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Cervical screening by high-risk HPV (hrHPV) testing requires additional risk stratification (triage), as most infections are transient and only a subset of hrHPV-positive women harbours clinically relevant disease. Molecular triage markers such as microRNAs (miRNAs) and DNA methylation markers are particularly promising, as they can be objectively tested directly on hrHPV-positive scrapes and cervicovaginal self-samples. Here, we evaluated the marker potential of 10 candidate miRNAs in 209 hrHPV-positive scrapes of women with underlying precancer (cervical intraepithelial neoplasia, grade 2–3 (CIN2-3)), cancer, or without disease (CIN0/1). A predictive miRNA classifier for CIN3 detection was built using logistic regression, which was compared to and combined with DNA methylation marker FAM19A4. Markers were correlated to histology parameters and hrHPV genotype. A miRNA classifier consisting of miR-149, miR-20a, and miR-93 achieved an area under the curve (AUC) of 0.834 for CIN3 detection, which was not significantly different to that of FAM19A4 methylation (AUC: 0.862, p = 0.591). Combining miRNA and methylation analysis demonstrated complementarity between both marker types (AUC: 0.939). While the miRNA classifier seemed more predictive for CIN2, FAM19A4 methylation was particularly high in HPV16-positive and histologically advanced CIN3, i.e. CIN3 with high lesion volume. The miRNA classifier, FAM19A4 methylation, and the miRNA/methylation combination were highest in cancer-associated scrapes. In conclusion, a panel of three miRNAs is discriminatory for CIN3 in hrHPV-positive scrapes and can complement DNA methylation analysis for the efficient detection of cervical disease. Combined analysis of the two marker types warrants further evaluation as triage strategy in hrHPV-based screening. [ABSTRACT FROM AUTHOR]

Recently, DNA methylation analysis of FAM19A4 in cervical scrapes has been shown to adequately detect high‐grade cervical intraepithelial neoplasia and cervical cancer (≥CIN3) in high‐risk HPV (hrHPV)‐positive women. Here, we compared the clinical performance of FAM19A4 methylation analysis to cytology and HPV16/18 genotyping, separately and in combination, for ≥CIN3 detection in hrHPV‐positive women participating in a prospective observational multi‐center cohort study. The study population comprised hrHPV‐positive women aged 18–66 years, visiting a gynecological outpatient clinic. From these women, cervical scrapes and colposcopy‐directed biopsies (for histological confirmation) were obtained. Cervical scrapes were analyzed for FAM19A4 gene promoter methylation, cytology and HPV16/18 genotyping. Methylation analysis was performed by quantitative methylation‐specific PCR (qMSP). Sensitivities and specificities for ≥CIN3 were compared between tests. Stratified analyses were performed for variables that potentially influence marker performance. Of all 508 hrHPV‐positive women, the sensitivities for ≥CIN3 of cytology, FAM19A4 methylation analysis, and cytology combined with HPV16/18 genotyping were 85.6, 75.6 and 92.2%, respectively, with corresponding specificities of 49.8, 71.1 and 29.4%, respectively. Both sensitivity and specificity of FAM19A4 methylation analysis were associated with age (p ≤ 0.001 each). In women ≥30 years (n = 287), ≥CIN3 sensitivity of FAM19A4 methylation analysis was 88.3% (95%CI: 80.2–96.5) which was noninferior to that of cytology [85.5% (95%CI: 76.0–94.0)], at a significantly higher specificity [62.1% (95%CI: 55.8–68.4) compared to 47.6% (95%CI: 41.1–54.1)]. In conclusion, among hrHPV‐positive women from an outpatient population aged ≥30 years, methylation analysis of FAM19A4 is an attractive marker for the identification of women with ≥CIN3. What's new? High‐risk human papillomavirus (hrHPV) infection is key to the development of cervical cancer. HrHPV DNA testing offers a very sensitive screening tool, but women who test positive require follow‐up testing to avoid over‐diagnosis and over‐treatment. This article is the first to compare the clinical performance of FAM19A4 methylation analysis for cervical (pre)cancer detection to cytology and HPV16/18 genotyping in a prospective observational multi‐center cohort study in a hrHPV‐positive gynecological outpatient population. Performance of methylation analysis to detect high‐grade disease is dependent on age. Among hrHPV‐positive women aged ≥30 years, methylation analysis appears a valuable alternative to cytology and HPV16/18 genotyping. [ABSTRACT FROM AUTHOR]