Cervical cancer (CC) is a significant global health concern, but effective screening programs have dramatically reduced its incidence and mortality. Current screening strategies often involve human papillomavirus (HPV) testing followed by cytology triage for positive cases. However, cytology's limitations, including low reproducibility and the need for frequent screening, necessitate the development of improved triage methods. DNA methylation (DNAme) offers a promising alternative, as aberrant methylation patterns are associated with CC development. This study focuses on the WID-qCIN test, a novel DNAme-based triage test that assesses methylation in the DPP6, RALYL, and GSXI genes. The aim was to evaluate the performance of WID-qCIN, in combination with HPV16/18 genotyping, as a triage strategy for HPV-positive women in a large, real-world setting compared to the standard cytology triage.

Extensive research supports the use of HPV testing as a primary screening method for CC, demonstrating higher sensitivity and longer screening intervals compared to cytology. However, HPV testing alone has limitations due to high prevalence in younger women and a subsequent need for triage. Current triage methods predominantly rely on cytology, which suffers from reproducibility issues and a need for short screening intervals. Several studies have explored DNAme as a triage marker for HPV-positive women. However, most previous studies were limited by small sample sizes or case-control designs, hindering their generalizability to real-world screening settings. The WID-qCIN test, developed by the authors, showed promising results in preliminary studies, offering a potential solution to these limitations. This study builds upon this prior research to assess the test's clinical utility in a large, real-world setting.

This population-based cohort study utilized data from 28,017 women aged ≥30 years who underwent cervical cancer screening in Stockholm between January and March 2017. The study focused on the 2,377 women who tested positive for HPV infection. Cervical samples from these women were analyzed using the optimized WID-qCIN test, which assesses DNAme levels in three genes (DPP6, RALYL, and GSXI). The performance of WID-qCIN, alone and in combination with HPV16/18 genotyping, was compared to cytology in terms of sensitivity, specificity, and predictive value for detecting prevalent and incident CIN2+ (cervical intraepithelial neoplasia grade 2 or worse) and CC cases. Statistical analyses included the Wilson method for confidence intervals of proportions, chi-squared tests, Kaplan-Meier estimators, Cox proportional hazards models, and a logistic Weibull mixture model to account for prevalent and interval-censored incident cases. The data on HPV status, cytology results, and histopathological diagnoses were obtained from the Swedish National Cervical Screening Registry and the Swedish National Quality Register for Gynecological Cancers, with discrepancies resolved through manual chart review whenever possible. Ethical approval for the study was obtained from the relevant ethical review authorities.

The combination of WID-qCIN and HPV16/18 genotyping demonstrated superior performance compared to cytology in detecting prevalent and incident CIN2+ cases. For prevalent CIN2+, the combined test showed a sensitivity of 85.9% (95% CI 81.3-89.6) compared to 98.4% for cytology (although cytology was used as a triage tool, creating a high sensitivity). For incident CIN2+, the combined approach predicted 69.4% (HR 3.55, 95% CI 2.73-4.63) of cases, significantly higher than cytology (18.2%). Remarkably, the WID-qCIN/HPV16/18 combination detected 100% of invasive CCs in both prevalent and incident settings (although the number of incident cancers was small). Compared to cytology, the combined test significantly reduced the number of colposcopy referrals needed to detect one case of CIN2+ (2.4 vs 4.1). The stand alone WID-qCIN test also performed better than HPV16/18 alone, with a sensitivity of 77.0% for prevalent CIN2+ and a predictive value of 46.3% for incident CIN2+. The specificity of the combined test was lower than cytology or WID-qCIN alone. However, the improvement in sensitivity and reduction in colposcopy referrals justify this trade-off.

This study demonstrates the potential of a DNAme-based triage strategy for HPV-positive women, providing a significantly improved approach compared to traditional cytology triage. The combination of WID-qCIN and HPV16/18 genotyping exhibited substantially higher sensitivity in detecting both prevalent and incident CIN2+ cases and 100% of CC cases, while simultaneously reducing the number of unnecessary colposcopy referrals. The results are particularly notable given the real-world setting, minimizing selection bias. This approach could improve efficiency in cervical cancer screening, potentially leading to earlier detection and treatment of precancerous lesions. The fact that the test works on DNA means it is suitable for self-sampling which may improve screening rates.

The WID-qCIN test, in combination with HPV16/18 genotyping, represents a significant advancement in cervical cancer screening triage. Its improved performance compared to cytology, coupled with its adaptability to self-sampling, makes it a promising tool for improving screening efficiency and early detection of CC. Future research should focus on prospective, randomized controlled trials to further validate these findings and assess the long-term impact on CC incidence and mortality. Exploration of the test's cost-effectiveness and implementation in diverse healthcare settings would also be valuable.

The study's retrospective nature and the shift in reporting of CIN2 and CIN3 as HSIL in Swedish pathology laboratories limit the analysis of incident CIN3 cases. The ascertainment bias inherent in the existing screening protocol, where only cytology-positive women underwent colposcopy, might affect the accurate evaluation of the proposed molecular triage approach. A prospective randomized trial is needed for more conclusive results on CC prevention. Further research is also needed to fully assess the potential of the WID-qCIN test in resource-limited settings.