ZNF671 Methylation as a Potential Biomarker to Identify Women with Cervical Intraepithelial Neoplasms Type 3 and Worse

Background: Cervical screening aims to identify women with cervical intraepithelial neoplasia 3 (CIN3) and squamous cell carcinoma (SCC), termed as CIN3+. Identification of women with CIN3+ ensure that they can receive optimal treatment and prevent overtreatment. Studies have suggested that the higher degree of cervical lesions, the more accumulation of DNA methylation (SCC > CIN3 > CIN2 > CIN1). DNA methylation analysis as a triage for women in cervical scrapings is feasible, but finding novel biomarkers with high sensitivity and high specificity is still a challenge. Zinc finger protein 671 (ZNF671), a member of the KRAB-ZFP family of mammalian transcriptional repressors, was hypermethylation of its promoter and downregulated in SCC. Methods: We investigated methylation of PAX1 or ZNF671 and genotyping of high-risk human papillomavirus (hrHPV) for detection of CIN3+ in 1006 women underwent Thinprep Cytologic test (TCT) and biopsy under colposcopy. Real-time methylation-specific PCR (qMSP) was used to detect the level of PAX1m and ZNF671m. Bisulfite pyrosequencing, the gold standard for detecting DNA methylation, was also used to measure the level of ZNF671m in 532 subjects. Findings: Sensitivities and specificities for CIN3+ were respectively, 75.69% and 77.54% for PAX1m, 79.52% and 85.76% for ZNF671m. High sensitivity (89.39%) but low specificity (60.63%) was observed for hrHPV. The sensitivities and specificities of PAX1m or hrHPV were 90.28% and 59.09%, of ZNF671m or hrHPV were 93.13% and 63.19%, of PAX1m or ZNF671m were 77.44% and 75.93%, respectively. The sensitivities and specificities of PAX1m(Algorithm 1) or ZNF671m (Algorithm 2) combined with hrHPV for CIN3+ were 98.61% and 51.57%, 76.77% and 88.42%, respectively. Interpretation: Compared with PAX1m, the level of ZNF671m could separate women more accurately into high risk groups either combined hrHPV genotyping or analysis alone. ZNF671mmay serve as a potential biomarker to identify women with CIN3+. And we propose that ZNF671m might be applied as a triage test alone or combined hrHPV genotyping and contribute to the globalization of cervical cancer screening. Funding Statement: This research was supported by Chinese National Major Project for New Drug Innovation (2019ZX09201-002-006), the National Natural Science Foundation (NNSF) of China (81973401) and the Natural Science Foundation for Young Scientist of Hunan Province, China (2018JJ3829). Declaration of Interests: The authors declare that they have no competing interests. Ethics Approval Statement: This study was undertaken in accordance with the Declaration of Helsinki, and the protocol was approved by the Ethical Committee of Xiangya Hospital of Central South University, China. All patients gave written informed consent before participation in this study.