p16INK4A flow cytometry of exfoliated cervical cells: Its role in quantitative pathology and clinical diagnosis of squamous intraepithelial lesions

• Published in: Clinical and translational medicine Vol. 13; no. 3; pp. e1209 - n/a
• Main Authors: He, Yifeng, Shi, Jun, Zhao, Hui, Wang, Yuefei, Zhang, Chi, Han, Sai, He, Qizhi, Li, Xiaolan, Li, Shangji, Wang, Wenjing, Yi, Muhua, Hu, Xiaoling, Xing, Zhihua, Han, Hao, Gao, Yinshuang, Zhou, Qing, Lu, Linlin, Guo, Jianfen, Cao, Hui, Lu, Caiping, Hou, Yanqiang, Chen, Dan, Yang, Fengyun, Lei, Ping, Di, Wen, Qian, Ji, Xia, Yi, Zhang, Youzhong, Deng, Yang, Zhu, Jianlong, Xu, Congjian
• Format: Journal Article
• Language: English
• Published: Heidelberg John Wiley & Sons, Inc 01.03.2023; John Wiley and Sons Inc; Wiley
• Subjects: Age; Cervical cancer; Clinical medicine; Cloning; flow cytometry; Genotype & phenotype; Hospitals; Human papillomavirus; immunostaining; Infections; Informed consent; Leukemia; Maternal & child health; p16INK4A; papanicolaou smear; Pathology; Physiology; quantification; squamous intraepithelial lesion; Womens health
• ISSN: 2001-1326; 2001-1326
• DOI: 10.1002/ctm2.1209

Background P16INK4A 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 P16INK4A FCM was built upon a novel antibody clone and a series of positive and negative (p16INK4A‐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 p16INK4A 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 p16INK4A 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 P16INK4A FCM detected a minimal ratio of 0.01% positive cells. The p16INK4A‐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 p16INK4A 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 p16INK4A‐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 p16INK4A quantification offers a better choice for conveniently and precisely monitoring the occurrence of HSIL+ and directing risk‐stratification‐based interventions. • The quantification detection system—p16INK4A FCM was established for triaging cervical high‐grade neoplasia/cancer. • 24 100‐women cross‐sectional studies revealed that HSIL+‐triaging efficacy of HPV and p16INK4A FCM co‐test is significantly higher than that of HPV and Pap co‐test. • 2‐year cohort studies indicated that HPV‐positive/Pap‐abnormal p16INK4A‐abnormal women would run a HSIL+‐risk > 4‐fold higher than those with a p16INK4A‐normal level.