p16 flow cytometry of exfoliated cervical cells: Its role in quantitative pathology and clinical diagnosis of squamous intraepithelial lesions.

Background: P16 is a surrogate signature compensating for the specificity and/or sensitivity deficiencies of the human papillomavirus (HPV) DNA and Papanicolaou smear (Pap) co-test for detecting high-grade cervical squamous intraepithelial lesions or worse (HSIL+). However, traditional p16INK4A immunostaining is labour intensive and skill demanding, and subjective biases cannot be avoided. Herein, we created a high-throughput, quantitative diagnostic device, p16INK4A flow cytometry (FCM) and assessed its performances in cervical cancer screening and prevention.

Methods: P16 FCM was built upon a novel antibody clone and a series of positive and negative (p16 -knockout) standards. Since 2018, 24 100-women (HPV-positive/-negative, Pap-normal/-abnormal) have been enrolled nationwide for two-tier validation work. In cross-sectional studies, age- and viral genotype-dependent expression of p16 was investigated, and optimal diagnostic parameter cut-offs (using colposcopy and biopsy as a gold standard) were obtained. In cohort studies, the 2-year prognostic values of p16 were investigated with other risk factors by multivariate regression analyses in three cervicopathological conditions: HPV-positive Pap-normal, Pap-abnormal biopsy-negative and biopsy-confirmed LSIL.

Results: P16 FCM detected a minimal ratio of 0.01% positive cells. The p16 -positive ratio was 13.9 ± 1.8% among HPV-negative NILM women and peaked at the ages of 40-49 years; after HPV infection, the ratio increased to 15.1 ± 1.6%, varying with the carcinogenesis of the viral genotype. Further increments were found in women with neoplastic lesions (HPV-negative: 17.7 ± 5.0-21.4 ± 7.2%; HPV-positive: 18.0 ± 5.2-20.0 ± 9.9%). Extremely low expression of p16 was observed in women with HSILs. As the HPV-combined double-cut-off-ratio criterion was adopted, a Youden's index of 0.78 was obtained, which was significantly higher than that (0.72) of the HPV and Pap co-test. The p16 -abnormal situation was an independent HSIL+ risk factor for 2-year outcomes in all three cervicopathological conditions investigated (hazard ratios: 4.3-7.2).

Conclusions: FCM-based p16 quantification offers a better choice for conveniently and precisely monitoring the occurrence of HSIL+ and directing risk-stratification-based interventions.