CAP TODAY Pathology/Laboratory Medicine/Laboratory Management
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Q. What is the recommended use of p16 immunostaining as an adjunct diagnostic biomarker in HPV-associated lesions of the lower anogenital tract?
A. Recommendations for the use of p16 were published recently by the LAST Project, cosponsored by the CAP and the American Society for Colposcopy and Cervical Pathology (ASCCP).1 The LAST Project recommended standardized terminology for all lower anogenital tract biopsies with noninvasive squamous pathology to follow the Bethesda System abbreviations of LSIL (low-grade squamous intraepithelial lesion) and HSIL (high-grade squamous intraepithelial lesion) in place of previously used mild-moderate-severe dysplasia or intraepithelial neoplasia grade 1, 2, or 3 (–IN 1–3). The lower anogenital tract (LAT) includes cervix, vagina, vulva, anus, perianal area, penis, and scrotum. The LAST Project also recognized p16 as a biomarker for E6/E7 oncogene activation in all HPV-related precancerous squamous lesions of the LAT. Overexpression of p16, as detected by immunohistochemistry, serves as a surrogate marker for cell-cycle dysregulation, a key step in HPV-mediated carcinogenesis.1,2 Additionally, Work Group 4 of the project issued specific recommendations for p16 to be used as an adjunct to standard morphology. When combined with the two-tiered grading system, this approach significantly improves diagnostic accuracy of the pathologic diagnosis of precancer1,3,4 and optimizes therapeutic management of patients for better clinical outcomes.1,5The summary of the LAST Project consensus recommendations, including p16 biomarker recommendations, is available at the CAP Web site: www.cap.org/apps/docs/membership/transformation/new/asccp_sum_last_recom.pdf.
Briefly, p16 staining is recommended whenever there is:
- Differential diagnosis between precancer (HSIL) and precancer mimics.
- Disagreement in interpretation of precancer.
- High risk for missing precancer (high-risk cytology with negative/LSIL biopsy findings).
- H&E morphologic pattern of –IN 2. This recommendation has proved to reduce the equivocal and poorly reproducible –IN 2 diagnostic category.3,4 Positive p16 staining supports classifying –IN 2 as definitive HSIL.
- Staining for p16 is not recommended for biopsies that are negative or show unequivocal LSIL or HSIL (–IN 3).
Positive p16 staining is defined as strong and diffuse (continuous nuclear or nuclear and cytoplasmic) staining of the basal cell layer that involves at least the lower third of the epithelial thickness with or without full-thickness extension.1-4 p16 should be used in conjunction with standard morphologic diagnosis and not as a replacement. Other biomarkers of viral transformation (for example, ProEx C, Ki-67) may be helpful in especially challenging cases, but they add no additional value to p16 staining.1
Application of p16 immunocytochemistry in triage of squamous atypia in cervical cytology remains controversial. Procedural and interpretation differences as well as a lack of standardized protocols make the analysis challenging.6,7 Recent meta-analysis from 17 studies in which p16 was used to predict HSIL (CIN 2+) showed higher specificities in ASC-US and LSIL groups with loss of sensitivity in LSIL category when compared with HPV testing.7 However, no consensus exists for cytologic application of p16 in standard clinical practice, pending further studies.
The use of p16 immunohistochemical staining when combined with other markers may help to distinguish endocervical from endometrial adenocarcinoma, though serous carcinomas can also be positive.8-10 Strong diffuse p16 staining is usually observed in HPV-associated preinvasive lesions of the endocervix (endocervical dysplasia and adenocarcinoma in situ) but not in benign mimics (tubal and squamous metaplasia), which may show focal or patchy staining.10
A p16 immunostain should always be interpreted in conjunction with morphologic findings, using previously validated antibodies and methods.
References
1. Darragh TM, Colgan TJ, Cox JT, et al. The Lower Anogenital Squamous Terminology Standardization Project for HPV-associated lesions: background and consensus recommendations from the College of American Pathologists and the American Society for Colposcopy and Cervical Pathology. Arch Pathol Lab Med. 2012;136(10):1266–1297.
2. Wentzensen N, von Knebel Doeberitz M. Biomarkers in cervical cancer screening. Dis Markers. 2007;23(4): 315–330.
3. Galgano MT, Castle PE, Atkins KA, et al. Using biomarkers as objective standards in the diagnosis of cervical biopsies. Am J Surg Pathol. 2010;34(8):1077–1087.
4. Bergeron C, Ordi J, Schmidt D, et al. Conjunctive p16INK4a testing significantly increases accuracy in diagnosing high-grade cervical intraepithelial neoplasia. Am J Clin Pathol. 2010;133(3):395–406.
5. Waxman AG, Chelmow D, Darragh TM, et al. Revised terminology for cervical histopathology and its implications for management of high-grade squamous intraepithelial lesions of the cervix. Obstet Gynecol. 2012;120(6):1465–1471.
6. Denton KJ, Bergeron C, Klement P, et al. The sensitivity and specificity of p16(INK4a) cytology vs HPV testing for detecting high-grade cervical disease in the triage of ASC-US and LSIL pap cytology results. Am J Clin Pathol. 2010;134(1):12–21.
7. Roelens J, Reuschenbach M, von Knebel Doeberitz M, et al. p16INK4a immunocytochemistry versus human papillomavirus testing for triage of women with minor cytologic abnormalities: a systematic review and meta-analysis. Cancer Cytopathol. 2012;120(5):294–307.
8. Kong CS, Beck AH, Longacre TA. A panel of 3 markers including p16, ProExC, or HPV ISH is optimal for distinguishing between primary endometrial and endocervical adenocarcinomas. Am J Surg Pathol. 2010;34(7):915–926.
9. Jones MW, Onisko A, Dabbs DJ, et al. Immunohistochemistry and HPV in situ hybridization in pathologic distinction between endocervical and endometrial adenocarcinoma: a comparative tissue microarray study of 76 tumors. Int J Gynecol Cancer. 2013;23(2):380–384.
10. Park KJ, Soslow RA. Current concepts in cervical pathology. Arch Pathol Lab Med. 2009;133(5):729–738.
Krzysztof Moroz, MD
Director, Cytology Laboratory
Tulane University Health Sciences Center
New Orleans
Member, CAP
Immunohistochemistry Committee