Objective triage strategies are required to prevent unnecessary referrals for colposcopy in population‐based screening programs using primary high‐risk human papillomavirus (hrHPV) testing. We have identified several DNA methylation markers with high sensitivity and specificity for detection of high‐grade cervical intraepithelial neoplasia or worse (CIN2+) in women referred for colposcopy. Our study assessed diagnostic potential of these methylation markers in a hrHPV‐positive screening cohort. All six markers (JAM3, EPB41L3, C13orf18, ANKRD18CP, ZSCAN1 and SOX1) showed similar association across histology in the hrHPV‐positive cohort when compared to the Dutch cohort (each p > 0.15). Sensitivity for CIN2+ was higher using methylation panel C13orf18/EPB41L3/JAM3 compared to the other 2 panels (80% vs. 60% (ANKRD18CP/C13orf18/JAM3) and 63% (SOX1/ZSCAN1), p = 0.01). For CIN3+ all three methylation panels showed comparable sensitivity ranging from 68% (13/19) to 95% (18/19). Specificity of SOX1/ZSCAN1 panel (84%, 167/200) was considerably higher compared to ANKRD18CP/C13orf18/JAM3 (68%, 136/200, p = 2 × 10−5) and C13orf18/EPB41L3/JAM3 (66%, 132/200, p = 2 × 10−7). High negative predictive value (NPV) (91–95% and 96–99%) was observed for CIN2+ and CIN3+, for all three methylation panels, while positive predictive value (PPV) varied from 25 to 40% for CIN2+ and 15–27% for CIN3+. Interestingly, 118/235 samples were negative for all six markers (including 106 controls (89.8%), 6 CIN1 (5.1%), 5 CIN2 (4.2%) and 1 CIN3 (0.8%)). Methylation results from both independent cohorts were comparable as well as high sensitivity for detection of cervical cancer and its high‐grade precursors in hrHPV‐positive population. Our study therefore validates these methylation marker panels as triage test either in hrHPV‐based or abnormal cytology‐based screening programs.
What's new? In cervical cancer screening, HPV testing provides greater sensitivity than cytology, but its lower specificity leads to some unnecessary treatment referrals. DNA methylation assays could potentially provide better specificity for identifying CIN2+ in women carrying high risk HPV. These authors investigated six previously identified CIN2+ methylation markers. They tested three different combinations of markers, and found high levels of sensitivity and specificity, making these markers potentially useful as part of a population‐based screening program.