DNA methylation triage of human papillomavirus-positive atypical squamous cells of undetermined significance in cervical cancer screening

Triage of human papillomavirus (HPV)-positive women with atypical squamous cells of undetermined significance (ASC-US) remains a longstanding clinical challenge in cervical cancer screening, as conventional strategies often lead to excessive colposcopy referrals. We conducted a diagnostic cohort study of 195 HPV-positive women who underwent ASC-US cytology. Multigene DNA methylation analysis was performed on residual cervical exfoliated cells, using histology from colposcopy-directed biopsy and/or endocervical sampling as the reference standard to minimise colposcopy referrals. Our results showed that methylation levels of PAX1, EPB41L3, and FAM19A4 increased progressively from ≤ CIN1 to CIN2, CIN3, and cervical cancer, with significant decreases in ΔCt values from ≤ CIN1 to CIN3 for all three genes (p < 0.05). For detection of CIN2+, the assay achieved a sensitivity of 85.19%, specificity of 96.43%, positive predictive value (PPV) of 79.31%, negative predictive value (NPV) of 97.59%, and an area under the receiver operating characteristic curve (AUC) of 0.91, thereby avoiding colposcopy referrals in 96.43% (162/168). For CIN3+, the assay demonstrated sensitivity, specificity, PPV, NPV, and AUC values of 94.44%, 93.22%, 58.62%, 99.4%, and 0.94, respectively. Stratified analyses showed high and comparable sensitivity (81.82% vs. 87.50%), specificity (96.92% vs. 96.12%), and AUC (0.89 vs. 0.92) in women with HPV16/18 compared with those with non-16/18 high-risk HPV. Optimal diagnostic accuracy was observed in women aged ≥30 years, with sensitivities ranging from 85.70% to 100%, specificities from 94.44% to 100%, and AUC values ranging from 0.93 to 0.97. In contrast, the performance was suboptimal in women aged