Identification and validation of CNTNAP2/PAX1 hypermethylation as an epigenetic biomarker panel for detection of cervical cancer and precancerous lesions in hrHPV-positive women

BACKGROUND: Cervical cancer remains a significant global health burden, and effective triage strategies are urgently needed for high-risk human papillomavirus (hrHPV)-positive women in screening programs. This study aimed to identify and validate DNA methylation markers for detecting cervical precancerous lesions in hrHPV-positive women. METHODS: Reduced representation bisulfite sequencing (RRBS) was performed on a discovery cohort (n = 17) to screen for differentially methylated candidate genes. Quantitative methylation-specific PCR (qMSP) was used to evaluate the performance of selected markers in an independent cohort of 347 hrHPV-positive women stratified into training (n = 154) and validation (n = 193) sets. Diagnostic accuracy was assessed using receiver operating characteristic (ROC) curves, sensitivity, specificity, and area under the curve (AUC). RESULTS: Among the candidates identified, CNTNAP2 and PAX1 methylation levels increased progressively with cervical disease severity (both P < 0.0001). In the independent validation set, the combined CNTNAP2/PAX1 panel achieved an AUC of 0.904 (95% CI: 0.853-0.942) for CIN2 + detection and 0.932 (95% CI: 0.887-0.963) for CIN3 + detection. The panel yielded a sensitivity of 73.8% and specificity of 95.5% for CIN2+, and a sensitivity of 89.2% and specificity of 87.8% for CIN3+. In comparison, HPV16/18 genotyping showed sensitivities of 54.1% for CIN2 + and 56.8% for CIN3+, with specificities of 81.1% and 76.3%, respectively. Cytology (≥ ASCUS) had higher sensitivity (CIN2+: 93.1%; CIN3+: 91.2%) but substantially lower specificity (CIN2+: 16.9%; CIN3+: 14.9%); notably, cytology specificity in this cohort was likely underestimated due to verification bias. The CNTNAP2/PAX1 panel outperformed both comparator methods in overall diagnostic accuracy. CONCLUSIONS: The CNTNAP2/PAX1 methylation panel demonstrates robust diagnostic performance for detecting cervical precancerous lesions in hrHPV-positive women, supporting its potential utility as an objective and effective triage tool in hrHPV-based cervical cancer screening.