Enhancing Triage of Non-16/18 High-Risk HPV Cervical Lesions Using FAM19A4, SOX1, and PAX1 Methylation Testing

Highlight

This study evaluates the clinical performance of DNA methylation markers—FAM19A4, SOX1, and PAX1—as a triage method in women aged 30 and older testing positive for non-16/18 high-risk human papillomavirus (hrHPV). The combined SOX1/PAX1 methylation assay shows comparable sensitivity but significantly higher specificity than cytology in detecting cervical intraepithelial neoplasia grade 3 or worse (CIN3+), potentially reducing unnecessary colposcopy referrals, especially in women with low-grade cytological findings.

Study Background

Cervical cancer remains a significant public health concern worldwide, predominantly caused by persistent infection with high-risk human papillomavirus (hrHPV) types. While HPV types 16 and 18 account for approximately 70% of cervical cancers, non-16/18 hrHPV types contribute substantially to high-grade cervical lesions and invasive disease. Current screening often employs cytology-based triage after hrHPV testing; however, cytology is limited by subjective interpretation and lower specificity, especially in women with ambiguous or low-grade cytological abnormalities such as atypical squamous cells of undetermined significance (ASC-US) or low-grade squamous intraepithelial lesion (LSIL). Over-referral to colposcopy leads to patient anxiety, increased healthcare costs, and overtreatment. There is an unmet clinical need for biomarker-based triage tools to enhance specificity without compromising sensitivity, particularly in women positive for non-16/18 hrHPV types.

Study Design

This prospective multicenter study enrolled 915 women aged 30 years and older who tested positive for non-16/18 hrHPV at three hospitals between January and December 2024. The diagnostic performance of methylation testing for the genes FAM19A4, SOX1, and PAX1 was compared head-to-head with cytology and their combination as triage tests for the detection of high-grade cervical lesions (CIN3+). Secondary analyses focused on patients with ASC-US/LSIL cytology to evaluate methylation testing’s ability to refine colposcopy referral decisions.

Key Findings

DNA methylation levels of FAM19A4, SOX1, and PAX1 showed a statistically significant positive correlation with CIN severity (P 0.05) but markedly greater specificity (82.29% to 93.51% versus 23.26%, P < 0.001). The combined SOX1/PAX1 methylation assay revealed optimal performance, with a sensitivity of 94.12% (95% CI: 85.83-97.69) and specificity of 89.96% (95% CI: 87.76-91.81), outperforming cytology and other methylation panels.

In women with ASC-US/LSIL cytology, incorporating methylation testing substantially optimized triage. Theoretical colposcopy referrals could be decreased from 100% to as low as 7.63%, and the number of referrals needed to detect one CIN3+ lesion could be reduced from 26.78 to as few as 3.11. Moreover, a positive methylation test conferred an immediate CIN3+ risk exceeding 16.39%, whereas negative methylation results reduced this risk to below 2.00%, enabling safe deferral of colposcopy in low-risk individuals.

Expert Commentary

These findings underscore the promising role of epigenetic biomarkers in cervical cancer screening, particularly for non-16/18 hrHPV-positive women where cytological triage lacks precision. FAM19A4, SOX1, and PAX1 methylation serve as objective molecular measures of aberrant host genomic regulation associated with progressive cervical neoplasia. The superior specificity observed with methylation-based triage addresses a critical limitation of cytology, offering a means to reduce unnecessary invasive procedures and overtreatment without sacrificing sensitivity for clinically significant lesions.

Nevertheless, while the study’s prospective multicenter design and substantial sample size strengthen its validity, certain limitations merit consideration. The population was restricted to women aged 30 years and older, and applicability in younger women remains to be determined. Longitudinal data assessing progression risk and clinical outcomes over time will be valuable to confirm the prognostic utility of these methylation markers. Additionally, implementation costs and laboratory standardization require careful evaluation for routine clinical uptake.

Conclusion

FAM19A4, SOX1, and PAX1 methylation testing offers an effective molecular triage strategy for women positive for non-16/18 hrHPV infections, particularly in the context of ambiguous cytology. The combined SOX1/PAX1 panel provides high sensitivity and significantly improved specificity compared with cytology, translating into fewer unnecessary colposcopic evaluations and improved patient management. Integration of methylation biomarkers into cervical screening protocols could facilitate stratified care, optimizing resource utilization while maintaining safety. Further studies are warranted to validate these findings in diverse populations and to explore real-world implementation pathways.

Funding and Registrations

The study was supported by institutional research grants from the participating hospitals. No clinical trial registration number was reported.

References

1. Feng P, Shi K, Zhao W, et al. Diagnostic value of FAM19A4/SOX1/PAX1 methylation in non-16/18 high-risk HPV cervical lesions. Gynecol Oncol. 2026;211:37-45. doi:10.1016/j.ygyno.2026.06.002. PMID: 42335608.
2. Wentzensen N, Sun C, Ghosh A, et al. Methylation markers for cervical cancer detection: systematic review. Cancer Epidemiol Biomarkers Prev. 2019;28(5):617-629.
3. Arbyn M, Castle PE. Offering self-sampling for HPV testing to reach women who do not attend cervical screening programs. Cancer Epidemiol Biomark Prev. 2015;24(5):769-772.
4. Dickson EL, Ronco G, Egawa N, et al. Molecular testing for cervical cancer prevention. Expert Rev Mol Diagn. 2020;20(12):1233-1246.