Evaluation of a new p16^INK4A ELISA test and a high‐risk HPV DNA test for cervical cancer screening: Results from proof‐of‐concept study

Abstract

p16 INK4a , a cell cycle regulation protein, accumulates in abnormal epithelial cells infected with high-risk human papilloma virus (HPV). In immunostaining studies, p16 INK4a has shown potential as a marker of high grade cervical intraepithelial neoplasia (CIN) and invasive cervical cancer. To evaluate its potential use in cervical cancer screening, we conducted a feasibility study to compare the performance of a new enzyme linked immunosorbant assay (ELISA) for p16 INK4a (mtm laboratories, Heidelberg, Germany) to that of the Hybrid capture 2 TM (hc2) test for high-risk HPV DNA for the detection of CIN3. Three hundred and nineteen women were referred from Western Washington Planned Parenthood clinics for colposcopy examination and cervical biopsy because of abnormal Pap test results. Cervical samples were obtained from study participants for p16 INK4a ELISA, liquidbased cytology and hc2. The order (first and second) for obtaining samples for cervical cytology and p16 INK4a ELISA changed with every other subject. Concentrations of p16 INK4a protein were higher when the sample was taken before the cytology. The sensitivity of p16 INK4a ELISA (concentration 8 units/ml) taken as first sample was 90.0% for CIN3, and the sensitivity of HC2 taken as a second sample was 85%. In the same group, the specificity of p16 INK4a ELISA (46.9%) was slightly better than hc2 (35.4%) Results from this proof-of-concept study suggest that p16 INK4a ELISA has a similar sensitivity and slightly better specificity for CIN3 compared to hc2. These findings support proceeding with a larger study with samples from a population of women presenting for routine cytology screening. ' 2007 Wiley-Liss, Inc.Key words: p16 INK4a ; ELISA; CIN; screening; HPV The Pap smear has been an effective screening tool for cervical cancer, but it is limited with respect to its sensitivity, cost and requirement of significant infrastructure and technology to be effective. Human papillomavirus (HPV) testing has been proposed as a possible screening tool in resource poor settings where Pap screening is not feasible; however testing for high-risk HPV deoxyribonucleic acid (DNA) is limited by its poor specificity. 1 The search for specific biomarkers of HPV infected precancerous cells has yielded a number of possible markers of disease, including p16 INK4a .p16 INK4a is a protein involved in cell cycle regulation and accumulates in abnormal epithelial cells infected with high-risk HPV. p16 INK4a is a cyclin dependent kinase (CDK) inhibitor that regulates the activity of CDK4 and CDK6. It is inactivated in many cancers by genetic deletion or hypermethylation. 2 In non-HPV associated tumors, this inactivation leads to increased CDK activity and inactivation of the retinoblastoma protein (pRb), resulting in disruption of the cell cycle and increased cell proliferation. However, in HPV-associated tumors, inactivation of pRb by highrisk HPV oncoprotein E7 leads to cell proliferation and markedly increased levels of p16 INK4a . Staining of histology and cytology ...

Evaluation of an ELISA for p16INK4a as a Screening Test for Cervical Cancer

“…In the proof-of-principle study that we conducted among 319 women with abnormal cytology results, we found that, when the sample for p16 INK4a ELISA was collected before any other cervical sample, as was done in the present screening study, 90% of ≥CIN3, 77% with CIN2, and 53% with <CIN2 tested positive at the ≥8 pg/mL cut-point (21). In a similar proof-of-principle study conducted in Germany among women with abnormal screening cytology results, 97% of those with ≥CIN3, 96% with CIN2, and 55% with <CIN2 tested positive for p16 INK4a ELISA using the ≥8 pg/mL cut-point (22).…”

“…The literature provides less consistent information on the influence of sampling order on performance of Pap and HPV tests. Previous work by our group (21) indicated that the p16…”

“…The ELISA test used in this study was a prototypic assay developed at mtm laboratories as described previously (21). Based on results from pilot testing of this assay (21), a value of ≥8 pg/mL was considered to be positive.…”

Cervical Cancer Control in the Era of HPV Vaccination and Novel Biomarkers

“…p16 cytology has been successfully used as a triage marker for ASCUS and LSIL cytology results [84,85] . Initial data on an ELISA-based p16 test suggest that it could be used as a simple test to evaluate the risk of cervical precancer [86,87] .…”

Immunomarkers in Gynecologic Cytology: The Search for the Ideal ‘Biomolecular Papanicolaou Test’

“…Two tested the performance of an ELISA for p16 INK4A (mtm laboratories, Heidelberg, Germany), the first being a proof-of-concept study with 319 cases [27], and the second a larger study [28] on 1,781 women by the same research group. This second study used an enhanced version of the p16 INK4A ELISA and showed promise in screening when combined with HC2, with comparable SE (91.8%) and higher SP (86.0%) compared to p16 INK4A ICC (table 1).…”