Aim: To evaluate the clinical performance of PAX1 methylation (PAX1m) and cytology for patients with non-HPV16/18 high-risk HPV (hrHPV) infection. Methods: Cervical exfoliated cells from 387 outpat... Tweetable abstract PAX1 methylation increased specificity and positive predictive value for cervical intraepithelial neoplasia (CIN) CIN2+/CIN3+ compared with cytology for women with non-HPV16/18 h...

Primary screening for high-risk human papillomavirus (hrHPV) with cytological triage for women with non-16/18 hrHPV-positive status has become popular in China. However, cytology relies on the subjective judgment of pathologists, leading to inconsistent clinical performance. A total of 657 hrHPV-positive women aged 25–64 years were enrolled in this cross-sectional study. All participants underwent colposcopic biopsy after cytology triage, with cytology residual specimens undergoing DNA methylation testing. CIN2+ and CIN3+ sensitivity and specificity were compared between the different triage strategies (n=487): PAX1 methylation (PAX1m) , Glycophorin C methylation (GYPCm), cytology, and combinations between them or with HPV16/18. The area under the receiver operating characteristic curves (AUCs) for PAX1m and GYPCm in detecting CIN2 or worse (CIN2+) were 0.867 (95% confidence interval [CI]: 0.796–0.937) and 0.873 (95% CI: 0.808–0.938), respectively. The sensitivities of PAX1m and GYPCm were consistent with those of cytology for both CIN2+ and CIN3+ detection. The relative specificities of PAX1m and GYPCm for CIN2+ detection compared to cytology were 2.83 (95% CI: 2.33–2.45) and 3.09 (95% CI: 2.40–3.98), respectively. The relative specificities of combining HPV 16/18 with PAX1m and GYPCm for CIN2+ detection compared to cytology were 3.38 (95% CI: 2.96–3.86) and 3.67 (95% CI: 3.15–4.27), respectively. Compared to low levels of DNA methylation, high levels of PAX1m and GYPCm resulted in odd ratios (ORs) of 57.66 (95% CI: 13.57–409.12, p < 0.001) and 23.87 (95% CI: 6.49–115.42, p < 0.001) for CIN3+, adjusted for HPV 16/18 and cytology results. PAX1m and GYPCm demonstrated superior ability to identify cervical precancerous lesions and cervical cancer, with AUC values exceeding 0.85. For detecting CIN2+/CIN3+ in women with hrHPV-positive status, DNA methylation (combined with HPV 16/18) showed higher specificity than cytology (combined with HPV 16/18) and is a potential molecular biomarker for detecting cervical (pre)cancer.

Effective risk stratification is crucial for managing cervical lesions. This study aimed to develop and evaluate machine learning models to improve the det

To evaluate PAX1 hypermethylation (PAX1m) as a triage strategy test for cervical intraepithelial neoplasia grade 3 or worse (CIN3+) in high-risk human papi

The triage of Atypical Squamous Cells (ASC) is pivotal in cervical cancer (CC) screening. This study aimed to evaluate the performance of paired box 1 gene methylation (PAX1^m) testing for triaging cervical intraepithelial neoplasia (CIN) grade 3 or worse (CIN3+) in an ASC population and to compare it with high-risk human papillomavirus (hrHPV) testing. This retrospective study was conducted from June 2023 to October 2025 and analyzed 1,305 women with ASC. Residual cervical cell samples were used for PAX1^m testing, and their hrHPV test results within two months were collected for comparison. All participants underwent colposcopy-biopsy to obtain the histopathological results. The area under the receiver operating characteristic curve (AUC), sensitivity, specificity of the two methods was calculated and compared. RESULTS PAX1M : difference of cross points (ΔCp) values showed a negative correlation with lesion severity (P _trend <0.001). For CIN3 + detection, PAX1^m (cut-off: ΔCp ≤ 11.74) demonstrated comparable sensitivity to hrHPV (94.6% vs. 94.6%), but with significantly higher specificity (66.0% vs. 10.4%, P < 0.001). Its AUC was also significantly higher to that of the hrHPV test. For CIN2 + detection, the AUC of PAX1^m (cut-off: ΔCp ≤ 11.74) was 0.716, which was significantly higher than that of the hrHPV test (0.544, P < 0.001). Furthermore, PAX1^m detected all cases of CC. In the triage of ASC, PAX1^m significantly improved the specificity for detecting CIN3 + while maintaining high sensitivity. These findings suggest that PAX1^m is a promising triage strategy with diagnostic performance superior to that of hrHPV, warranting further prospective validation of its clinical impact and cost-effectiveness.

Effective triage of high-risk human papillomavirus (hrHPV)-positive women is critical for cervical cancer prevention. This study evaluated and compared PAX