HPV E6/E7 mRNA vs. HPV DNA in co-testing with ThinPrep cytology for cervical cancer screening: a prospective observational diagnostic accuracy study of methylation biomarkers.

Human papillomavirus (HPV) gene transcription activity and methylation could be promising tools for cervical cancer (CC) screening. This study compared HPV E6/E7 messenger RNA (mRNA) + ThinPrep cytology test (TCT) vs. HPV genotyping + TCT in screening CC and explored the relationship between gene methylation and cervical lesions. Women who visited gynecology outpatient clinics between January 2023 and November 2024 were enrolled and assigned to 2 screening strategies based on their wishes: HPV DNA + TCT or HPV E6/E7 mRNA + TCT. Methylation testing of FAM19A4, ZNF582, EPHA7, SOX17, PAX1, and JAM3 genes was performed on the biopsy specimens from 100 patients. Ultimately, 1,399 and 991 women underwent HPV DNA + TCT and HPV E6/E7 mRNA + TCT screening, respectively. The sensitivity, specificity, and accuracy in diagnosing high-grade squamous intraepithelial lesion (HSIL)+ were 72.09%, 83.05%, and 81.7% for HPV DNA + TCT, and 77.97%, 86.37%, and 85.37% for HPV E6/E7 mRNA + TCT, respectively. The areas under the receiver operating characteristic curves were 0.777 for HPV DNA + TCT and 0.822 for HPV E6/E7 mRNA + TCT. No HSIL+ lesions were observed in 65 patients with HPV 16/18 (+) but HPV E6/E7 mRNA (-). In cases with HPV 16/18 (+) and HPV E6/E7 mRNA (+), the immediate risks for cervical intraepithelial neoplasia (CIN) 2+ and CIN3+ were 64.06% and 50%, respectively. Methylation levels of FAM19A4, ZNF582, EPHA7, SOX17, PAX1, and JAM3 genes increased with lesion severity. The sensitivity and specificity of HPV E6/E7 mRNA + TCT appeared higher than HPV DNA + TCT. Simultaneous positivity for HPV 16/18 and HPV E6/E7 mRNA suggests a high immediate risk of CIN2+ and CIN3+. These results may improve CC screening.