The Role of HPV Genotyping, Cytology, and Methylation in the Triage of High-Risk HPV-Positive Patients

<b>Background/Objectives</b>: This study evaluates the diagnostic performance of DNA methylation testing, alone and in combination with cervical cytology, for high-grade squamous intraepithelial lesion (HSIL) detection. <b>Methods</b>: A prospective study was conducted on 170 high-risk HPV (hr-HPV)-positive women. DNA methylation (QIAsure^®) and cervical cytology were performed prior to cervical large loop excision of the transformation zone (LLETZ). Sensitivity, specificity, and area under the curve (AUC) metrics were calculated, including stratified analyses for HPV16/18 and other hr-HPV genotypes. <b>Results</b>: DNA methylation alone achieved a sensitivity of 69.7%, specificity of 79%, and an AUC of 0.796 for HSIL detection. The combination of cervical cytology and DNA methylation improved sensitivity to 94.7%, specificity to 76.3%, and AUC to 0.860. Stratification by HPV genotype revealed that in HPV16/18-positive cases, DNA methylation alone reached an AUC of 0.790, while the combination with cytology significantly enhanced performance to 0.917. DNA methylation alone demonstrated an AUC of 0.744 for other hr-HPV types, and the combined approach achieved an AUC of 0.849. Specificity for the combined approach was notably higher in HPV16/18-positive women (88.9%) than in other hr-HPV cases (72.4%), whereas the sensitivity of the combined approach was significantly higher in both groups (94.5% vs. 95%, respectively). <b>Conclusions</b>: The integration of DNA methylation with cervical cytology significantly enhances diagnostic accuracy for CIN2+ lesions, especially in HPV16/18-positive cases. However, the comparatively lower AUC and specificity observed in other hrHPV types suggest the need for further optimization to enhance accuracy in non-16/18 infections. These findings support the integration of methylation-based testing with cytology as a valuable triage strategy for improving cervical cancer screening and patient management.