The ABCs of TBS. A Novice's Guide to the Bethesda System

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1 CE U P D A T E W O M E N ' S HEALTH III Julia Woodruff Wildes, MD The ABCs of TBS A Novice's Guide to the Bethesda System This is the third and final article in a three-part series on women's health. The exam follows this article. On completion of this series, the reader will be able to describe laboratory findings associated with osteoporosis, list factors contributing to cardiovascular disease in women, and explain aspects of the Bethesda System for reporting Pap smears. From the Oregon Health Sciences University and Portland Veterans Affairs Medical Center. Reprint requests to Dr Wildes, Pathology and Laboratory Medicine Service (P-2-PATH), Department of Veterans Affairs Medical Center, 370 S W US Veterans Hospital Rd, Portland, OR 9720; or wildes.julia_w@port land.va.gov The role of the Pap smear in decreasing deaths due to cervical carcinoma is one of the great success stories of modern medicine. From 97 to 98, the incidence and mortality rates for invasive cervical cancer declined 70% to 7% in the United States. This dramatic decrease seems to be due almost entirely to increased detection and therapy of precursor lesions, because in developing countries, where such screening is much less common, the death rate has shown little change during this period.2 by guest 6 VOLUME 29, NUMBER 9 Anatomy and Histologic Features of the Uterine Cervix An appreciation of the anatomy and histologic features of the uterine cervix is required to understand disorders of this organ (Fig ). The uterine cervix is the portion of the uterus immediately adjacent to the vagina. A portion of the cervix, the portio vaginalis, projects into the vagina and, as a consequence, is readily accessible for visual examination, Pap smear, and biopsy. The external part of the cervix, or ectocervix, is covered by squamous epithelium, a tough, protective epithelium found in other sites such as the skin and oral cavity. The internal portion of the cervix, or endocervix, is covered by mucus-secreting glandular (columnar) epithelium. The point at which squamous and glandular epithelium meet, the squamocolumnar junction, or transformation zone, is where most serious abnormalities arise. In younger women, the transformation zone tends to be located on the external portion of the cervix or in the lower part of the endocervical canal and, thus, is easily sampled. As a woman becomes older, however, this important zone recedes up the endocervical canal and may not be as amenable to sampling (Fig 2). Detection of Potential Precursors of Malignancy: The Most Important Function of the Pap Smear Although the Pap smear detects numerous minor and serious abnormalities, its most important function is detection of conditions that can progress to invasive squamous cell carcinoma. These so-called precursor lesions, as well as squamous cell carcinoma itself, are highly associated with infection with specific serotypes of human by guest on November 208 The Pap smear is the most successful cancer screening method known, playing a pivotal role in the dramatic decrease in incidence of and mortality from invasive cervical cancer. The Bethesda System, sometimes called TBS, for reporting cytologic diagnoses of cervicovaginal samples originated from a conference held by the National Cancer Institute in 988. The Bethesda System provides a means of communicating Pap smear diagnoses to clinicians in a standardized manner and has largely supplanted previous systems, such as the Papanicolaou classification. This article provides basic information about premalignant and malignant lesions involving the cervix and their classification under the Bethesda System. A brief overview of relevant cervical anatomy and histologic and cytopathologic features is included.

2 UtariMtafca Ovary Extarnal cervical m Fig. Normal female internal genital organs. From Patton SF Jr. Anatomy and histology. In: Keebler CM, Somrak T M, eds. The Manual of Cytotechnology. Chicago, III: ASCP Press; 993:. Fig 2. Location of the transformation (squamocolumnar junction) zone in younger (A) and older (B) women. The heavy violet line indicates glandular (columnar) epithelium; the red line indicates squamous epithelium. by guest on November 208 papillomavirus (HPV), a sexually transmitted DNA virus. Infection by HPV may result in morphologic changes in cervical epithelial cells that may be detected by Pap smear or cervical biopsy. These morphologic changes form a continuum. The earliest changes may imply HPV infection only (condyloma). As the changes progress, atypical cells populate the cervical epithelium. The severity of the changes is estimated by the appearance of the abnormal cells in conjunction with the fraction of the total thickness of epithelium involved by these cells (Fig 3). Currently, three systems of terminology are used to describe these potentially serious abnormalities occurring in squamous cells. The oldest of these systems, which dates back to the 90s, uses the terms mild, moderate, and severe dysplasia and carcinoma in situ to describe a progression in severity of abnormalities.3' Abnormal cells involve only the lower third of the squamous epithelium in mild dysplasia, the lower and middle thirds in moderate dysplasia, and the lower, middle, and upper thirds in severe dysplasia. In carcinoma in situ, severely abnormal cells populate the full thickness of the epithelium. During the 970s, the concept of cervical intraepithelial neoplasia (CIN) was introduced to emphasize the continuity of these changes. This system, which still is used widely in cervical biopsy diagnosis, is very similar to the oldest system; CIN and mild dysplasia are basically equivalent, as are CIN 2 and moderate dysplasia. CIN 3, however, encompasses severe dysplasia and carcinoma in situ. The third system of terminology is the twotiered Bethesda System. The category low-grade squamous intraepithelial lesion (LSIL) includes changes previously categorized as condyloma, mild dysplasia, and CIN. High-grade squamous intraepithelial lesion (HSIL) includes moderate and severe dysplasia, carcinoma in situ, and CIN 2 and CIN 3. These abnormalities were grouped together because the entities included in LSIL may be difficult to distinguish from each other cytologically, as may those included in HSIL. In addition, although many HPV serotypes may be seen in LSIL and HSIL, HPV types 6 and tend to predominate in LSIL, whereas other serotypes, including HPV type 6, tend to predominate in HSIL. Likewise, the entities included in LSIL c 3 E o 0 Category Classification System 0 IS (Year Introduced) Bethesda (988) Normal Cervical Normal HSIL LSIL Condyloma CIN z e CIN3 Intraepithelial Neoplasia (970s) Traditional (90s) Normal! Fig 3. Diagram of human papillomavirus-associated changes in the cervix using several systems of terminology. As dysplasia becomes more severe, a greater proportion of the epithelial thickness is involved by abnormal cells. Modified from Wright TC, Kurman RJ, Ferenczy A. Precancerous lesions of the cervix. In: Kurman RJ, ed. Blaustein's Pathology of the Female Genital Tract. th ed. New York, NY: Springer-Verlag NY; 99:26. LSIL indicates low-grade squamous intraepithelial lesion; HSIL, high-grade squamous intraepithelial lesion; CIN, cervical intraepithelial neoplasia. by guest VOLUME 29, NUMBER 9 7

3 exhibit a lesser rate of progression to more serious abnormalities if left untreated than do those included in HSL. Although this terminology was developed for use in cytologic diagnosis, it seems to reflect fundamental biologic differences and, thus, is used by some pathologists in biopsy interpretation. The original Bethesda System for Reporting Cervical/Vaginal Cytologic Diagnoses arose from a workshop held in 988 at the National Cancer Institute in Bethesda, Md. The purpose of the workshop was to devise a means for clear and standardized communication of Pap smear diagnoses to clinicians.6 At the time of the workshop, the Papanicolaou classification, which consisted of five diagnostic classes, was in widespread use. Although of historic significance, the Papanicolaou system was deemed unacceptable for four reasons: Test time! Look for the CE Update exam on Women's Health (806) in this issue of Laboratory Medicine. Participants will earn 3 CMLE credit hours.. The classification as a whole did not reflect contemporary understanding of cervical and vaginal neoplasia. 2. The Papanicolaou classes had no consistent relationship to terminology used in biopsy diagnosis. 3. Abnormal but benign entities were largely omitted from the classification.. The original Papanicolaou classes had been modified to the extent that they no longer had the same significance in all institutions. Inclusion of a statement about adequacy of the specimen for diagnostic purposes is an important part of the Bethesda System because not all cervicovaginal smears are equally satisfactory for providing a definitive cytologic diagnosis. A specimen may be characterized as satisfactory for evaluation, satisfactory for evaluation but limited, or unsatisfactory for evaluation. The category "satisfactory for evaluation but limited (by some technical factor)" (originally "less than optimal") indicates that although a diagnosis may be given, the diagnosis may not be entirely valid owing to some technical factor. For example, if a smear taken from a premenopausal woman exhibits adequate numbers of well-preserved and wellvisualized squamous cells but with no cytologic evidence that the transformation zone has been sampled, the specimen is considered "satisfactory for evaluation but limited." The categories "satisfactory for evaluation" and "unsatisfactory for evaluation" are self-explanatory, with the understanding that a specimen is not considered unsatisfactory if abnormal cells are detected despite severe technical deficiencies. Four factors are considered when evaluating the adequacy of a specimen:. Patient and specimen identification 2. Pertinent clinical information 3. Technical interpretability. Cellular composition and sampling of the transformation zone Although there are obvious reasons that correct patient and specimen identification are required, correct identification also facilitates finding previous material from the patient for review if needed. Age of the patient and date of the last menstrual period, the minimum clinical information required, are essential for accurate In the Bethesda System, the format of the cervi- interpretation because the same cytologic finding covaginal cytopathology report consists of three may have different implications in different clinical settings. For example, the presence of elements: endometrial cells in the Pap smear is normal dur. A statement on the adequacy of the specimen for ing the first half of the menstrual cycle in a prediagnostic purposes ("satisfactory," "satisfactory menopausal woman, but in a postmenopausal but limited," or "unsatisfactory") woman, presence of endometrial cells requires 2. An optional general categorization of the diag- additional investigation because serious endomenosis ("within normal limits," "benign cellular trial disease, such as endometrial hyperplasia or changes," or "epithelial cell abnormality") 3. The descriptive diagnosis (ie, a more specific description of any abnormalities present) In 99, a second workshop was held to evaluate clinical use of the Bethesda System and make necessary revisions. The system widely used today for reporting Pap smear diagnoses is the product of this workshop.7'8 by guest 8 VOLUME 29, NUMBER 9 by guest on November 208 Origin of t h e Bethesda S y s t e m Adequacy of the Specimen for Evaluation

4 Fig. A ball of endometrial glandular and stromal cells (Papanicolaou stain, high power). Depending on the clinical setting, this may be a normal finding or may indicate the need for further workup. From Bigner SH, Erozan YS, Giant MD. Cyfofesf: A Practice Examination in Cytopathology. Chicago, III: ASCP Press; 99:-. General Categorization The second element of the Bethesda System is assignment of the diagnosis to one of three general categories, ie, within normal limits, benign cellular changes (used for infections and reactive changes), and epithelial cell abnormality (used for potentially premalignant entities, such as LSIL and HSIL, and malignant entities). Assignment to a general category is optional and is used for triage and for recording statistical data. If a specimen is assigned to the category "within normal limits," no further explanation is required. Assignment to either of the other categories mandates elaboration under descriptive diagnosis. Descriptive Diagnosis Descriptive diagnoses fall within four categories. The two major categories are benign cellular changes, in which infections and benign abnormalities of squamous and glandular cells are more fully characterized, and epithelial cell abnormality, in which the exact nature of the abnormality and the cell type affected are given, when they are possible to determine (see "Epithelial Cell Abnormality"). Other categories are other malignant neoplasms (ie, the rare malignant neoplasms that are not of squamous or glandular origin, such as lymphoma or sarcoma) and hormonal evaluation, which indicates whether the cellular composition of the smear is compatible Fig. The large polygonal cells with small round nuclei are normal superficial and intermediate squamous epithelial cells (Papanicolaou stain, high power). The small pear-shaped blue organisms are Trichomonas. From Bigner SH, Erozan YS, Giant MD. Cyfofesf: A Practice Examination in Cytopathology. Chicago, III: ASCP Press; 99:-. with the patient's age and history. The latter two categories will not be discussed in this article; for additional information, see Kurman and Solomon's text on the Bethesda System9 and DeMay's The Art and Science of Cytopathology.0 Benign Cellular Changes The "benign cellular changes" category, an area not well addressed by the Papanicolaou classification, includes cervical and vaginal infectious agents exhibiting morphologic features sufficiently distinctive that they at least can be suggested by microscopic examination. Trichomonas vaginalis may be identified definitively on Pap smear (Fig ). by guest VOLUME 29, NUMBER 9 9 by guest on November 208 adenocarcinoma, may be present (Fig ). The criterion for technical interpretability requires that enough well-preserved and well-visualized squamous cells be present for adequate evaluation (see reference 9 for specific criteria). Such factors as obscuring blood, inflammation, areas where the smear is too thick, poor fixation, and air-drying artifact may compromise interpretation or, if too extensive, completely preclude it. The transformation zone must be sampled because most serious abnormalities detected by Pap smear tend to involve this region. The presence of endocervical glandular or squamous metaplastic cells is considered evidence that the transformation zone is represented. However, this zone may not be accessible for sampling in postmenopausal women because of its location. Thus, in some circumstances, an optimal specimen may contain neither endocervical glandular nor squamous metaplastic cells.

5 However, because cytologic examination is not the reference method for identification of most organisms, a qualifying phrase is often added. Thus, the presence of budding yeasts, pseudohyphae, and true hyphae is reported as "fungal organisms morphologically consistent with Candida spp." Viral infections may manifest by typical morphologic changes in epithelial cells. For herpes simplex virus, these changes include nuclei exhibiting a ground-glass quality with peripheral margination of chromatin, eosinophilic intranuclear inclusions, and multinudeation with nuclear molding (Fig 6). These cytopathologic effects are reported as "cellular changes associated with herpes simplex virus." Viral cytopathic effects of HPV, on the other hand, are reported under the heading "epithelial cell abnormality" because of the association of HPV with premalignant and malignant entities. Also included among benign cellular changes are those that are reactive in response to inflammation, atrophy (with or without inflammation), radiation, and intrauterine contraceptive devices. These morphologic changes are benign and welldefined; abnormalities exceeding these criteria are encompassed by the general category "epithelial cell abnormality." Epithelial Cell Abnormality Epithelial cell abnormalities include those arising in squamous and glandular cells. These abnormalities span a spectrum from lesions that are not by guest 0 VOLUME 29, NUMBER 9 by guest on November 208 Fig 6. Cellular changes associated with herpes simplex virus (Papanicolaou stain, high power). Note nuclear molding, best appreciated in cells located at 9 o'clock. From Bigner SH, Erozan YS, Giant MD. Cytotest: A Practice Examination in Cytopathology. Chicago, III: ASCP Press; 99:-. unequivocally benign but that cannot be characterized more definitively (atypical squamous or glandular cells of uncertain significance) to frankly invasive squamous cell carcinoma or adenocarcinoma. The most important function of the Pap smear is detection of the abnormalities that are potential precursors of invasive squamous cell carcinoma so that biopsy confirmation and treatment can be initiated. As previously mentioned, the category LSIL includes mild dysplasia (CIN ) and HPV cytopathic effect (condyloma). The inclusion of both entities within the same category has been one of the more controversial aspects of the Bethesda System, because some experts have regarded HPV effect as merely indicative of infection and not necessarily a precursor lesion. However, similarities in cytomorphologic features, rate of progression to more serious abnormalities, and associated HPV serotypes support the inclusion of HPV cytopathic effect in the LSIL category. The entities included in HSIL seem closely related to each other for similar reasons. Nuclear morphologic abnormalities are needed to make a diagnosis of SIL. The nuclei of cells that raise suspicion of abnormality are compared with those of normal intermediate squamous cells found in the patient's smear. In general, dysplastic squamous cells exhibit enlarged nuclei with darker chromatin staining (hyperchromasia), irregular nuclear contours, and a higher nuclear/cytoplasmic ratio than normal cells (Figs 7-0). Some abnormal cells may be multinucleated, and others may exhibit a clear perinuclear zone rimmed by dense cytoplasm (koilocytosis) (Fig 7). Abnormalities in LSIL and HSIL constitute a spectrum, with HSIL generally showing greater hyperchromasia, more irregular nuclear outlines, more granular chromatin, and a higher nuclear/cytoplasmic ratio than LSIL (for example, compare the cells in Figs 7 and 8 with those in Figs 9 and 0). Invasive squamous cell carcinoma may exhibit features of HSIL; however, other features, such as macronucleoli, markedly irregular chromatin distribution, and a

6 #. / i Fig 8. Low-grade squamous intraepithelial lesion (Papanicolaou stain, high power). These dysplastic cells exhibit abundant cytoplasm and a relatively low nuclear/cytoplasmic ratio. The nuclei, however, are large and darkly staining (hyperchromatic). From Bigner SH, Erozan YS, Giant MD. Cytotest: A Practice Examination in Cytopathology. Chicago, III: ASCP Press; 99:-2. > by guest on November 208 Fig 7. Low-grade squamous intraepithelial lesion (Papanicolaou stain, high power). Three binucleated koilocytes, exhibiting human papillomavirus cytopathic effect. In addition to prominent perinuclear clearing, the nuclei of these cells are larger and more darkly staining than those of normal squamous cells. From Bigner SH, Erozan YS, Giant MD. Cytotest: A Practice Examination in Cytopathology. Chicago, III: ASCP Press; 99:-9. $\ \ t # r^^ & ^ Fig 9. High-grade squamous intraepithelial lesion (Papanicolaou stain, high power). Note the higher nuclear/cytoplasmic ratio and greater hyperchromasia of these cells (moderate dysplasia, cervical intraepithelial neoplasia grade 2) than is found in a low-grade squamous intraepithelial lesion. From Bigner SH, Erozan YS, Giant MD. Cytotest: A Practice Examination in Cytopathology. Chicago, III: ASCP Press; 99:-37. tumor diathesis composed of blood and necrotic debris, may be present. In addition, if the tumor produces keratin (keratinizing squamous cell carcinoma), the size and shape of malignant cells and their nuclei may vary greatly (Fig ). Pap smear examination also may detect abnormalities of glandular cells. In this situation, the diagnostic statement includes not only the degree of abnormality present (eg, adenocarcinoma), but also the probable site of origin. The latter is important because glandular abnormalities may arise in the endocervix or endometrium (the lining of the uterus). In addition, adenocarcinoma metastatic from an extrauterine primary neoplasm rarely may be seen in the Fig 0. High-grade squamous intraepithelial lesion (Papanicolaou stain, high power). These cells are from a patient with squamous carcinoma in situ (cervical intraepithelial neoplasia grade 3). They have scant cytoplasm, a very high nuclear/cytoplasmic ratio, and coarsely granular chromatin. From Bigner SH, Erozan YS, Giant MD. Cytotest: A Practice Examination in Cytopathology. Chicago, III: ASCP Press; 99:-38. r " : w i -.! r t ( m v Fig. Invasive keratinizing squamous cell carcinoma (Papanicolaou stain, high power). Note the irregular shapes, dense orange or pink cytoplasm, and irregular darkly staining nuclei of these cells. In the background is necrotic debris ("tumor diathesis") associated with invasion. From Bigner SH, Erozan YS, Giant MD. Cytotest: A Practice Examination in Cytopathology. Chicago, III: ASCP Press; 99:-36. by guest VOLUME 29. NUMBER 9

7 Please let us know your opinion of the Women's Health (806) series.. The series met the objectives stated in the abstract. 2. The series provided useful technical data or original ideas. 3. The information provided in the series was new and timely. 2. Technical points were explained clearly and were easy to comprehend. 2» 3. The text was organized logically. Conclusion The Pap smear is the most successful cancer screening method known. The Bethesda System provides a means of communicating Pap smear diagnoses in a concise, reproducible, and readily understood format Illustrations, charts, and tables helped explain text and added to series value. Comments: (Attach additional pages, if necessary.) References. Devesa SS, Silverman DT, Young JL, et al. Cancer incidence and mortality trends among whites in the United States, / Natl Cancer Inst. 987;79: Pisani P. Parkin DM, Ferlay J. Estimates of the worldwide mortality from eighteen major cancers in 98: implications for prevention and projections of future burden. Int ] Cancer. 993;: Reagan JW, Seideman JL, Saracusa Y. The cellular morphology of carcinoma in situ and dysplasia or atypical hyperplasia of the uterine cervix. Cancer. 93;6: Reagan JW, Hamonic MJ. Dysplasia of the uterine cervix; part IV: the cytology of early cancer. Ann N Y Acad Set. 96;63: Richart RM. Cervical intraepithelial neoplasia. Pathology Annual. 973;8: National Cancer Institute. The 988 Bethesda System for Reporting Cervical/Vaginal Cytologic Diagnoses. Acta Cytol. 989;33: National Cancer Institute. The revised Bethesda System for Reporting Cervical/Vaginal Cytologic Diagnoses: report of the 99 Bethesda workshop. Acta Cytol. 992;36: National Cancer Institute. The Bethesda System for Reporting Cervical/Vaginal Cytologic Diagnoses. Acta Cytol. 993;37: Kurman RJ, Solomon D. The Bethesda System for Reporting Cervical/Vaginal Cytologic Diagnoses: Definitions, Criteria, and Explanatory Notes for Terminology and Specimen Adequacy. New York, NY: Springer-Verlag; DeMay RM. 77ie Art and Science of Cytopathology. Chicago, III: ASCP Press; 996. by guest 2 VOLUME 29. NUMBER 9 Thank you for your input. Mail this form (or a photocopy) alone or with your exam to: Laboratory Medicine, 200 W Harrison St, Chicago, IL by guest on November 208 Pap smear. Obviously, the nature of any additional diagnostic procedures required will depend on the site involved. Endocervical cell abnormalities range from findings that are too atypical for the cytopathologist to be certain that they are only reactive but that are not diagnostic of a premalignant or malignant entity (atypical glandular cells of uncertain significance, or AGUS) to frankly invasive adenocarcinoma. Endometrial cell abnormalities also span a spectrum. At one end of the spectrum are cells that appear completely normal but that are found at times when their presence alone suggests the need for further investigation to exclude serious disease (see "Adequacy of the Specimen for Evaluation"). At the other end of the spectrum are obvious adenocarcinomas.