BACKGROUND Human papillomavirus (HPV)-based cervical cancer screening requires triage markers to decide who should be referred to colposcopy. p16/Ki-67 dual stain cytology has been proposed as a biomarker for cervical precancers. We evaluated the dual stain in a large population of HPV-positive women. METHODS One thousand five hundred and nine HPV-positive women screened with HPV/cytology cotesting at Kaiser Permanente California were enrolled into a prospective observational study in 2012. Dual stain cytology was performed on residual Surepath material, and slides were evaluated for dual stain-positive cells. Disease endpoints were ascertained from the clinical database at KPNC. We evaluated the clinical performance of the assay among all HPV-positive women and among HPV-positive, cytology-negative women. We used internal benchmarks for clinical management to evaluate the clinical relevance of the dual stain assay. We evaluated sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of the dual stain compared with Pap cytology. All statistical tests were two-sided. RESULTS The dual stain had lower positivity (45.9%) compared with cytology at an ASC-US threshold (53.4%). For detection of CIN2+, the dual stain had similar sensitivity (83.4% vs 76.6%, P = .1), and statistically higher specificity (58.9% vs 49.6%, P < .001), PPV (21.0% vs 16.6%, P < .001), and NPV (96.4% vs 94.2%, P = .01) compared with cytology. Similar patterns were observed for CIN3+. Women with a positive test had high enough risk for referral to colposcopy, while the risk for women with negative tests was below a one-year return threshold based on current US management guidelines. CONCLUSION Dual stain cytology showed good risk stratification for all HPV-positive women and for HPV-positive women with normal cytology. Additional follow-up is needed to determine how long dual stain negative women remain at low risk of precancer.

Abstract Background: Human papillomavirus (HPV) testing has been approved as a primary strategy for cervical cancer screening, either alone or in combination with cytology (co-testing), based on its high sensitivity and long-term reassurance against cervical precancer following a negative test result. However, nearly twice as many women will screen positive for HPV compared with cytology-based screening. Thus, effective management of HPV-positive women requires triage markers to distinguish those at high-risk who should be referred to colposcopy from those with benign infections who can safely return to routine screening. p16/Ki-67 dual stain cytology has previously shown good risk stratification for triage of HPV-positive women; however, studies with follow-up extending beyond 3 years are lacking. We evaluated the long-term risk prediction of p16/Ki-67 for detection of cervical precancer (cervical intraepithelial neoplasia grade 3 or worse, CIN3+) in a large population of HPV-positive women. Methods: 1,588 HPV-positive women screened with HPV/cytology co-testing were enrolled in 2012 at Kaiser Permanente Northern California. p16/Ki-67 cytology was performed on residual Surepath material and slides were evaluated for p16/Ki-67 positivity. Cervical histology endpoints were ascertained from the clinical database with follow-up through 2017. We conducted a Kaplan Meier analysis to estimate risk of CIN3+ by p16/Ki-67 and cytology (atypical squamous cells of undetermined significance or worse, ASC-US+, versus normal cytology). Risks were compared to internal benchmarks for colposcopy referral and for a one year return interval. Results: In women testing p16/Ki-67 positive at baseline, the 2-year risk of CIN3+ was 14.3%, compared with 2.2% in p16/Ki-67-negative women. For ASC-US+, the risk was 12.6% and 2.9% for normal cytology. The 5-year risk of CIN3+ in p16/Ki-67-positive women was 21.6% and 5.0% in p16/Ki-67-negatives. The 5-year risk of ASC-US+ was 17.1% compared to 8.2% for normal cytology. Among p16/Ki-67-negatives, the risk remained below the colposcopy referral threshold for 5 years while in women with normal cytology, the colposcopy referral threshold was crossed after year 3. Conclusion: In the first study evaluating long-term risk stratification of p16/Ki-67 dual staining, p16/Ki-67- negativity provided strong reassurance against CIN3+ for at least five years. In contrast, the risk in women with normal cytology crossed the colposcopy referral threshold after three years. These data support use of p16/Ki-67 for triage of HPV-positive women with the possibility of extending surveillance intervals in p16/Ki-67-negative women. Citation Format: Megan A. Clarke, Barbara Fetterman, Mark Schiffman, Philip E. Castle, Eric Stiemerling, Diane Tokugawa, Nancy Poitras, Walter Kinney, Thomas Lorey, Nicolas Wentzensen. Long term risk prediction of p16/Ki-67 dual stain in triage of HPV-positive women [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2202.

BACKGROUNDDual‐stain cytology for p16 and Ki‐67 has been proposed as a biomarker in cervical cancer screening. The authors evaluated the reproducibility and accuracy of dual‐stain cytology among 10 newly trained evaluators.METHODSIn total, 480 p16/Ki‐67–stained slides from human papillomavirus‐positive women were evaluated in masked fashion by 10 evaluators. None of the evaluators had previous experience with p16 or p16/Ki‐67 cytology. All participants underwent p16/Ki‐67 training and subsequent proficiency testing. Reproducibility of dual‐stain cytology was measured using the percentage agreement, individual and aggregate κ values, as well as McNemar statistics. Clinical performance for the detection of cervical intraepithelial neoplasia grade 2 or greater (CIN2+) was evaluated for each individual evaluator and for all evaluators combined compared with the reference evaluation by a cytotechnologist who had extensive experience with dual‐stain cytology.RESULTSThe percentage agreement of individual evaluators with the reference evaluation ranged from 83% to 91%, and the κ values ranged from 0.65 to 0.81. The combined κ value was 0.71 for all evaluators and 0.73 for cytotechnologists. The average sensitivity and specificity for the detection of CIN2+ among novice evaluators was 82% and 64%, respectively; whereas the reference evaluation had 84% sensitivity and 63% specificity, respectively. Agreement on dual‐stain positivity increased with greater numbers of p16/Ki‐67–positive cells on the slides.CONCLUSIONSGood to excellent reproducibility of p16/Ki‐67 dual‐stain cytology was observed with almost identical clinical performance of novice evaluators compared with reference evaluations. The current findings suggest that p16/Ki‐67 dual‐stain evaluation can be implemented in routine cytology practice with limited training. Cancer (Cancer Cytopathol) 2014;122:914–920. © 2014 American Cancer Society.