Denton K, Rana DN, Lynch MA, Desai MS. Bland dyskaryosis: a new pitfall in liquid-based cytology. Cytopathology. 2008;19(3):162-6.

7

CHAPTER

Abnormality of Glandular Cells

Atypical glandular cell (AGC) abnormality is classified by TBS 2001/TBS 2014 as: • Atypical – Endocervical in origin – Endometrial in origin – Glandular cell, not otherwise specified • Atypical – Endocervical cells, favor neoplastic – Glandular cell favors neoplastic, not otherwise specified • Endocervical adenocarcinoma in situ • Adenocarcinoma – Endocervical – Endometrial – Extra uterine – Not otherwise specified. TBS 2001/2014 encouraged to determine the origin of the atypical glandular cell (AGC) such as endocervical or endometrial. If it is not possible to determine the exact origin of the atypical cells then the terminology should be kept just as atypical cells, not otherwise specified. The terminology “Atypical glandular cell of undetermined significance” has been eliminated in TBS 2001/2014. Endocervical adenocarcinoma in situ has been kept as an individual entity in TBS 2001/2014. Due to increased use of cervical brush technique, the more number of cases of AGC are recognized on cervical smear. Overall number of AGC consists of less than 1% of cervical smears.1

ATYPICAL ENDOCERVICAL CELLS Atypical endocervical cells show cytological atypia exceeding that of reactive endocervical cells but fall short the cytological features of adenocarcinoma in situ or adenocarcinoma. The smears show endocervical cells with overlapping

102 Handbook of Cervical Cytology: Special Emphasis on Liquid-based Cytology or crowded nuclei. The cytoplasm of the cells show vacuolation. The nuclei of the cells are enlarged five times that of normal endocervical cells. The nuclei show mild pleomorphism and prominent nucleoli. The criteria of diagnosis of atypical endocervical cells are (Figures 7.1–7.5): • Cells are in sheets or small overcrowded groups • Cytoplasm is abundant, vacuolated with distinct cell border • Nuclei of the cells show: – Mild enlargement: Three to five times the area of normal endocervical nuclei – Mildly pleomorphic – Hyperchromatic – Small nucleoli – Rare mitotic figures.

Figure 7.1: Atypical endocervical cells: Loose cluster of endocervical cells with mild nuclear enlargement

Figure 7.2: Atypical endocervical cells: The nuclei of endocervical cells are three times enlarged than normal endocervical cells

Abnormality of Glandular Cells

Figure 7.3: Atypical endocervical cells: The endocervical cells show nuclear enlargement and pleomorphism

Figure 7.4: Atypical endocervical cells: Higher magnification shows nuclear overlapping of endocervical cells

Figure 7.5: Atypical endocervical cells: Clusters of endocervical cells with nuclear crowding

103

104 Handbook of Cervical Cytology: Special Emphasis on Liquid-based Cytology Liquid-based Cytology LBC smears show hyperchromatic crowded group of cells with significant nuclear overlapping. The other cytoplasmic and nuclear features are identical in CP and LBC.

ATYPICAL GLANDULAR CELL, ENDOCERVICAL ORIGIN, FAVORS NEOPLASIA In this group the smears show cytologi- Box 7.1: Atypical glandular cell, cal features that are almost similar to endocervical origin, favors neoplasia adenocarcinoma in situ (AIS) or adeno• Overlapping crowded group of carcinoma but the features fall short to cells label as AIS or adenocarcinoma. The • Nuclear abnormality is more than nuclear features of the cells are more atypical glandular cells however severe compared to AGC. In addition, less than that of adenocarcinoma the smears also show hyperchromatic • In addition, occasional rosetting or feathering appearance. crowded groups, feathering and rosette like structures (Box 7.1). The criteria of diagnosis of atypical endocervical cells, favours neoplasia include (Figure 7.6–7.11): • Hyperchromatic crowded group of cells • Occasional sheets of cells • Occasional rosetting or feathering appearance of the cell cluster • Cytoplasm: abundant and vacuolated • Nuclei of the cells are enlarged, hyperchromatic, pleomorphic with irregular margin • High N/C ratio • Rare mitotic figures are also seen.

Liquid-based Cytology In LBC preparation the frequent clusters of crowded cells (hyperchromatic crowded group) are seen. Rosetting or feathering appearance of the cell clusters are less frequent in LBC than CP.2

ATYPICAL ENDOMETRIAL CELLS In this lesion, the smears show multiple Box 7.2: Atypical endometrial cells small cohesive clusters of endometrial • Small tight clusters of cells cells. The cytoplasm of the cell is scanty. • Small cells with scanty cytoplasm The nuclei are round, enlarged and • Mild nuclear pleomorphism. mildly pleomorphic. The amount of these atypical cells and degree of severity of the nuclear change are much less to qualify them as endometrial carcinoma (Box 7.2).

Abnormality of Glandular Cells

Figure 7.6: Atypical endocervical cells, favour neoplastic: Small clusters of cells with overlapping nuclei

Figure 7.7: Atypical endocervical cells, favour neoplastic: Higher magnification showing enlarged pleomorphic cells

Figure 7.8: Atypical endocervical cells, favour neoplastic: Endocervical cells with overcrowded nuclei

105

106 Handbook of Cervical Cytology: Special Emphasis on Liquid-based Cytology

Figure 7.9: Atypical endocervical cells, favour neoplastic: The cells show moderate amount of vacuolated cytoplasm and moderately pleomorphic nuclei

Figure 7.10: Atypical Endocervical cells, favour neoplastic: Cohesive cluster of moderately pleomorphic cells with overlapping nuclei. Note peripheral irregularity giving feathering appearance

Figure 7.11: Atypical Endocervical cells, favour neoplastic: Hyperchromatic crowded group with irregular margin

Abnormality of Glandular Cells

The criteria of the atypical endometrial cells include (Figures 7.12–7.15): • The cells are present in small tight cohesive clusters and also singly • The cells contain scanty cytoplasm. The margin of the cells is ill defined • Nuclei are mildly enlarged compared to normal endometrial cells • Mild hyperchromasia • The nucleoli are small.

Liquid-based Cytology The cells are in tight clusters with relatively monomorphic nuclei. The nucleoli are more prominent in LBC preparation than CP. Nuclear hyperchromasia is relatively more in LBC (Box 7.3).

Box 7.3: Liquid-based cytology of atypical endometrial cells • • • • •

Tight clusters Small nuclei Monomorphic More prominent nucleoli More hyperchromatic

Figure 7.12: Atypical endometrial cells: Tight small clusters of cells

Figure 7.13: Atypical endometrial cells: The cells with scanty ill-defined cytoplasm. The nuclei are hyperchromatic with high N/C ratio

107

108 Handbook of Cervical Cytology: Special Emphasis on Liquid-based Cytology

Figure 7.14: Atypical endometrial cells: Small cells with hyperchromatic pleomorphic nuclei

Figure 7.15: Atypical endometrial cells: Bag of polyps

ENDOCERVICAL ADENOCARCINOMA IN SITU Adenocarcinoma in situ (AIS) lesions of cervix is the precursor lesion of adenocarcinoma and is much less common than CIN. AIS constitutes only 1% of all in situ carcinoma of cervix. AIS are now increasingly detected because of the improved screening devices of endocervical region by endocervical brush, broom, etc and better characterization of glandular lesions of cervix. TBS 2001 has kept AIS as a definite category. This is an uncommon entity and the diagnosis of AIS is a challenge to the cytopathologists.3,4 Both architectural and cytological features should be considered for the diagnosis of AIS (Figure 7.16). The cells are present in hyperchromatic crowded group, small strips and rosette. Characteristically the background of the smear is clean and no tumor diathesis is present. The individual cells are columnar in shape with enlarged round nuclei. The nuclei are more than twice the size of normal endocervical cells. The nuclear pleomorphism is moderate.

Abnormality of Glandular Cells

Figure 7.16: Histopathology of adenocarcinoma in situ showing markedly atypical cells with multilayering of the glandular lumen

Figure 7.17: Adenocarcinoma in situ: The hyperchromatic crowded group of cells with feathered irregular margin

The chromatin is coarse with prominent Box 7.4: Adenocarcinoma in situ nucleoli. Occasionally the periphery of • All the cytological features of the clusters of cells show nuclear and adenocarcinoma except tumor cytoplasmic protrusion which is known diathesis as feathering effect (Box 7.4). • Strips of cells and feathering The characteristic cytological feafrequent. tures of AIS include (Figures 7.17 and 7.20): • Hyperchromatic crowded groups of cells • Rosettes or gland like arrangement of cells • Strips of cells • The cells show feathering where nuclei and cytoplasm are protruding in the periphery of the cluster • More columnar looking cells

109

110 Handbook of Cervical Cytology: Special Emphasis on Liquid-based Cytology

Figure 7.18: Adenocarcinoma in situ: Strip of cells with nuclear protrusion

Figure 7.19: Adenocarcinoma in situ: Long strip of tumor cells

Figure 7.20: Adenocarcinoma in situ: Cytoplasmic and nuclear sprouting

Abnormality of Glandular Cells

• Nuclei are enlarged (more than twice the size of normal endocervical cells), hyperchromatic with coarse granular chromatin • Small to inconspicuous nucleoli • Frequent mitosis and apoptosis are noted • No tumor diathesis.

Liquid-based Cytology

Box 7.5: Liquid-based cytology of adenocarcinoma in situ • • • •

More cellular Many single cells Bird tail like appearance Frequent feathering and rosetting arrangement • Frequent hyperchromatic crowded groups of cells • Coarse to fine granular nuclear chromatin • Prominent nucleoli.

Cellularity is usually more in LBC (Box 7.5). The characteristics peripheral feathering of cells and strips of cells with pseudostratified nuclei are more frequently seen. The arrangement of the cells often simulate “bird tail like” appearance. The hyperchromatic crowded groups of cells (HCG) are frequently seen. The margin of HCG has relatively regular outline. The nuclear chromatin is coarse to granular with prominent nucleoli.

ENDOCERVICAL ADENOCARCINOMA Endocervical adenocarcinoma comprises of 15% of all cervical carcinomas. Histopathological features are characteristic of adenocarcinoma of cervix. The section shows multiple glands lined by malignant cells (Figure 7.21). The patients may present with vaginal bleeding, discharge, and pain. The cytological features of AIS and adenocarcinoma are often similar and overlapping except the presence of tumor diathesis in adenocarcinoma. Moreover, AIS often coexist with adenocarcinoma. The smears show frequent hyperchromatic crowded group of cells, rosetting, and pseudostratified strips of cells. The cells are columnar in shape with vacuolated cytoplasm. The nuclei are enlarged and more than twice the size of normal endocervical cells.

Figure 7.21: Histopathology section of adenocarcinoma of cervix showing malignant glands lined by tall endocervical cells

111

112 Handbook of Cervical Cytology: Special Emphasis on Liquid-based Cytology Nuclear pleomorphism is moderate and Box 7.6: Endocervical adenocarcinoma nuclei show coarsely granular chromatin • Hyperchromatic crowded groups with macro nucleoli (Box 7.6). with scalloped border The characteristic cytological fea- • Nuclear enlargement and tures of endocervical adenocarcinoma pleomorphism with prominent nucleoli include (Figures 7.22–7.32): • Tumor diathesis • Highly cellular smear • Cells are present in tight cohesive three dimensional clusters and sheets, HCGs are more common in LBC • Well defined scalloped borders of the cluster • Columnar cells with scanty and vacuolated cytoplasm. • Nuclei are enlarged, pleomorphic, hyperchromatic with coarse chromatin • Prominent nucleoli and often macro nucleoli are present.

Figure 7.22: Adenocarcinoma: Multiple clusters and strips of cells

Figure 7.23: Adenocarcinoma: Discrete cells with enlarged moderately pleomorphic nuclei having prominent nucleoli

Abnormality of Glandular Cells

Figure 7.24: Discrete cells with columnar appearance. The nuclei are basally placed, enlarged and pleomorphic

Figure 7.25: Adenocarcinoma: Higher magnification of the columnar cells showing large pleomorphic nuclei

Figure 7.26: Adenocarcinoma: Sheet of cells with nuclear overlapping

113

114 Handbook of Cervical Cytology: Special Emphasis on Liquid-based Cytology

Figure 7.27: Adenocarcinoma: Strip of tumor cells

Figure 7.28: Adenocarcinoma: Hyperchromatic crowded group with feathery margin

Figure 7.29: Adenocarcinoma: Linear strip of cells

Abnormality of Glandular Cells

Figure 7.30: Adenocarcinoma: Bird tail strip of cells

Figure 7.31: Adenocarcinoma: Clinging tumor diathesis

Figure 7.32: Clinging tumor diathesis is attached with tumor cells

115

116 Handbook of Cervical Cytology: Special Emphasis on Liquid-based Cytology Liquid-based Cytology

Box 7.7: Liquid-based cytology of

The single dispersed malignant cells are endocervical adenocarcinoma more frequent in LBC (Box 7.7). The • Frequent single dispersed malignuclei of the cells are smaller in size nant cells compared to CP. The nuclei of the cells • Small nuclei show vesicular chromatin and the cells • Nuclei show vesicular chromatin with prominent nucleoli may look less malignant. The nucleoli are more prominent. The cells show similar • Certain features of nuclear abnormalities remain same: such nuclear pleomorphism and nuclear as nuclear pleomorphism and margin irregularity as that are seen in CP. irregular nuclear margin In LBC, the typical necrotic background • Clinging tumor diathesis. may be missing and the tumor cells are attached with necrotic debris showing clinging tumor diathesis. 5

ENDOMETRIAL ADENOCARCINOMA The cells are overall lesser in amount in endometrial carcinoma than endocervical carcinoma. The cells are commonly present in small tight cohesive clusters. There is nuclear overlapping in the cluster of cells. Rarely papillary structures or glands are identified in the smear. Individual cells are large and pleomorphic with prominent nucleoli. However, nuclear pleomorphism depends on the differentiation of the endometrial carcinoma. The characteristic cytological features of endometrial adenocarcinoma include (Figure 7.33 to 7.39): • Relatively cellular smear • Cells are present discretely or in tight clusters • The cells are round with enlarged hyperchromatic and pleomorphic nuclei having multiple nucleoli.

Figure 7.33: Endometrial adenocarcinoma: Cluster of cells with scanty to moderate amount of cytoplasm

Abnormality of Glandular Cells

Figure 7.34: Endometrial adenocarcinoma: Cells with moderate nuclear pleomorphism

Figure 7.35: Endometrial adenocarcinoma: Cells show enlarged nuclei with occasional prominent nucleoli

Figure 7.36: Endometrial adenocarcinoma: Discrete cells with moderate amount of vacuolated cytoplasm

117

118 Handbook of Cervical Cytology: Special Emphasis on Liquid-based Cytology

Figure 7.37: Endometrial adenocarcinoma: Tumor diathesis in endometrial carcinoma

Figure 7.38: Endometrial adenocarcinoma: Loose clusters of endometrial cells with significant nuclear pleomorphism

Figure 7.39: Endometrial adenocarcinoma: Higher magnification showing nuclear pleomorphism

Abnormality of Glandular Cells

• Cytoplasm of the cells is scanty and vacuolated. The cytoplasm contains many polymorphs giving “bag of poly” appearance • Tumor diathesis is finely granular and looks like watery tumor diathesis.

Liquid-based Cytology The LBC preparation shows frequent tight clusters and papillary configuration of cells. Overall the cells are larger in size compared to CP. Tumor diathesis is present as thin watery finely granular (Box 7.8).

Box 7.8: Liquid based cytology of endometrial adenocarcinoma • • • • •

Tight three dimensional clusters Papillary like arrangement Relatively large cell Opened chromatin Watery granular tumor diathesis.

NOTES Atypical glandular cells of endocervical origin frequently show hyperchromatic crowded group of cells. This finding is more evident in LBC preparation. Hyperchromatic crowded group (HCG) is defined as tight ball like three dimensional cluster of cells with hyperchromatic nuclei in the cervical smear. Various benign and malignant lesions may show HCG and this group of cells may be a common source of mistake (Figure 7.40 and 7.42). The common sources of benign condition that may show HCG include endometrial cells, endocervical cells, tubal metaplastic cells, cells in atrophic smears. The malignant lesions that show HCG include endocervical adenocarcinoma, endometrial adenocarcinoma, squamous cell carcinoma, and also metastatic carcinoma. It is important to note that the vast majority of HCG are benign in nature.6 Malignant HCG is distinguished from the benign one by the following features: • Irregular frayed margin in malignant HCG compared to regular margin in benign HCG

Figure 7.40: Hyperchromatic crowded group: benign endocervical cells. Note the regular margin of the group

119

120 Handbook of Cervical Cytology: Special Emphasis on Liquid-based Cytology

Figure 7.41: Hyperchromatic crowded group: Cells of HSIL making tight clusters of cells

Figure 7.42: Hyperchromatic crowded group: Tight clusters of cells in adenocarcinoma of cervix. Note the irregular margin of the cluster

• Loss of polarity of the cells in malignant HCG • Frequent mitosis in malignant HCG. The common conditions that may mimic AGC include: • Reactive endocervical cells • Tubal metaplasia • HSIL • Intrauterine contraceptive device • Radiation induced changes • Microglandular hyperplasia • Endocervical polyp.

Abnormality of Glandular Cells

(a) Reactive Endocervical Cells (Figure 7.43) Box 7. 9: Reactive endocervical cells Reactive endocervical cells often show Mimicking features of AGC: enlarged nuclei with single to multiple Enlarged nuclei with single to prominent nucleoli. These cells may be multiple prominent nucleoli mistaken as AGC. However, reactive cells Characteristic of endocervical are present in flat sheet with minimal to no cells: overlapping of nuclei. The nuclei of these • Two dimensional flat sheet cells are bland with fine granular chromatin • Minimal nuclear overlapping • Round regular nuclear margin (Box 7.9). (b) Tubal Metaplasia (Figure 7.44) Tubal metaplasia is frequently seen in the upper part of the endocervical canal

• Pleomorphism absent • Fine chromatin • Small nucleoli.

Figure 7.43: Reactive endocervical cells: Nuclear pleomorphism in endocervical cells

Figure 7.44: Tubal metaplasia: Cluster of endocervical cells showing cilia

121

122 Handbook of Cervical Cytology: Special Emphasis on Liquid-based Cytology (Box 7.10). This is also commonly seen Box 7.10: Tubal metaplasia after conization of cervix. The cells are Mimicking features of AGC: commonly seen as strips of columnar Enlarged and even mildly epithelium with terminal plate in one end pleomorphic along with tuft of cilia. The nuclei of the Characteristic features: cells may be enlarged and even mildly • Strips of cells or singly present pleomorphic. The nuclear chromatin is • Maintained polarity finely granular. Occasional mitosis may • Columnar epithelium with terminal plate also be seen in the cell clusters. The • Cilia present recognition of terminal plate, cilia and • Enlarged mildly pleomorphic fine nuclear chromatin are important to nuclei distinguish these cells from AGC. • Fine granular chromatin.

(c) Intrauterine Contraceptive Devices Recently intrauterine contraceptive Box 7.11: Intrauterine contraceptive devices (IUCD) are increasingly used. devices The cells in IUCD are present as tight Mimicking features of AGC: cohesive clusters with enlarged nuclei, Enlarged, mildly pleomorphic with prominent nucleoli and cytoplasmic prominent nucleoli vacuoles (Box 7.11). These cells are often Characteristic features: mistaken as AGC. The history of using • Tight cohesive clusters IUCD, presence of psammoma bodies, • Round cells with cytoplasmic vacuoles giving bubble gum apand frequent presence of actinomycosis pearance help in distinguishing such cells from • Enlarged pleomorphic nuclei AGC. • Prominent nucleoli • Associated actinomycosis • Occasional psammoma bodies.

(d) HSIL Involving Endocervical Glands HSIL involving endocervical glands may show features that simulate glandular abnormalities (Box 7.12). These cells are Box 7.12: HSIL involving glands versus present as HCG. The cells are smaller atypical glandular cells in size with cytoplasmic vacuoles. The • The central dense group of cells nuclei are enlarged with fine chromatin in hyperchromatic crowded group and often prominent nucleoli. The central dense group of cells, absence of rosetting, • Absence of rosetting poorly formed glandular structure, fine • Poorly formed glandular structure chromatin and the other evidences of • Fine chromatin squamous intraepithelial lesion help to • Features of squamous intraepiidentify HSIL. thelial lesion.

(e) Radiation Changes Radiation changes show cytomegaly with nuclear enlargement and pleomorphism. The degenerative changes of nucleus, more or less preserved N/C ratio, other changes of radiation and history of radiation help to distinguish these cells from AGC.

Abnormality of Glandular Cells

(f) Endocervical Polyp (Figures 7.45 and 7.46) Box 7.13: Endocervical polyp Endocervical polyp may be ulcerated on the • Hyperchromatic crowded surface and the cells may show marked reacgroups of cells tive changes (Box 7.13). The smear from such • Necrosis and inflammatory polyp shows necrosis, inflammatory backbackground ground, hyperchromatic crowded groups of • Enlarged mildly pleomorphic reactive endocervical cells cells and reactive endocervical cells. These • “Bag of polys” characterized cells may show nuclear enlargement and by cells with vacuolated pleomorphism. The smears may show “bag of cytoplasm containing many polys” characterized by cells with vacuolated polymorphs. cytoplasm containing many polymorphs. It is almost impossible to differentiate such smears from abnormal glandular cells.

Figure 7.45: Endocervical polyp: Cluster of glandular cells in endocervical polyp

Figure 7.46: Endocervical polyp: Higher magnification of the clusters of glandular cells

123

124 Handbook of Cervical Cytology: Special Emphasis on Liquid-based Cytology Atypical endometrial cells are present in tight small round clusters and also singly. The cytoplasm of the cell is variable and may be scanty to moderate with vacuolation. The nuclei of the cells are enlarged than normal endocervical cells and may show irregular nuclear contour and prominent nucleoli. These nuclear changes are lesser in degree than the cells of endometrial adenocarcinoma. The various conditions that simulate atypical endometrial cells include: chronic endometritis, endometrial hyperplasia, endometrial carcinoma and IUCD effect. Menstrual endometrial cells also may be mistaken as atypical endometrial cells particularly in LBC preparation as the nuclei are more enlarged in LBC compared to CP. The differentiation of AIS and endocervical adenocarcinoma on cytology smear is very difficult due to overlapping cytological features in these two conditions. The presence of tumor diathesis along with large pleomorphic cells, hyperchromatic nuclei having prominent nucleoli indicate adenocarcinoma. However, in LBC preparation the characteristic tumor diathesis may be subtle and invasive carcinoma is difficult to diagnose. The differential diagnosis of AIS and endocervical adenocarcinoma are same as that of AGC such as atypical repair, tubal metaplasia, HSIL, IUCD, radiation induced atypia and endocervical polyp. If possible, endocervical adenocarcinoma should be differentiated from endometrial adenocarcinoma (Table 7.1). Endometrial adenocarcinoma is usually seen in post-menopausal patient. The cells of endometrial carcinoma are usually seen in tight spherical clusters and the cells are round to oval compared to columnar looking cells in endocervical carcinoma. N/C ratio is relative high in endometrial cells. In addition, many histiocytes are present in the background of endometrial cells.

Table 7.1: Endometrial versus endocervical adenocarcinoma Features

Endocervical adenocarcinoma

Endometrial adenocarcinoma

Cellularity

Abundant

Scanty

Cell arrangement

Discrete and clusters

Tight cohesive cluster

Cell shape

Columnar

Round

Cell size

Large

Small

N/C ratio

Relatively low

High

Nucleoli

Multiple prominent

Less prominent

Chromatin

Coarsely granular

Fine granular

Background

Hemorrhagic tumor diathesis Watery diathesis

Histiocytes

Less

Many

Abnormality of Glandular Cells

REFERENCES 1. Schnatz PF, Guile M, O’Sullivan DM, Sorosky JI: Clinical significance of atypical glandular cells on cervical cytology. Obstet Gynecol. 2006;107(3):701-8. 2. Belsley NA, Tambouret RH, Misdraji J, Muzikansky A, Russell DK, Wilbur DC. Cytologic features of endocervical glandular lesions: comparison of SurePath, ThinPrep, and conventional smear specimen preparations. Diagn Cytopathol. 2008;36(4):232-7. 3. Bousfield L, Pacey F, Young Q, et al. Expanded cytologic criteria for the diagnosis of adenocarcinoma in situ of the cervix and related lesions. Acta Cytol. 1980;24:284-96. 4. Ayer B, Pacey F, Greenberg M, Bousfield L. The cytologic diagnosis of adenocarcinoma in situ of the cervix uteri and related lesions. I. Adenocarcinoma in situ. Acta Cytol. 1987; 31(4):397-411. 5. Wilbur DC, Dubeshter B, Angel C, Atkison KM. Use of thin-layer preparations for gynecologic smears with emphasis on the cytomorphology of high-grade intraepithelial lesions and carcinomas. Diagn Cytopathol. 1996;14(3):201-11. 6. Demay RM. Hyperchromatic crowded groups: pitfalls in pap smear diagnosis. Am J Clin Pathol. 2000;114 Suppl:S36-43.

125

8

Chapter

The Presence of Endometrial Cells

In 2014 TBS, the presence of benign looking endometrial cells above 45 year of age is kept in “other” and the presence of atypical endometrial cells is kept in glandular cell abnormality.1 Majority of the endometrial carcinomas occur in post-menopausal patients. It has been noted that the patients of endometrial carcinoma may be asymptomatic and may present as only benign looking endometrial cells in the cervical smear. Therefore, TBS 2001, recommended to mention the presence of endometrial cells in the cervical cytology smear. The exact rate of the presence of endometrial cells in the postmenopausal women is difficult to recognize. However, the prevalence of endometrial cells in the women 40 years or older varies from 0.5 to 1.1%.2,3 The prevalence of atypical endometrial cells is much lower and it is 0.1% of all cervical smears.4

Endometrial cells The cytological characteristics of endometrial cells are (Figures 8.1 and 8.2): •• The cells are seen as ball like tight spherical clusters. Single discrete endometrial cells are rare •• The cells are same size as that of nuclei of the intermediate cells •• Round cells with scanty basophilic cytoplasm •• Cytoplasmic vacuolation may be present •• Nuclei are small and round or bean shaped •• Nuclear molding or angulated contour •• Fine granular chromatin •• No nucleoli or inconspicuous nucleoli.

Liquid-based Cytology In LBC preparation the smear shows frequent three dimensional cell clusters along with many discrete single cells. Single cell necrosis or apoptosis is seen within the cell clusters. The individual cells show bean shaped nuclei with details chromatin pattern. Unlike CP, the smear is relatively free from blood or any necrotic debris as the menstrual blood is mostly removed in LBC (Box 8.1).

The Presence of Endometrial Cells

Figure 8.1: Endometrial cells: Small tight clusters of endometrial cells.

Figure 8.2: Endometrial cells: Higher magnification of the endometrial cells.

The characteristic features of endo- Box 8.1: Liquid-based cytology of endometrial cells metrial stromal cells are: •• Clusters of round cells •• Frequent three dimensional cell •• Histiocyte like cells with bean shaped clusters are present nuclei •• More frequent discrete single cells •• Vacuolated cytoplasm • • Single cell necrosis or apoptosis The characteristic features of deeper within the cell clusters stromal cells include: •• Bean shaped nuclei •• Clusters of cells •• Chromatin detail better seen •• Round to spindle shaped cells with •• Clean background and free from oval nuclei blood or any necrotic debris. •• Nuclear chromatin is coarse.

127

128 Handbook of Cervical Cytology: Special Emphasis on Liquid-based Cytology notes Endometrial cells are exfoliated in several conditions such as: •• Menstruation •• Endometritis •• Endometriosis •• Endometrial polyp •• Submucosal leiomyoma •• Intrauterine instrumentation •• Tamoxifen therapy •• Endometrial adenocarcinoma. The endometrial cells are normally exfoliated during menstruation. The cells are found on cervical smear from 6th to 10th day of the menstrual cycle. The presence of endometrial cells after 12th day of the menstrual cycle is unusual and commonly associated with endometritis or endometrial polyp. The endometrial cells during menstrual cycle appears as exodus. The cytological features of exodus include: •• Ball like tight clusters of cells •• Central dense stromal cells •• Peripheral relatively larger endometrial cells. It has been noted that Tamoxifen therapy due to breast carcinoma increases the prevalence of endometrial cells in the cervical smears. 5 The endometrial cells should always be distinguished from endocervical cells (Table 8.1). The presence of tight clusters of cells with overlapping nuclei, scanty cytoplasm and dense chromatin help to differentiate endometrial from endocervical cells. The endocervical cells are larger than endometrial cells and are columnar in appearance (Figure 8.3). They are often present in honey comb sheet. The other cells that should be distinguished from endometrial cells include: •• Lymphocytes (Figure 8.4): These cells are usually discrete and lymphocytes have coarse chromatin. •• Histiocytes: Histiocytes are commonly seen as discrete cells or in small loose clusters. The cells show bean shaped nuclei with cytoplasmic debris.

Table 8.1: Distinguishing features of endometrial and endocervical cells Feature

Endometrial

Endocervical

Distribution

Three dimensional cohesive Loose two dimensional cells. clusters Honey comb appearance

Cell cytoplasm

Scanty

Moderate

N/C ratio

High

Low

Nuclear chromatin

Dense

Fine

Nucleoli

Absent

Small, inconspicuous

The Presence of Endometrial Cells

Figure 8.3: Endocervical cells: Compared to endometrial cells these cells have more cytoplasm. The cells are columnar in shape.

Figure 8.4: Lymphocytes occasionally simulate endometrial cells. However the cells are discrete.

•• Parabasal cells: Parabasal cells are present in small two dimensional flat sheet or discrete. The cells have relatively more amount of dense cytoplasm. •• HSIL (Figure 8.5): The dysplastic cells of HSILs are larger than endometrial cells with hyperchromatic nuclei and irregular nuclear margin. These cells are arranged discretely or in loose clusters compared to tight spherical arrangement of endometrial cells. •• Atypical glandular cells (Figure 8.6 and 8.7): Atypical endometrial cells are usually present in strips. The cells show nuclear enlargement and pleomorphism. However, benign endometrial cells may show nuclear pleomorphism in LBC preparation and the differentiating between these two entities may be challenging. •• Adenocarcinoma (Figure 8.8 and 8.9): The presence of relatively large cells with nuclear pleomorphism, hyperchromatic nuclei, prominent nucleoli, and tumor diathesis (watery diathesis) indicate adenocarcinoma.

129

130 Handbook of Cervical Cytology: Special Emphasis on Liquid-based Cytology

Figure 8.5: HSIL simulates endometrial cells: The close look of the cells reveal the pleomorphic hyperchromatic cells.

Figure 8.6: Atypical glandular cells may simulate endometrial cells.

Figure 8.7: Atypical glandular cell of endometrial origin.

The Presence of Endometrial Cells

Figure 8.8: Adenocarcinoma: The cell cluster has irregular margin. Nuclei are much more pleomorphic than that of endometrial cells.

Figure 8.9: Endometrial adenocarcinoma: The cells of endometrial carcinoma shows enlarged pleomorphic nuclei.

In case of well differentiated adenocarcinoma the nuclear enlargement and pleomorphism is minimal and differentiation from normal endometrial cells is difficult. At times abraded endometrial cells of lower uterine segment (LUS) may be noted. These cells are always present in tight cohesive clusters. The cells may be arranged as tubules. The nuclei of the cells are kidney shaped. In addition, round to spindle shaped stromal cells and histiocytes are also seen. Rarely mitosis and apoptosis are also seen in the smears taken from LUS.

131

132 Handbook of Cervical Cytology: Special Emphasis on Liquid-based Cytology

References 1. Nayar R, Wilbur DC. The Pap test and Bethesda 2014. Cancer Cytopathol. 2015;123(5): 271-81. 2. Mount SL, Wegner EK, Eltabbakh GH, Olmstead JI, Drejet AE. Significant increase of benign endometrial cells on Papanicolaou smears in women using hormone replacement therapy. Obstet Gynecol. 2002;100:445-50. 3. Saad RS, Mahdavy M, Takei H, Ruiz B. Endometrial cells in cervical specimens of women 940 years of age (abstract). Acta Cytol. 2004;48:678-9. 4. Thrall MJ, Kjeidahl KS, Savik K, Gulbahce HE, Pambuccian SE. Significance of benign endometrial cells in Papanicolaou Tests from women aged 940 years. Cancer Cytopathol. 2005;105:207-16. 5. Abadi MA, Barakat RR, Saigo PE. Effects of tamoxifen on cervicovaginal smears from patients with breast cancer. Acta Cytol. 2000;44:141-6.

9

Chapter

Other Malignant Tumors

The cervix may be rarely involved by uncommon cervical malignancies or metastatic malignant tumors. These tumors should be labeled as other malignant tumors in the TBS 2001.

Cervical malignancies Small Cell Carcinoma Small cell carcinoma of cervix is uncommonly encountered in cervical smear. Unlike other areas cervical small cell carcinoma is associated with HPV 18. 1 The characteristic features of small cell carcinoma of cervix include (Figures 9.1 and 9.2): •• Loose clusters and discrete cells •• Cell are small in size with scanty cytoplasm •• Nuclei are round with hyperchromatic nuclei •• Frequent nuclear moulding present •• Fragile cells may produce nuclear smearing.

Malignant Melanoma Melanoma of cervix is very rare and cytologists rarely encounter malignant melanoma on cytology smear. The cytology features of melanoma is similar as that of other sites. The characteristic cytological features of melanoma include: • Discrete round to oval cells. Occasional spindle cells may be seen • The nuclei are enlarged with large prominent nucleoli • Macro nucleoli • Cytoplasmic melanin pigment may be seen.

Carcinosarcoma Carcinosarcoma of the cervix is exceedingly rare entity. The tumor is composed of both malignant glandular and sarcoma component. 2,3

134 Handbook of Cervical Cytology: Special Emphasis on Liquid-based Cytology

Figure 9.1: Small cell carcinoma: Discrete monomorphic round cells with hyperchromatic nuclei

Figure 9.2: Small cell carcinoma: Higher magnification shows cells with round enlarged nuclei and condensed chromatin

The smears show: •• Clusters of cuboidal to columnar epithelial cells with enlarged pleomorphic nuclei and prominent nucleoli •• Sarcoma element show clusters and discrete spindle cells with moderate pleomorphism. These elements may not be evident in cytology smear •• Dirty necrotic background.

Metastatic Tumors Uncommonly various malignant tumors from different sites may metastasize in cervix. The common primary sites are ovary, fallopian tube, colon, and breast. The half of the extra uterine metastatic malignancies are of gynecological origin particularly from ovary and fallopian tubes. The other 50% of

Other Malignant Tumors

metastatic sites are from colon and breast. The metastatic tumors usually show clean background compared to dirty tumor diathesis in cervical carcinomas and watery diathesis in uterine carcinomas. Metastatic Serous Ovarian Carcinoma The characteristic cytological features of metastatic ovarian carcinoma include (Figures 9.3–9.6): •• Multiple tight cohesive clusters of cells •• Cells with variably vacuolated cytoplasm •• Enlarged nuclei with prominent nucleoli •• Occasionally psammoma bodies may be seen •• The smear shows clean background.

Figure 9.3: Metastatic ovarian adenocarcinoma: Small tight cluster of cells in a clean background

Figure 9.4: Metastatic ovarian adenocarcinoma: Small tight cluster of cells in a clean background

135

136 Handbook of Cervical Cytology: Special Emphasis on Liquid-based Cytology

Figure 9.5: Metastatic ovarian adenocarcinoma: Higher magnification shows cells with scanty cytoplasm and round nuclei with prominent nucleoli

Figure 9.6: Metastatic ovarian adenocarcinoma: Papillary cluster of cells

Breast Carcinoma The cytology smears of metastatic lobular breast carcinoma show: •• Rows of single cells •• Nuclear moulding •• Cells are small in size with scanty to moderate cytoplasm. Colonic Carcinoma The smears show: •• Clusters of cells •• Columnar cells with enlarged nuclei •• Moderate nuclear pleomorphism •• Prominent nucleoli.

Other Malignant Tumors

References 1. Stoler MH, Mills SE, Gersell DJ, Walker AN: Small cell neuroendocrine carcinoma of the cervix: A human papillomavirus type 18-associated cancer. Am J Surg Pathol. 1991;15:2832. 2. Costa MJ, Tidd C, Willis D. Cervicovaginal cytology in carcinosarcoma [malignant mixed mullerian (mesodermal) tumor] of the uterus. Diagn Cytopathol. 1992;8(1):33-40. 3. Snyder MJ, Robboy SJ, Vollmer RT, Dodd LG. An abnormal cervicovaginal cytology smear in uterine carcinosarcoma is an adverse prognostic sign: analysis of 25 cases. Am J Clin Pathol. 2004;122(3):434-9.

137

10

Chapter

Screening, Quality Control and Management of Cervical Lesion

Screening means the identification of an individual with a precursor lesion that has high probability to develop the disease. The screening is therefore primarily targeted in the asymptomatic population. A good screening test should be accurate, cheap, easily available, relatively easy to do, and acceptable. Cervical cytology bears most of the characteristics of a good screening test. There are different cervical screening programs in different countries.

Screening strategy Start Age of the Target Population World Health Organization (WHO) recommends that start age of the screening program should be 30 year1 (Table 10.1). Below this age the screening program is not cost effective as most of the females with HPV infection do not develop cancer and unnecessary detection of many such cases will overburden the health load. However, American Society for Colposcopy and Cervical Pathology (ASCCP) recommends to start screening from 21-year of age. Table 10.1: Cervical screening strategy American Society for Colposcopy World Health Organization1 and Cervical Pathology (ASCCP)2 Initiation

21 years

30 years

Frequency

Every 3 years

Every 3 to 5 years

Stop age

65 years

65 years

Mode of screening

21 to 30 years: Only cervical cytology 30 to 65 years: Cytology every 3 year with HPV testing as triage of ASUCS cases/primary HPV testing every 3 year or HPV co-testing every 5 year

30 years onwards by cervical cytology or HPV high risk testing or visual inspection with acetic acid (VIA)

Screening, Quality Control and Management of Cervical Lesion

Stop of Age The stop age of the screening depends on the national program. However, the minimum end age should be at least 50 years. The most of the cervical screening program consider 65-year as stop age (if the female has previous normal cervical cytology report).

Frequency Frequency of screening is recommended as 3 years interval.

Mode of Screening ASCCP recommends that between 21–30 years the women should be primarily screened by cervical cytology alone. 2 The screening of women between 31–65 years should be done by either by cervical cytology 3 yearly with HPV testing as a triage of ASCUS patients, or by cervical cytology with HPV co-testing (5 yearly).

Different screening methods for cervical pre-cancer The different screening modalities of cervical pre-cancer include: •• HPV testing •• Visual inspection under acetic acid •• Conventional cytology •• Liquid based cytology.

HPV Testing HPV Testing is used to detect DNA of high risk HPV such as HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 59, and 68. The positive test indicates the presence of HPV infection. However, all the high risk HPV infective patients may not develop cervical preneoplastic lesions. In fact the virus is eliminated from the body in most of the patients under 30 years of age. Therefore, HPV DNA testing is not recommended below 30 years of age. Sample for HPV testing is easy to take and it can be taken by the patient herself or by the health care attendant. A small brush (1–1.5 cm) is inserted into the cervical os and the bristles of the brush should touch the ectocervix. Brush should be rotated gently and then withdrawn. The brush is then put into the transport tube containing liquid preservative. The sample is transported to the laboratory. Advantages: •• Highly sensitive method to detect HPV infection •• Sample is easy to collect •• A single sample can be processed both for LBC and HPV testing •• No special technical skill is needed.

139

140 Handbook of Cervical Cytology: Special Emphasis on Liquid-based Cytology Limitations: •• High cost of the test •• The testing kit may not be easily accessible to remote areas •• The result of the test takes time and point of care test is still not available •• The positive test does not mean preneoplastic lesion Three main ways to do HPV screening include: •• ASCUS triage: Primary cytology followed by HPV testing as a triage in ASCUS cases. Ultimately it reduces colposcopic burden. •• Cotesting of cytology and HPV testing: To do both cervical cytology and HPV testing. This reduces the frequency of the visit in both negative cases. •• Primary HPV testing: Here HPV testing is used as primary screening test and cytology is done only in HPV positive cases. More than 193 commercially available tests are available for HPV testing. However, only a few HPV tests for screening cervical preneoplastic lesion have been approved in USA by FDA (Box 10.1). These tests are approved as the combination of cervical cytology to avoid unnecessary colposcopic examination. For primary cervical cancer screening, FDA advisory panel recommended only Cobas 4800 system as it is more sensitive and specific than Hybrid capture 2.3 a. Hybrid capture (HC): The use of this test is approved by FDA along with routine Papanicolaou’s stained smear of the women over the age of 30 years. Hybrid capture 2-test is able to quantify the HPV subtypes. Principle: In this test the specimen containing patient’s DNA is mixed with a specific RNA cocktail probes of both high risk and low risk HPV virus. The RNA-DNA hybrid is formed if the patient’s specimen has specific HPV DNA. The RNA-DNA hybrid is then captured on the surface of microplate well that is coated with anti- RNA-DNA antibody (Figure 10.1). The immobilised RNA-DNA complex is then reacted with antibody against DNA-RNA hybrid conjugated with alkaline phosphatase. Subsequently the reaction is demonstrated by chemiluminescent substrate. The emitted light in the reaction is measured. b. Cervista HPV HR: This is also FDA approved and this test identifies 14 high risk HPV subtypes including HPV 16 and 18. Principal (Figure 10.2): This is a qualitative in vitro diagnostic test that uses nucleic acid signal amplification method to detect specific nuclei acid sequence of the HPV DNA. Two types of isothermal reaction occurs: Primary reaction: Combination of oligonucleotide probes into target DNA sequence. Secondary reaction: Generation of fluorescent signals. Primary reaction happens in the targeted DNA sequence of HPV. Two types of sequence specific oligonucleotide probes (oligonucleotide probe and invader probe) against the target DNA are used. An invasive structure

Screening, Quality Control and Management of Cervical Lesion Box 10.1: HPV tests for cervical preneoplastic lesions screening DNA based test HC2 Test (Qiagen, Redwood City, CA) •• FDA approved in 1999 •• DNA-probe hybridization assay •• Quantitative •• Detects 13 high risk HPV •• Analytical sensitivity: 1000 to 5000 copies/reaction. Limitations: –– Lacks internal control for specimen adequacy –– Cross reactive with untargeted HPV types. Cervista HPV HR (Hologic Gen-Probe, Inc., san Diego, CA, USA) : •• FDA approved in 2009 •• Signal amplification •• Qualitatively detects 14 high risk HPV •• Analytical sensitivity: 1250 to 2500 copies/reaction Limitations: –– Cross reactivity –– False negativity. Cobas 4800 System (Roche Molecular Systems, Inc., Pleasanton, CA, USA): •• FDA approved in 2011 •• Multiplex Real time PCR based assay •• It detects 14 high risk HPV •• Analytical sensitivity: 150 copies/ml RNA based test Aptima mRNA (Hologic Gen-Probe, Inc. San Diego, CA): •• FDA approved 2012. •• RNA based technique. •• It detects 14 high risk HPV subtypes •• Analytical sensitivity: 24- 488 copies/reaction Advantages –– No cross reactivity –– More specific than hybrid capture 2

is formed when the probes combine at least one of the base pair of the target DNA sequence. This invasive structure behaves as a substrate of the cleavase enzyme that breaks the flap of the probe. Subsequently the cleaved flap combines to a universal hairpin fluorescence resonance energy transfer (FRET) oligonucleotide. This produces another invasive structure which acts as a substrate of Cleavase enzyme and fluorescent signals are created.

141

142 Handbook of Cervical Cytology: Special Emphasis on Liquid-based Cytology

Figure 10.1: Schematic diagram of hybrid capture 2-test

Figure 10.2: Schematic diagram of Cervista HPV HR

Screening, Quality Control and Management of Cervical Lesion

c. Cobas 4800 System: This is a very sensitive polymerase chain reaction (PCR) based technique that can detect a total of 14 high risk oncogenic HPV subtypes. Principle: Cobas 4800 System applies real time PCR based method to detect HPV DNA in the target sample. With the help of type-specific DNA primers the specific region of DNA is amplified and then measured in the fully automated system. d. Aptima mRNA: This test targets mRNA of E6/E7 of high risk HPV. This test is also FDA approved and has 100% sensitivity with 84% specificity. 4 Principal: Aptima mRNA test is a qualitative nucleic acid amplification test. The test detects E6/E7 mRNA of high risk HPV. This test has predominantly three steps: 1. Target capture by oligomers: In this step the target mRNA is released from the clinical sample and then it is captured by the oligomers containing specific complementary sequence of mRNA. Thereby an oligomer-target complex is generated. 2. Transcription mediated amplification (TMA) of the target: The high risk HPV mRNA is amplified with the help of TMA. In this step the high risk HPV mRNA at first is converted to DNA by the help of MMLV reverse transcriptase. This DNA copy contains a promoter sequence for T7 RNA polymerase. With the help of T7 RNA polymerase many copies of the RNA is generated from the DNA copy template. 3. Hybridization Protection Assay: In this step hybridization Protection Assay is used to detect the RNA amplicon. The chemiluminescentlabelled single-stranded nucleic acid probes containing the complementary sequence of the amplicon are used. The emitted light of the hybrid RNA-DNA complex is estimated in relative light unit. Overall HPV tests show 100% sensitivity. The specificity of HPV test varies from 75 to 84%.4

Visual Inspection Under Acetic Acid (VIA) This test helps to identify the early changes of cervical lesions with simple naked eye examination by applying dilute acetic acid over the cervix. Well trained health personnel is needed to interpret the positive result. Method of Testing With the help of cotton swab 3–5% acetic acid is applied to the cervix. After one minute the presence of any acetowhite area is noted. The presence of acetowhite area with well-defined border in the transformation zone is considered as positive test. Advantages •• Easy to perform in remote areas

143

144 Handbook of Cervical Cytology: Special Emphasis on Liquid-based Cytology •• Cheap •• The health personnel can be trained easily to do this test •• Immediate result Limitations •• It has low specificity •• Interpretation of VIA test is subjective and needs lot of supervision and quality assurance

Conventional and Liquid-based Cytology Detailed discussion of these techniques have been done in earlier chapters.

Quality control of cervical cytology Quality control is a process by which desired level of quality of a test is verified and maintained by predetermined series of measures.5 The process of quality control begins from the specimen receiving to final delivery of the report. It also includes the slide and report storage, retrieval and further verification of the report in future. The quality control is divided into three main parts (Box 10.2): •• Preanalytic phase •• Analytic phase •• Post analytic phase.

Preanalytic Phase This includes receiving of the sample, preparation of the sample and staining of the smear. Periodic maintenance of the processing quality, staining quality, stock registration of consumable, and nonconsumable substances are needed in this stage.

Analytic Phase This is the most important part of the quality control process. It is recommended that the cervical cytology should only be screened by a qualified cytotechnologist or cytopathologists. The primary screener should not screen more than 100 slides in a single day.6 All the positive slides must be finally reported by the consultant cytopathologists. The discordant cases between the cytotechnologist and the cytologist should be recorded.

Box 10.2: Quality control in cervical cytology •• Preanalytic phase: Sample processing, staining, submission, stock registration •• Analytic phase: –– Reporting slides and generation of report –– Only trained and qualified cytotechnologist and cytologist can do screening –– Final report should be signed by cytopathologists –– Rescreening of negative slides by rapid review, selected rescreening, 10% rescreening or automated screening. •• Post analytic phase –– Review all cytology cases with available histopathology External quality assurance: Continuous medical education program, repeated performance test of the technologist and cytopathologists.

Screening, Quality Control and Management of Cervical Lesion

Re-screening of negative cases a. 10% screening of negative slide: This means to screen randomly of 10% negative slides. This is not a very effective method. b. Rapid review: Rapid review means to screen all the negative slides rapidly within 30 seconds. It is an effective method to detect LSIL and ASCUS cases. c. Selective rescreening: In this method only the smears of high risk individuals are screened such as the previously diagnosed LSIL or ASCUS cases or the patients with abnormal bleeding etc. d. Automated screening: In this rescreening process the machine rescreens the slide and identifies the potential positive slides for re-evaluation by the cytologist.

Post Analytic Phase This is also very important for further follow up of the reported cases by correlating cytology-histology and clinical follow up. All the histology cases with previous cytopathology should be reviewed thoroughly. Discordant cases should be carefully reviewed. The discordant cases may be seen by a third person or the case may be inserted in the routine reporting slides.

Indicators of good screening program There are several indicators of a good Box 10.3: Indicators of screening screening programme (Box 10.3) such as: •• •• •• •• ••

Sensitivity

Sensitivity of the test Specificity of the test Positive predictive value Negative predictive value False negative fraction.

It indicates the ability of the test to recognize the disease. Therefore sensitivity means the detection of truly positive cases among all the positive cases. A good screening test should have high sensitivity. Unfortunately, cervical cytology is not so much sensitive test and the sensitivity varies from 30 to 87% and on an average just 50%.7,8

Sensitivity =

True positive True positive + False negative

× 100%

Specificity Specificity indicates the ability of the test to eliminate the presence of disease. Therefore, it means the percentage of true negative cases among the all the negative cases. Papanicolaou’s smear for cervical cytology has good specificity (86 to 100%).7

Specificity =

True negative True negative + False positive

× 100%

145

146 Handbook of Cervical Cytology: Special Emphasis on Liquid-based Cytology Positive Predictive Value Positive predictive value (PPV) indicates the reliability of a test to predict the presence of disease.

         True positive Positive predictive value =         True positive + False positive

× 100%

Negative Predictive Value         True negative cases Negative predictive value =         True negative cases + False negative cases

× 100%

False Negative Cervical Cytology False negative cytology is an important indicator of successful cervical cytology screening program. It means the negative cytology report in a truly diseased patient. False negative cytology is defined as originally a negative cervical cytology report that shows sufficient number of dysplastic or carcinoma cells on review.9 The false negative fraction is defined as        Estimated false negative results False negative fraction = × 100%         (True positive results + estimated false negative results)

Management If cytology reported as negative for intraepithelial lesions: These cases should undergo to routine screening without any early repeat. If cytology reported as unsatisfactory: These cases should always be repeated irrespective of HPV report. Atypical squamous cells: The cases of ASC is classified as ASC-US and ASC-H. The probability of HSIL or invasive carcinoma in ASC-US is low compared to ASC-H. Therefore, the further management is different in ASCUS and ASC-H cases.

Management of ASC-US Cases (Figure 10.3) Three approaches are recommended in these cases:10 a. Two repeat cervical cytology at 6 monthly intervals b. HPV testing c. Single colposcopic examination. In 2012 guideline of consensus management conference,11 it was recommended that it is preferable to do HPV DNA testing (HPV 16 and 18) in such cases and further management will depend according to HPV DNA test report such as: a. HPV DNA test negative cases: Repeat cytology and HPV test after one year b. HPV DNA test positive cases: Colposcopy

Screening, Quality Control and Management of Cervical Lesion

Figure 10.3: Schematic diagram of ASCUS management

If repeat cytology and HPV DNA test both are negative after one year then the patient should undergo routine screening program.

Management of ASC-H

•• Colposcopy is recommended in all ASC-H patient •• If colposcopic examination is negative for CIN 2 or CIN 3 then high risk HPV DNA test can be done •• Alternately cytology smear examination can be done 6 month and 12 month interval •• If HPV DNA test is positive or any of the cytology test shows ASC or above then repeat colposcopy should be done.

Management of LSIL Cases (Figure 10.4)

•• Colposcopy is recommended in LSIL cases if no HPV test is done or HPV test is positive •• LSIL with negative HPV test can be followed up and repeat cervical cytology and HPV test are preferred after one year •• If the repeat test is positive for HPV or cervical cytology shows ASC or above then colposcopy is recommended •• If there is no lesion in colposcopy, unsatisfactory colposcopy, or the patient is not pregnant then endocervical sampling is preferred •• If there is no evidence of CIN 2 or 3 then HPV DNA testing or repeat cervical cytology should be done •• If colposcopic guided biopsy shows CIN 2 or 3, then surgical ablation could be done.

147

148 Handbook of Cervical Cytology: Special Emphasis on Liquid-based Cytology

Figure 10.4: Schematic diagram of LSIL and HSIL management

Management of HSIL Cases (Figure 10.4)

•• Colposcopy with endocervical assessment should be done in all cases of HSIL •• If colposcopy confirms CIN 2/3, then surgical excision is done •• If colposcopy is negative then following measures can be taken: (1) a diagnostic excisional biopsy (2) repeat cervical smear 6 monthly twice, (3) repeat colposcopy •• If repeat cervical cytology smear shows HSIL then the diagnostic excisional procedure should be taken •• If two subsequent cytology smears at 6 monthly interval negative along with negative colposcopy and endocervical sampling then the patient can go to routine cervical cytology program.

Management of AGCUS

•• Colposcopy with endocervical sampling •• Woman with atypical endometrial cells should have endometrial sampling •• If no abnormality is identified then repeat cervical smears at 4 to 6 months interval should be done until 4 consecutive samples are negative.

References 1. WHO. Guidelines for screening and treatment of precancerous lesion for cervical cancer prevention. 2013. www.who.int/reproductivehealth/publications/cancers/ screening_and_treatment_of_precancerous_lesions/en/).

Screening, Quality Control and Management of Cervical Lesion 2. Saslow D, Solomon D, Lawson HW, Killackey M, Kuasingam SL, Cain J, Garcia FA, Moriarty AT, Waxman AG, Wilbur DC, Wentzensen N, Downs LS, Jr, Spitzer M, Moscicki AB, Franco EL, Stoler MH, Schiffman M, Castle PE, Myers ER, ACS-ASCCP-ASCP Cervical Cancer Guideline Committee. 2012. American Cancer Society, American Society for Colposcopy and Cervical Pathology, and American Society for Clinical Pathology screening guidelines for the prevention and early detection of cervical cancer. CACancer J Clin. 2012: 62:147-72. 3. Food and Drug Administration. FDA news release. FDA approve first human papillomavirus test for primary cervical cancer screening. 2014. www.fda.gov/newsevents/ newsroom/pressannouncements/Ucm 394773. htm. 4. Cubie HA, Canham M, Moore C, et al. Evaluation of commercial HPV assays in the context of posttreatment follow-up: Scottish Test of Cure Study (STOCS-H). J Clin Pathol. 2014;67:458-63. 5. Cervical cytology practice guidelines. Acta Cytol. 2001;45:201-26. 6. Clinical laboratory improvement amendments of 1988. Final rule federal register. 1992; 57:493.1257(b). 7. Nanda K, McCrory D, Myers E, et al. Accuracy of the Papanicolaou test in screening for and follow up of cervical cytologic abnormalities: a systematic review. Annals of Internal Medicine. 2000;132(10):810-9. 8. Fahey M, Irwig L, Macaskill P. Meta-analysis of Pap test accuracy. American Journal of Epidemiology. 1995.141:680-9. 9. Dolinar J, Ollayos CW, Tellado M, Ali I, Stevens A, Paquette C, Brodbelt S. False-negative results in cervical cytologic studies.Mil Med. 1999;164(6):410-1. 10. 2006 consensus guidelines for the management of women with abnormal cervical cancer screening tests. Wright TC Jr, Massad LS, Dunton CJ, Spitzer M, Wilkinson EJ, Solomon D; 2006 American Society for Colposcopy and Cervical Pathology-sponsored Consensus Conference. Am J Obstet Gynecol. 2007;197(4):346-55. 11. 2012 updated consensus guidelines for the management of abnormal cervical cancer screening tests and cancer precursors. Massad LS, Einstein MH, Huh WK, Katki HA, Kinney WK, Schiffman M, Solomon D, Wentzensen N, Lawson HW; 2012 ASCCP Consensus Guidelines Conference. Obstet Gynecol. 2013;121(4):829-46.

149

11

CHAPTER

Sample Cases with Answers

Case 11.1 History: A 37 year female with routine cervical screening.

Figure 11.1

Ans. Satisfactory for evaluation: Endocervical cells present. Interpretation: Negative for intraepithelial lesion or malignancy.

Sample Cases with Answers

Case 11.2 History: A 31 year female with routine cervical screening.

Figure 11.2

Ans. Unsatisfactory for evaluation: Specimen processed but unsatisfactory because of insufficient squamous cells.

Case 11.3 History: A 33-year female with routine cervical screening.

Figure 11.3

Ans. Unsatisfactory for evaluation: Specimen processed but unsatisfactory because of obscuration by inflammatory cells.

151

152 Handbook of Cervical Cytology: Special Emphasis on Liquid-based Cytology Case 11.4 History: A 35 year female with routine cervical screening.

Figure 11.4A

Figure 11.4B

Figure 11.4C

Sample Cases with Answers

Figure 11.4D

Ans. Satisfactory for evaluation Interpretation: Negative for intraepithelial lesion or malignancy. Optional comment: Inflammatory smear.

Case 11.5 History: A 39 year female with routine cervical screening.

Figure 11.5A

153

154 Handbook of Cervical Cytology: Special Emphasis on Liquid-based Cytology

Figure 11.5B

Ans. Satisfactory for evaluation Interpretation: Negative for intraepithelial lesion or malignancy. Comment: Trichomonas vaginalis identified. Advise: Repeat cervical smear after treatment of trichomonas vaginalis.

Case 11.6 History: A 49 year female with routine cervical screening.

Figure 11.6A

Sample Cases with Answers

Figure 11.6B

Ans. Satisfactory for evaluation Interpretation: Negative for intraepithelial lesion or malignancy. Comment: Atrophic smear.

Case 11.7 History: A 38 year female with routine cervical screening.

Figure 11.7A

155

156 Handbook of Cervical Cytology: Special Emphasis on Liquid-based Cytology

Figure 11.7B

Figure 11.7C

Figure 11.7D

Ans. Satisfactory for evaluation Interpretation: Epithelial cell abnormality: Atypical squamous cell of undetermined significance (ASC-US). Advise: High risk HPV testing.

Sample Cases with Answers

Case 11.8 History: A 40 year female with routine cervical screening.

Figure 11.8A

Figure 11.8B

Figure 11.8C

157

158 Handbook of Cervical Cytology: Special Emphasis on Liquid-based Cytology

Figure 11.8D

Ans. Satisfactory for evaluation Interpretation: Atypical squamous cell: high grade squamous intraepithelial lesion cannot be excluded (ASC-H). Advise: High risk HPV testing.

Case 11.9 History: A 40 year female with routine cervical screening.

Figure 11.9A

Sample Cases with Answers

Figure 11.9B

Figure 11.9C

Figure 11.9D

Ans. Satisfactory for evaluation Interpretation: Epithelial cell abnormality: LSIL (koilocytotic changes). Advise: High risk HPV testing or colposcopic examination.

159

160 Handbook of Cervical Cytology: Special Emphasis on Liquid-based Cytology Case 11.10 History: A 33 year female with routine cervical screening.

Figure 11.10A

Figure 11.10B

Figure 11.10C

Sample Cases with Answers

Figure 11.10D

Ans. Satisfactory for evaluation Interpretation: Epithelial cell abnormality: HSIL. Advise: Colposcopic examination followed by biopsy.

Case 11.11 History: A 38 year female with post coital bleeding.

Figure 11.11A

161

162 Handbook of Cervical Cytology: Special Emphasis on Liquid-based Cytology

Figure 11.11B

Figure 11.11C

Figure 11.11D

Ans. Satisfactory for evaluation Interpretation: Epithelial cell abnormality: Squamous cell carcinoma. Advise: Colposcopic examination followed by biopsy.

Sample Cases with Answers

Case 11.12 History: A 40 year female with routine cervical screening.

Figure 11.12A

Figure 11.12B

Figure 11.12C

163

164 Handbook of Cervical Cytology: Special Emphasis on Liquid-based Cytology

Figure 11.12D

Ans. Satisfactory for evaluation Interpretation: Epithelial cell abnormality: Atypical glandular cells of cervical origin favors neoplasia. Advise: Colposcopy and endocervical sampling.

Case 11.13 History: A 43 year female with routine cervical screening.

Figure 11.13A

Sample Cases with Answers

Figure 11.13B

Figure 11.13C

Figure 11.13D

Ans. Satisfactory for evaluation Interpretation: Epithelial cell abnormality: Adenocarcinoma.

165

Index Page numbers followed by f refer to figure and t refer to table Air drying artifact 77 American Society for Colposcopy and Abnormal cells 17, 32, 63, 79, 83 Cervical Pathology 138 Abnormal shaped cells 74 Anaerobic organism 51 Abnormality of glandular cells 101–124 Anucleated squamous cells 8 atypical endocervical cells 101–105f Apoptosis 111, 126, 127, 131 criteria of diagnosis of 102 Artefacts 74 liquid based cytology 104 Artifact producing changes 61 atypical endometrial cells 104, 107– Atrophic 108f criteria of 107 changes 54 liquid based cytology pattern 66 Abundant smear 8, 31, 41, 54f, 83, 99, 100, 155 lymphocytes 44 abundant parabasal cells 54f pale green cytoplasm 7 in higher magnification 100 parabasal cells 54f parabasal cells and metaplastic squamous cells 54f thin pink 6 Atypia 39, 57, 61 Acellular material 16, 17f inflammation causing 62f Actinomyces spp. 35 repair of 98 Actinomycosis 52, 53f infection 44, 46 therapy induced 61 Adenocarcinoma 30, 36, 129, 131, 165 Atypical endocervical cells 101, 105 discrete cells 112 criteria of diagnosis 102, 104 hyperchromatic crowded group with favour neoplastic feathery margin 114 cohesive cluster of moderately in situ pleomorphic cells 106 cytoplasmic and nuclear endocervical cells with sprouting 110 overcrowded nuclei 105 histopathology 109 higher magnification enlarged pleomorphic cells 105 hyperchromatic crowded group of cells 109f moderate amount of vacuolated cytoplasm 106 long strip of tumor cells 110 small clusters of cells with strip of cells with nuclear overlapping nuclei 105 protrusion 110f linear strip of cells 114 Atypical endometrial cells 104 multiple clusters 112 bag of polyps 108f strip of tumor cells 114 liquid-based cytology of 107 Adequate cervical smear 37f small cells with hyperchromatic pleomorphic nuclei 108 Adjunctive testing 36

A

168 Handbook of Cervical Cytology: Special Emphasis on Liquid-based Cytology Atypical glandular cells 39, 129, 101, 130 cervical origin 164 simulating 67 undetermined significance 101 Atypical immature metaplasia 63 Atypical metaplastic squamous cells 59 Atypical parakeratotic squamous cells 59 Atypical squamous cells 51, 56, 59, 146 setting of atrophy 59 undetermined significance 57, 156 Automated scanning system 40 Automated screening devices 31 BD FocalPoint GS imaging system 31 HOLOGIC ThinPrep imaging system 31

B Bacterial vaginosis 31, 35, 49, 50f Bag of poly 119 Benign and dysplastic cells 18 cellular changes 38 endometrial cells 13 hCG 119 Bethesda System 2014 34, 35t general categorization 35 organisms 35 reporting cervical cytology 33 satisfactory for evaluation 35 specimen type 35 unsatisfactory for evaluation 35 Bi-nucleated cell 76f Bird tail strip of cells 115 Bizarre nucleus 54 Bland dyskaryosis 87, 88 Blue blobs 55 British Society of Clinical Cytology 33 Bubble gum cell 44, 46

C Candida 31, 50, 51f Candida spp. 35 Candidal pseudohyphae 51 Cannon ball 48 Carbowax 24 Cell counting 36 Cell cytoplasm 83, 128

Cells of adjacent organ cervical cytology 16 colonic cells 16 dysplastic cell 17 urothelial cells 16 Cellularity 124 Central dense stromal cells 128 Central pyknotic nuclei 6 Cervical intraepithelial lesions grade 1 69 grade 2 69 grade 3 69 neoplasia 33 different screening methods 139 lesion indicators of good screening program 145 management 146 quality control of 144 screening strategy 138t malignancies 133 carcinosarcoma 133 malignant melanoma 133 metastatic tumors 134 small cell carcinoma 133 smear different devices 23f overall approach of reporting 34 reporting 33f sampling fixative 23 instrument 23 laboratory form 23 squamous epithelium 70 Cervix 1, 2, 3f histology of 3 transformation zone 4 Chromatin pattern 18-20, 30 Chronic inflammation 44 Clinging diathesis 30, 97 Clinging tumor diathesis 97, 98, 115f, 116 Clitoris 1, 2 Clue cells 31, 49, 50 Clump of actinomycosis 52f

Index Clusters of atypical cells 74 cells 80, 136 cuboidal 134 endometrial cells 67f Coarsely granular 124 Coccobacilli 49 Cohesive cluster of endometrial cells 85f Colon 134 Colposcopic examination 140, 146, 147, 159, 161, 162 Colposcopy with endocervical assessment 148 Columnar cell 2, 4, 5, 10f, 11f, 16, 112, 113f, 136 endocervical cells 11f, 46, 55 Computer-assisted interpretation of cervical cytology 36 Conventional smear 34 Corn flakes 6 Corn flakes cells 7f Cotton ball 52 Cytology 5 endocervical cells 9 endometrial cells 13 in LBC compared to CP background 29 cell crowding 30 cell size 29 cytoplasmic and nuclear detail 30 hyperchromasia 30 inflammatory cells 14 metaplastic squamous cells 12 others 14 giant cells 14 lactobacilli 14 ova of parasites 14 spermatozoa 14 reported as negative for intraepithelial lesions 146 unsatisfactory 146 squamous cells 6 Cytomegalovirus 35 Cytomegaly 122 Cytoplasmic fragments 48

melanin pigment 133 orangeophilia 7 vacuolation 61,126 Cytotrophoblasts 16

D Decidual cells 61 Dense cytoplasm 7 orangeophilic 57 cytoplasm 59 Density gradient sedimentation 28 Device score 31 Discrete cells 81f dysplastic cells 18f intermediate cells 65 Dorsolithotomy 24 Drying artifact 35 Dysplastic cell 8, 18, 19f, 21f, 70, 79, 83

E Ectocervix 2, 3, 24 Endocervical adenocarcinoma 101, 111 characteristic cytological features of 112 in situ 108 liquid-based cytology of 116 cells 10f, 36, 41, 62, 101, 119 characteristic features of 11 distinguishing features of 128t columnar lining epithelial cells 4f glands 79 nuclei 102 polyp 120, 123 cluster of glandular cells in endocervical polyp 123 higher magnification of the clusters of glandular cells 123 sampling 148, 164 Endocervix 4 Endometrial adenocarcinoma 116, 131 cells enlarged nuclei 117 cells with moderate nuclear pleomorphism 117

169

170 Handbook of Cervical Cytology: Special Emphasis on Liquid-based Cytology

characteristic cytological features of 116 cluster of cells 116 discrete cells 117 higher magnification nuclear pleomorphism 118 liquid-based cytology of 119, 127 loose clusters of 118 tumor diathesis 118 and endocervical cells, distinguishing features of 128t cells 30, 84, 99, 119, 126 cytological features of 13 higher magnification of 127 liquid-based cytology 126 small tight clusters of 127 tight clusters of round cells 14 polyp 128 sampling 148 Enlarged hyperchromatic nuclei 80 pleomorphic nuclei 131 Eosinophilia 44 Eosinophilic parabasal cells 55 Epithelial cell abnormalities 39 atypical glandular lesion 39 atypical squamous cells 39 automated review and ancillary testing 40 endocervical adenocarcinoma in situ 40 squamous cell carcinoma 39 squamous intraepithelial lesion 39 Epithelioid cell granulomas 44 Excess neutrophils 41 parabasal cells 44, 61 External quality assurance 144

F Fallopian tube 1, 2, 5, 134 False hyperchromasia 84 koilocytosis 78 Female genital tract 1f anatomy of 1 cervix 3 histology of 3

transformation zone 4 fallopian tubes, tubular structure of 5 ovaries, mature ovum 5 uterus 2 histology of body and fundus 2 inner basalis layer outer functionalis layer 2 parts of 2 body 2 cervix 2 fundus 2 vagina, histology of 2 vulva 1 clitoris 2 labia majora 2 labia minora 2 mons pubis 2 Female sex hormones estrogen 5 Fertilization 5 Fiber cells 74, 98 endometrial stromal cells 98 fibroblasts 98 smooth muscle cells 98 stromal cells in atrophic smear 98 Fibromuscular tube 2 Fine granular chromatin 57, 126 Folic acid deficiency 61, 63f, 77, 79f Follicular cervicitis 31, 44 Food and Drug Administration 24 Frayed edges of the cells 74

G Giant cell multinucleated 15 Glandular cell atypical 36 status post-hysterectomy 55 Granular debris 99 Granular nuclear chromatin 83 Granulomatous inflammation 44 Group of metaplastic cells 65f

H Herpes simplex virus 35, 41, 52, 53f High grade intraepithelial lesions 79 High grade squamous intraepithelial lesion 13, 33, 69, 158 Histiocytes 44, 83, 128 Homogenous bland chromatin 19

Index Hormone status 17 Human papilloma virus (HPV) 8, 139, 140, 143, 146 ASC-US with equivocal changes of 61 changes Bi-nucleated cell 76f increased number of parakeratotic cells 76 f multi-nucleated cell 76 f nuclear enlargement of cell 75f cytological features of infection 74 DNA test 147 high risk of 69, 159 infection 59, 74, 138 low risk of 60 screening 140 subtypes 140 testing 139, 141 Hyperchromasia 19, 55, 59, 63, 66, 70 Hyperchromatic crowded group 66, 87, 88, 108, 111, 119 nuclei 59, 61, 64, 71, 97, 99, 133 Hyperkeratosis 9f

I Improperly fixed smear 61 Inflammatory atypia 61 cells 151 exudate 38f, 61 smear 42f, 153 Intermediate cells 7 Intracellular keratin 7 keratinization 93 Intraepithelial lesion 38, 39, 41, 150, 154, 155 Intrauterine contraceptive device (IUCD) 17, 35, 44, 120, 122 changes 45f cell with high N/C ratio 45 multinucleated cells 46 Invasive carcinoma 40, 69, 98 squamous cell carcinoma 69 Irregular clumped chromatin 66



coarse chromatin 93 contour of nuclei 93 nuclear margin 70 nuclear membrane 59

J Judgement in borderline cellularity 38

K Karyolysis 41, 42, 61 Karyorrhexis 41, 42, 44, 61 Keratinizing high grade squamous intraepithelial lesion 89 differential diagnosis 89 glandular involvement 89 Keratohyaline granules 6f Keratotic changes 35 Koilocytosis 51, 70, 74, 75 histopathology of 72f, 73f Koilocytotic changes 59, 159

L Labia majora See Female genital tract, vulva Labia minora See Female genital tract, vulva Lactobacilli 49 Rod like structures 15 Large polygonal cells 6, 7 Leptothrix 49, 51, 52f Lesser screening 29 Loose cluster of cells 71, 72f Low grade intraepithelial lesions 70 squamous intraepithelial lesion 21 Lower uterine segment 131 Low-grade squamous intraepithelial lesion 69 Lubricant jelly 24 Lymphocytes 128, 129f

M Macronucleoli 93, 133 Malignancy 38 Malignant glandular 133 tumors 133 Melanoma of cervix 133 Menstrual cycle, exodus 128

171

172 Handbook of Cervical Cytology: Special Emphasis on Liquid-based Cytology Menstruation 128 Metaplastic cells 60f, 65, 82 endocervical cells 12f, 36 squamous cells 13, 34, 54f, 59, 84 Metastatic ovarian adenocarcinoma 135 carcinoma 135 tumors breast carcinoma 136 colonic carcinoma 136 metastatic serous ovarian carcinoma 135 Mimic glandular cells 66 Mimickers of ASC-H 67 atypical glandular cells 67 endometrial cells 67 histiocytes 67 Mimickers of HSIL 83 Minimal hyperchromasia 74 Mitosis 88, 89, 111,120, 122, 131 Moderate nuclear pleomorphism 136 Monolayer cell 29 Mononuclear histiocytes 83 Mons pubis See Female genital tract, vulva Mosaic 12 Multinucleated giant cell 16f, 46, 48, 54 Multiple nucleoli 116 tight cohesive clusters of cells 135

N Naked large nuclei 61 Navicular cells 7 Necrotic debris 116 Neoplasia 164 Neutrophils 85 Non-specific perinuclear halos 77 Nuclear atypia 51 chromatin 59 contour 20 enlargement 20, 66 hyperchromasia 72f membrane 21 moulding 136 pyknosis 61

Nuclei of the cells 70 Nucleocytoplasmic ratio 44 Nucleoli 63 Nucleus 63 Nucleus of the cell 58 Nulliparous woman 3

O Occasional necrosis 41 Orangeophilic cytoplasm 6, 9, 91f Organism 39, 48 Organisms Trichomonas vaginalis 48 Outer connective tissue layer 2 Overstained cells 84

P Pale cell dyskaryosis 85 mucinous material 55 Papanicolaou’s 6, 24, 28, 140 Para nuclear vacuoles 42 Parabasal cells 3, 8f, 55, 129 cytological features of 7 Parakeratosis 8, 74 Parakeratotic cells 9f Perinuclear halo 50, 51, 61, 73 vacuolation 41 Pleomorphic hyperchromatic cells 130 nuclei 41, 116 Pleomorphism 59 Polymorphs 44 Post coital bleeding 161 Posterior vaginal fornix 2 Preanalytic phase 144 Prepstain density reagent 27 Procedure of sample collection 24 Progesterone 5 Prominent nucleoli 79, 112, 135, 136 Psammoma bodies 46, 135 Pus ball 48 Pyknosis 41, 44 Pyknotic nuclei 7

R Radiation changes 47f

Index

effect causing nuclear enlargement 77 induced changes 120 with squamous cell carcinoma 48 Reactive cellular changes 46 inflammation 41 in endocervical cells 77 endocervical cells 43f, 44, 78, 121 nuclear pleomorphism 121 inflammatory changes 77 Red cytoplasmic granules 48 Re-screening of negative cases automated screening 145 rapid review 145 selective rescreening 145 Round regular nuclear margin 121 Routine cervical screening 150, 152, 154, 160, 163

S Sample cases with answers 150 Sarcoma element show clusters 134 Satisfactory for evaluation 153, 164 Scanty cellularity in cervical smear 37f Shish Kebab 31, 50 Single multinucleated cell 58 Small cell carcinoma 133 discrete monomorphic round cells with hyperchromatic nuclei 134 higher magnification cells with round enlarged nuclei 134 Small cell pattern 63 severe dyskaryosis 85, 86 Small pyknotic nuclei 7 Sparse cell 87f dyskaryotic cell 85 Specific changes in LBC 30 Specimen adequacy 34 Speckled chromatin 85 Sperms with tail 15f Spindle cells 98f shaped stromal cells 131

Squamocolumnar junction 5f Squamous cells carcinoma 30, 84, 91, 162 cytological features of 93 discrete and loose clusters of malignant cells 94 discrete tumor cells 95f fibre cells with elongated nuclei 96f histopathology of 93 lower magnification showing tumor diathesis 96f markedly enlarged nuclei 94 markedly pleomorphic nuclei 95f tadpole cell with one sided nuclei 96f Squamous epithelial cells 2, 77 layers 2 Squamous epithelium changes of hyperkeratosis 8 pseudoparakeratosis 8 Squamous intraepithelial lesion 33, 35, 69, 122 Squamous lining epithelium histology of 3f Squamous metaplastic cells 83 cytological features of 12 Stripped out cytoplasm 61 Submucosal leiomyoma 128 Superficial cells and intermediate cells 6f Superficial squamous cells 8 SurePath basic principle of 26f preparation technique cell enrichment 26 resuspension 27 vortexing 26 Syncytial aggregates 79

T Tadpole cells 93 Tamoxifen therapy 128 Tampon 24 ThinPrep basic principle of 25f preparation technique cell collection 25

173

174 Handbook of Cervical Cytology: Special Emphasis on Liquid-based Cytology cell transfer 25 dispersion 25 versus SurePath, comparison of 29t Transformation zone components 35, 36 Trichomonas infection 51 Trichomonas vaginalis 31, 35, 49f, 74, 154 Trophoblastic cells 54, 61 True koilocytosis 75 Tubal metaplasia 11f, 35, 120, 121 cluster of endocervical cells, cilia 121 Tubal metaplastic cells 119 Tumor diathesis 46, 84, 92, 93, 97, 108, 119

U Unsatisfactory for evaluation 37, 151 Uterine metastatic malignancies 134

V Vacuolated cytoplasm 46, 67, 127 Vacuolation 44 Vaginal cream 24 Variably enlarged nuclei 74 Vesicular chromatin 67 Vestibule 1 Viral infection 16 Visual inspection under acetic acid 143 advantages 143 limitations 144 method of testing 143 Vortexing 27

W Water soluble lubricant 24 Wooly body 52 World Health Organization 91, 138

Y Yeast form 50