Novel triple-target panels utilizing methylation-sensitive restriction enzyme-based quantitative PCR for detecting advanced cervical precancers and cancers among high-risk HPV-positive women
Optimal triage option for high-risk HPV-positive (hrHPV) women remains uncertain. We aimed to utilize methylation-sensitive restriction enzyme-based quantitative PCR (MSRE-qPCR) technique and develop triple-target human gene methylation panels to improve detection of advanced cervical precancers and cancers (CIN3 +) among hrHPV women. Eighteen candidate genes were detected by MSRE-qPCR in cervical samples from hrHPV women. All possible triple-target panels from these genes were generated by logistic regression models with repeated ten-fold cross-validation on a training set of 1223 women (180 CIN3 +; 1043 < CIN3). Panels with the top two AUCs for CIN3 + on a validation set of 937 women (69 CIN3 +; 868 < CIN3) were ultimately selected for qPCR reconstruction, retesting, and retraining. Triage performance, screening efficiency and risk stratification of the selected panels were then compared with traditional triage strategies (cytology [ASCUS as the threshold, atypical squamous cells of undetermined significance], HPV16/18 genotyping, HPV16/18 genotyping combined with cytology [ASCUS]) within the validation set. Two panels were finally identified and validated for CIN3 + detection. Panel 1 includes JAM3, PCDHGB7, and SORCS1; while Panel 2 consists of PAX1, ZNF671, and ASCL1. Compared to traditional triage strategies, both panels demonstrated superior AUCs (Panel 1: 0.799; Panel 2: 0.790; Cytology: 0.532; HPV16/18 genotyping: 0.589; HPV16/18 + Cytology: 0.515; all P Two triple-target human gene methylation panels were successfully developed, each integrated into a single MSRE-qPCR system for one-tube detection. Both panels outperformed current triage strategies, indicating their potential as alternatives, though external validation among diverse settings is needed before clinical application.