Evaluation of p16INK4a immunostaining for the detection of high-grade changes in cervical cytology.

Since its introduction in Australia in 2007, the human papillomavirus (HPV) vaccine has led to a markedly lower prevalence of vaccine targeted HPV genotype infections as well as HPV disease including genital warts and histologically confirmed high-grade (HG) cervical abnormalities. To increase the ability to identify abnormal cells in lower prevalence, adjunct markers can be incorporated to improve the sensitivity and specificity of cytology test. One such marker is p16(p16), which is detectable in cells expressing the E7 oncogene encoded by high-risk HPVs (HR-HPV). In this study, the sensitivity and specificity of p16 immunostaining in detection of underlying HG lesions was evaluated in a cohort of 454 women undergoing surgical treatment for biopsy proven cervical dysplasia. Overall, p16 positive cells were detected in 321 (71%) of cytology preparations evaluated. Comparison of p16 staining on cytological preparations to histology diagnosis available on 212 patients, showed 26 (54%), 41 (78%) and 80 (90%) of cytology preparations to be p16 positive in women with CIN1, CIN2 and CIN3, respectively (p < 0.005). HPV16 and 18 were the most prevalent genotypes in HG lesions and were highly correlated with p16 staining. p16 staining provides an additional marker which can assist in better detecting underlying HG lesion in cytology smears with low disease prevalence.

Evaluation of cervical cone biopsies for coexpression of p16INK4a and Ki-67 in epithelial cells.

Diffuse overexpression of p16(INK4a) in basal and parabasal cells of cervical epithelium is a hallmark of human papillomavirus-mediated transformation. Focal p16(INK4a) expression is occasionally observed in nondysplastic epithelium. In normal cells, expression of p16(INK4a) triggers cell cycle arrest. However, cells undergoing transformation in intraepithelial lesions actively proliferate. To prove that the different expression patterns of p16(INK4a) , i.e., focal versus diffuse, reflect biologically different entities, we hypothesized that p16(INK4a) -positive cells in epithelia displaying focal p16(INK4a) expression pattern do not coexpress proliferation-associated Ki-67 protein, while p16(INK4a) -positive cells in lesions with diffuse p16(INK4a) expression may do. A total of 138 cervical cone biopsies were stained for the expression of p16(INK4a) and Ki-67 using a primary antibody cocktail. All metaplastic lesions (n = 21) displayed focal staining for p16(INK4a) , and in all of these lesions p16(INK4a) -positive cells were found to be negative for Ki-67 expression. Diffuse expression of p16(INK4a) was observed in 12/21 (57.1%) cervical intraepithelial neoplasia (CIN) 1 lesions, all of them simultaneously showed Ki-67 immunoreactivity in a large proportion of p16(INK4a) -positive cells. Seventeen of 23 (73.9%) CIN2 lesions and all 27 (100%) CIN3/carcinoma in situ (CIS) as well as all 46 (100%) carcinoma cases displayed diffuse and combined expression of p16(INK4a) and Ki-67. Coexpression of Ki-67 and p16(INK4a) in the same cell is entirely restricted to cervical lesions displaying diffuse p16(INK4a) expression, whereas in lesions with focal p16(INK4a) expression, p16(INK4a) -expressing cells are negative for Ki-67. Thus, diffuse expression of p16(INK4a) reflects lesions with proliferation-competent cells, while p16(INK4a) -expressing cells associated with focal expression patterns are cell cycle arrested.

Performance of p16INK4a-cytology, HPV mRNA, and HPV DNA testing to identify high grade cervical dysplasia in women with abnormal screening results.

OBJECTIVE: The prognostic value of dysplastic lesions of the uterine cervix cannot be adequately determined by Pap cytology alone. Detection of HPV DNA increases the diagnostic sensitivity. However, due to the very high prevalence of transient HPV infections, HPV DNA testing suffers from poor diagnostic specificity. Biomarkers that highlight the shift from self limited transient to potentially dangerous transforming HPV infections may improve the accuracy of cervical cancer screening. We evaluated HPV E6/E7 mRNA detection (APTIMA), p16(INK4a)-immunocytology (CINtec), and HPV DNA testing (HC2) to identify women with high grade cervical neoplasia in a disease-enriched cross-sectional cohort. METHODS: Liquid based cytology specimens were collected from 275 patients. All assays were performed from these vials. Detection rates of each test were evaluated against conventional H&E based histopathology alone and stratified by p16(INK4a)-immunohistochemistry (IHC). RESULTS: All assays yielded a high sensitivity for the detection of CIN3+ (96.4% (95% CI, 90.4-98.8) for HC2, 95.5% (89.2-98.3) for APTIMA and CINtec) and CIN2+ (91.5% (85.8-95.1) for HC2, 88.4% (82.3-92.7) for APTIMA, 86.6% (80.2-91.2) for CINtec). The specificity to detect high grade dysplasia was highest for CINtec p16(INK4a)-cytology (60.6% (52.7-68.0) in CIN3+ and 74.8% (65.5-82.3) in CIN2+), followed by APTIMA (56.4% (48.4-64.0) in CIN3+ and 71.2% (61.7-79.2) in CIN2+) and HC2 (49.1% (41.3-56.9) in CIN3+ and 63.4% (53.7-72.1) in CIN2+). All tests had higher sensitivity using p16(INK4a)-IHC-positive CIN2+ lesions as endpoint. CONCLUSIONS: Biomarkers that detect HPV induced dysplastic changes in the transforming stage are promising tools to overcome the current limitations of cervical cancer screening.

Validation of p16INK4a as a marker of oncogenic human papillomavirus infection in cervical biopsies from a population-based cohort in Costa Rica.

Due to the high prevalence of cancer-associated types of human papillomavirus (HPV) and the poorly reproducible histologic classification of low-grade lesions, identifying infected women at highest risk for cancer prior to neoplastic progression remains a challenge. We therefore explored the utility of p16INK4a immunostaining as a potential diagnostic and prognostic biomarker for cervical neoplasia using paraffin-embedded tissue blocks (punch biopsies and loop electrosurgical excision procedures) obtained from women referred to colposcopy during the enrollment phase of the Guanacaste Project (1993 to 1994). All blocks from 292 women selected by HPV status (HPV negative, nononcogenic HPV positive, or oncogenic HPV positive) and representing the diagnostic spectrum of the population [normal to precancer: cervical intraepithelial neoplasia (CIN) 3] were immunostained for p16INK4a using the p16INK4a research kit based on the monoclonal antibody clone E6H4 (MTM Laboratories, Heidelberg, Germany). For CIN3, the sensitivity of diffuse p16INK4a immunostaining was 100% and the specificity was 95%. For CIN2, the sensitivity and specificity for diffuse staining were 81.1% and 95.4%, respectively. Generalized to the 10,000-woman cohort, this translated to positive predictive value and negative predictive value of 13.9% and 100% for CIN3, respectively, and 20.4% and 99.7% for CIN2 or CIN3, respectively. Of women with an initial diagnosis of less than CIN2 for whom follow-up data for up to 5 to 7 years were available, 44% with diffuse staining developed persistent infection (CIN2 or CIN3). Whereas our data support the diagnostic potential for p16INK4a, further prospective studies with detailed follow-up determining the prognostic capacity of this marker are needed.