| Abstract|| |
The terminology for reporting human papillomavirus-associated squamous lesions in the cervix, both in tissue samples and cytology specimens, has suffered from many changes throughout the last years creating confusion in interpreting cervical biopsy and cytology reports by clinicians. This review presents a summary and discussion of the current terminology for reporting results of cervical biopsies and cytology with emphasis in the lower anogenital squamous terminology consensus recommendations for tissue specimens and the 2001 Bethesda Workshop for reporting cytology results. Microscopic features of cervical lesions in tissue samples and cytology specimens are presented. Biomarkers, including p16 and Ki-67, are discussed and how they can help the pathologist when dealing with difficult cases.
Keywords: High-grade squamous intraepithelial lesion, human papillomavirus, Ki-67, low squamous intraepithelial lesion, p16
|How to cite this article:|
Reyes MC, Cooper K. Cervical cancer biopsy reporting: A review. Indian J Pathol Microbiol 2014;57:364-8
| Introduction|| |
Dysplasia of the cervix is caused by human papillomavirus (HPV) infection of low or high oncogenic risk type. With low-risk HPV infection, squamous epithelium is altered secondary to virus production and replication. This will give rise to lesions that have been referred to in different ways throughout the last couple of decades: Atypical hyperplasia, low-grade squamous lesion, mild dysplasia, and condyloma acuminatum. The majority of these lesions will regress if left untreated, with only 30% persisting and 10% progressing to high-grade lesions.  Infection with high-risk HPV types gives rise to lesions that are precursors of invasive squamous cell carcinoma, and various terminologies have been used to in this respect: Moderate/severe dysplasia, carcinoma in situ (CIS) or high-grade squamous lesion.
Since the terminology of squamous lesions in the cervix and other lower anogenital tract sites differ among pathologists and other medical subspecialties, a consensus process was convened sponsored by the College of American Pathologists and the American Society for Colposcopy and Cervical Pathology and, as a result, the lower anogenital squamous terminology (LAST) was published and recommendations were implemented in January 2013. ,
Reporting of cytologic abnormalities in cervical samples also suffered from terminology problems before the implementation of The Bethesda System (TBS),  which resulted in the creation of a standard reporting system in cytology and that now correlates with the LAST recommendations in histologic samples.
| Historical development of terminology in histologic cervical preinvasive lesions|| |
In the early 1900's the term CIS was described in the cervix;  lesion in which atypical cells occupy the whole thickness of the cervical epithelium with no invasion into underlying stroma. It was later understood that there were other atypical lesions that did not fulfill the criteria for CIS or progressed to invasive cancer as CIS did. The term "atypical hyperplasia" of the cervix appeared in 1952 and subsequently, in 1953, the term dysplasia was introduced by Reagan et al. and graded as mild, moderate and severe. , Until then there had been no mention of HPV infection and the relationship with cervical lesions. It was not until the late 1900's when atypia related with the squamous cervical epithelium (enlarged, hyperchromatic irregular cells with a halo) was linked to infection with HPV. , In 1969, cervical intraepithelial neoplasia (CIN) terminology was introduced by Richart.  He recognized CIN as a spectrum of disease: From mild dysplasia (CIN1), to moderate dysplasia (CIN2) evolving to severe dysplasia (CIN3). During this time, because the biology of HPV was not understood, all patients were radically treated regardless of grade of dysplasia with cryotherapy and CO 2 laser ablation resulting in over-treatment of patients.  Subsequently, the inter-observer variability in reporting cervical biopsies with the three-tier grading system was recognized,  leading to the acceptance that a two-tier grading system with low and high-grade intraepithelial lesions, was more reproducible than the three-tier grading system. In addition, the biology of HPV and oncogenesis in cervical epithelium was understood as well. However, the two-tiered grading system in histological sections was never consistently adopted due to the lack of support. In parallel to this development, changes in cytology reporting was developing resulting in TBS supporting the low and high-grade reporting terminology in cytology samples in 1988. 
In the 1990's, there was a clear difference in the management of patients with CIN1 versus those with CIN2/CIN3. The natural history of lesions in the first group of patients is to regress, so they were followed. The second group of patients (CIN2/CIN3) started being treated with loop electrosurgical excision procedure.
In 2013, the recommendations of the LAST consensus conference emerged with terminology for HPV associated squamous lesions of the cervix and other lower anogenital tract sites as "low-grade squamous intraepithelial lesion" (LSIL) and "high-grade squamous intraepithelial lesion" (HSIL), which can be further classified by the applicable - IN sub-categorization in parenthesis, e.g., LSIL (CIN1) or HSIL (CIN2). , This subcategorization is particularly useful for clinicians who are willing to follow a young patient with a HSIL/CIN2 diagnosis instead of actively treating an HSIL/CIN3 diagnosis.
Regarding early invasive squamous cell carcinoma of the cervix various terminologies and definitions have emerged during the last several years. Depth of invasion, largest horizontal tumor extension and in some cases presence or absence of lymphovascular invasion (LVI), have all been used for the definition of this entity. Historically terminologies such as microinvasive carcinoma, minimally invasive carcinoma and early invasive carcinoma have been used. The LAST consensus recommends the use of superficially invasive squamous cell carcinoma (SISCCA) (see below).
| Squamous Intraepithelial Lesions (Low-Grade Squamous Intraepithelial Lesion/High-Grade Squamous Intraepithelial Lesion )|| |
Cervical intraepithelial neoplasia is classified on a three-tiered grading system (CIN1, 2 and 3) based on the level of immature neoplastic basaloid cells involving the epithelium, cytologic atypia and mitotic activity: LSIL/CIN1 and HSIL/CIN2/3. Knowledge of the HPV biology does not support the three-tiered grading system of mild, moderate, severe dysplasia/CIS, but rather a two-tier system where low-grade lesions are understood to be transient, whereas high-grade lesions may persist and progress to cancer. Studies have shown great variability in grading cervical lesions among pathologists.  However, variability between observers has been shown to improve with the two-tier grading system. ,, In addition, ancillary immunohistochemical stains including p16 and Ki-67 have shown to be useful in decreasing the variability. ,,
| Low-Rade Squamous Intraepithelial Lesion (Low-Grade Squamous Intraepithelial Lesion/Cervical Intraepithelial Neoplasia 1)|| |
Low-grade squamous intraepithelial lesion/CIN1 is characterized by a proliferation of immature basal/parabasal cells that extend no higher than one-third of the thickness of the epithelium [Figure 1]a. There is preserved maturation in the upper two-thirds of the epithelium, and mitotic figures are limited to the lower third. In addition, the surface cells may show nuclear enlargement and irregularity, multinucleation and perinuclear halos [Figure 1]b (HPV cytopathic changes or koilocytic effect).
|Figure 1: Low-grade squamous intraepithelial lesion (LSIL). (a and b) H and E LSIL (cervical intraepithelial neoplasia [CIN1]). (c) Focal-weak p16 staining. (d) Increased proliferati on acti vity above basal-layer with Ki-67. Condyloma acuminatum showing papillary appearance (e) and koilocytes (f). LSIL cytology, CIN1 (g) and koilocyte (h)|
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Condyloma acuminatum, although not very common in the cervix as in vulva and vagina, is a low-grade lesion with a warty and papillary appearance, both grossly and microscopically. The papillae are lined by atypical cells with enlarged hyperchromatic nuclei [Figure 1]e-f. Lesions usually show the hyperkeratosis, dyskeratosis and parakeratosis. The majority of these lesions are caused by HPV types 6 and 11. These lesions should be designated LSIL with the optional designation of condyloma acuminatum in parenthesis.
| High-Grade Squamous Intraepithelial Lesion (High-Grade Squamous Intraepithelial Lesion/Cervical Intraepithelial Neoplasia 2, 3)|| |
High-grade squamous intraepithelial lesion is characterized by increased proliferation of immature basal squamous cells that extend above the lower one-third but not beyond the upper third of epithelial thickness (CIN2) [Figure 2]a or involvement of greater than two-thirds of the epithelial thickness (CIN3) [Figure 2]b. Mitotic figures can be found in the middle third and/or superficial thirds of the epithelium. Since viral DNA has been integrated into the host DNA and is no longer a productive viral infection, typical viral cytopathic changes as in LSIL are not always encountered. In addition, there is a particular form of HSIL characterized by multinucleation (5 or more nuclei) and enlarged nuclei (>5 times the size of a parabasal cell) which must be recognized  [Figure 3]a. In this entity, and unlike classic HSIL, cells have abundant cytoplasm, and a diagnosis of LSIL may be entertained. If in doubt, p16 immunohistochemical positivity (see below) in this circumstance supports the diagnosis of HSIL [Figure 3]b.
|Figure 2: High-grade squamous intraepithelial lesion (HSIL). (a) H and E HSIL (cervical intraepithelial neoplasia [CIN2]). (b) H and E HSIL (CIN3). (c) Diffuse band-like p16 positivity. (d) Increased proliferati on activity with Ki-67; whole epithelial thickness. (e) Cytology HSIL. (f) Cytology atypical squamous cells cannot exclude HSIL|
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|Figure 3: High-grade squamous intraepithelial lesion. (a) Multinucleation and markedly enlarged nuclei. (b) Diffuse band-like p16 positivity|
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| Biomarkers (P16 And KI-67) and in Situ Hybridization|| |
There are two immunohistochemical stains that have proved to be the most useful to help distinguish between dysplasia and benign mimics: p16iNK4a and Ki-67/Mib-1. P16 INK4a is a tumor suppressor gene that encodes a protein involved in cell cycle regulation. It is a cyclin-dependent kinase 4 inhibitor which binds to p16 thereby disabling the phosphorylation of pRb and preventing cell cycle. When high-risk HPV DNA integrates into the host genome, viral oncoprotein E7 binds to pRb rendering it inactive.  As a result, p16 is over-expressed which manifests as diffuse, strong cytoplasmic and nuclear staining in lesions associated with high-risk HPV infection [Figure 2]c.  On the other hand LSIL, will have weak and patchy or negative p16 staining [Figure 1]c.
Mib-1 antibody targets Ki-67, cellular proliferation marker, detected during the active phases of the cell cycle. In normal squamous epithelium, Ki-67 labeling is confined to the parabasal layer.  Ki-67 can be used to detect increased proliferation activity related to HPV infection. LSIL will show a cluster of at least two strongly stained epithelial cell nuclei above the basal-layer [Figure 1]d. , HSIL will show increased Ki-67 labeling either up to 2/3 or whole epithelial thickness [Figure 2]d.
Because parabasal cells and inflammatory cells normally label with Ki-67, there are two pitfalls in the interpretation of Ki-67. One is the presence of intraepithelial inflammatory cells which can be present throughout the epithelium, mimicking SIL. Second, tangential sections through basal-layer of squamous epithelium can mimic diffuse Ki-67 labeling, mimicking HSIL.
Performing p16 immunostaining is currently recommended when the morphologic differential diagnosis is between HSIL and a mimic, , such as metaplasia, reactive change and atrophy. It is also recommended when there is professional disagreement in biopsy interpretation. Finally, LAST consensus recommends against using p16 as an adjunct to morphologic assessment in cases that are favored to be benign or LSIL, unless patient has a prior cytology sample read as HSIL, atypical squamous cells cannot exclude high-grade (ASC-H) or atypical glandular cell. [Table 1] shows the staining patterns of Ki-67 and p16 immunohistochemical stains in HSIL, LSIL, benign squamous atypia, atrophy, immature squamous metaplasia and tangentially sectioned squamous epithelium.
|Table 1: Staining patterns of p16 and Ki-67 immunohistochemical stains in different settings|
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In situ, hybridization is a direct assay that detects individual HPV DNA fragments within the infected squamous cells and can be used in formalin fixed paraffin embedded tissue. However, this method has a low sensitivity and specificity for HPV detection and, therefore, this test is not widely used for detecting high-grade lesions and invasive carcinomas.
| Superficially Invasive Squamous Cell Carcinoma|| |
Cervical carcinomas that are minimally or focally invasive are recommended to be referred to as "SISCCA". , It is defined as a lesion fulfilling all the following criteria: Microscopic lesion that invades ≤3 mm (from the basement membrane of the point of origin), horizontal spread of ≤7 mm and has been completely excised. LVI and pattern of invasion were not included in the definition of the entity. However, it is advised that the pathology report includes the presence or absence of LVI in the biopsy specimen. If the size of the invasive component fulfills the criteria for SISCCA, but there is carcinoma at a resection margin, these cases can be referred as "at least SISCCA."
| Current Terminology for Reporting Results in Cervical Cytology (Bethesda 2001)|| |
The Bethesda System for reporting cervical/vaginal cytological diagnosis was originally developed in 1988 and was intended to create an uniform system for reporting cervical cytology reports.  In 1991, a second workshop was held, and TBS was modified and later implemented in 2001 as a modification.  The revised TBS reports have a standardized format: Statement of specimen adequacy, an optional general characterization and a descriptive diagnosis. The descriptive diagnosis includes benign, cellular changes (reactive changes and infections) and epithelial cell abnormalities (glandular and squamous). Similar to the LAST recommendations, cytological squamous abnormalities are divided into two categories: LSIL and HSIL. LSIL category includes HPV-associated cellular changes, mild dysplasia and CIN1. On the other hand, HSIL includes moderate/severe dysplasia, CIS and CIN2/CIN3. The criteria for each category is dependent on comparison of the size of the nuclei in question with the size of an intermediate cell nuclei.  Abnormalities with borderline nuclear change including ASCs of undetermined significance, ASCs cannot exclude HSIL (ASC-H), and atypical glandular cells of undetermined significance are recognized.
Low-grade squamous intraepithelial lesion is characterized by cells with nuclear enlargement (>3 times are of a normal intermediate cell nucleus), irregular nuclear membranes and chromatin abnormalities [Figure 1]g. Macrocytes, abnormal keratinization, binucleation or multinucleation and koilocytes [Figure 1]h are also part of LSIL.
High-grade squamous intraepithelial lesion is characterized by parabasal-sized cells with high nuclear to cytoplasmic rations, nuclear membrane irregularity and hyperchromasia [Figure 2]e.
Atypical squamous cells of undetermined significance is characterized by mature intermediate or superficial squamous cells with minimal nuclear enlargement (2-3 times normal intermediate nucleus). It is a category intended for those abnormalities that are more marked than those attributable to reactive changes, but fall short for LSIL diagnosis. ASC-H is a category used when there are cells that are suggestive of HSIL but lack criteria for definitive interpretation (immature metaplastic cells with minimal nuclear enlargement) [Figure 2]f. This category reflects a mixture of true HSIL and mimics and is thought to include approximately 5-10% of ASC cases. 
| Conclusion|| |
Recommendations of terminology used in reporting squamous lesions in the cervix and other lower anogenital sites, were implemented in January 2013. , Only two categories are recommended for noninvasive squamous proliferations: LSIL- and HSIL. If the pathologist and/or the clinician desires, further characterization of the lesions with the CIN subcategorization in parenthesis is recommended. This novel implementation will improve communication between clinicians and pathologists as well as adopting and being parallel to the two-tier system of Bethesda reporting in cytology samples.
Immunohistochemistry for p16 and Ki-67 can be used when the differential diagnosis is HSIL and a mimic of lesion, like metaplasia, atrophy and reparative changes.
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Maria Carolina Reyes
Department of Pathology and Laboratory Medicine, University of Pennsylvania, 3400 Spruce Street, 6 Founders, Philadelphia, PA
Source of Support: None, Conflict of Interest: None
[Figure 1], [Figure 2], [Figure 3]