Offering self-sampling of cervico-vaginal material for high-risk human papillomavirus (hrHPV) testing is an effective method to increase the coverage in cervical screening programs. Molecular triage directly on hrHPV-positive self-samples for colposcopy referral opens the way to full molecular cervical screening. Here, we set out to identify a DNA methylation classifier for detection of cervical precancer (CIN3) and cancer, applicable to lavage and brush self-samples. We determined genome-wide DNA methylation profiles of 72 hrHPV-positive self-samples, using the Infinium Methylation 450K Array. The selected DNA methylation markers were evaluated by multiplex quantitative methylation-specific PCR (qMSP) in both hrHPV-positive lavage ( = 245) and brush ( = 246) self-samples from screening cohorts. Subsequently, logistic regression analysis was performed to build a DNA methylation classifier for CIN3 detection applicable to self-samples of both devices. For validation, an independent set of hrHPV-positive lavage ( = 199) and brush ( = 287) self-samples was analyzed. Genome-wide DNA methylation profiling revealed 12 DNA methylation markers for CIN3 detection. Multiplex qMSP analysis of these markers in large series of lavage and brush self-samples yielded a 3-gene methylation classifier ( and ). This classifier showed a very good clinical performance for CIN3 detection in both lavage (AUC = 0.88; sensitivity = 74%; specificity = 79%) and brush (AUC = 0.90; sensitivity = 88%; specificity = 81%) self-samples in the validation set. Importantly, all self-samples from women with cervical cancer scored DNA methylation-positive. By genome-wide DNA methylation profiling on self-samples, we identified a highly effective 3-gene methylation classifier for direct triage on hrHPV-positive self-samples, which is superior to currently available methods. .
Widespread adoption of primary human papillomavirus (HPV)‐based screening has encouraged the search for a triage test which retains high sensitivity for the detection of cervical cancer and precancer, but increases specificity to avoid overtreatment. Methylation analysis of
FAM19A4
and
miR124‐2
genes has shown promise for the triage of high‐risk (hr) HPV‐positive women. In our study, we assessed the consistency of
FAM19A4
/
miR124‐2
methylation analysis in the detection of cervical cancer in a series of 519 invasive cervical carcinomas (
n
= 314 cervical scrapes,
n
= 205 tissue specimens) from over 25 countries, using a quantitative methylation‐specific PCR (qMSP)‐based assay (QIAsure Methylation Test®). Positivity rates stratified per histotype, FIGO stage, hrHPV status, hrHPV genotype, sample type and geographical region were calculated. In total, 510 of the 519 cervical carcinomas (98.3%; 95% CI: 96.7–99.2) tested
FAM19A4/miR124‐2
methylation‐positive. Test positivity was consistent across the different subgroups based on cervical cancer histotype, FIGO stage, hrHPV status, hrHPV genotype, sample type and geographical region. In conclusion,
FAM19A4/miR124‐2
methylation analysis detects nearly all cervical carcinomas, including rare histotypes and hrHPV‐negative carcinomas. These results indicate that a negative
FAM19A4/miR124‐2
methylation assay result is likely to rule out the presence of cervical cancer.
Urine samples provide a potential alternative to physician-taken or self-collected cervical samples for cervical screening. Screening by primary hrHPV testing requires additional risk assessment (so-called triage) of hrHPV-positive women. Molecular markers, such as DNA methylation, have proven most valuable for triage when applied to cervical specimens. This study was set out to compare hrHPV and DNA methylation results in paired urine and cervical scrapes, and to evaluate the feasibility of DNA methylation analysis in urine to detect cervical cancer. Urine samples (n = 41; native and sediment) and paired cervical scrapes (n = 38) from cervical cancer patients, and urine from 44 female controls, were tested for hrHPV and 6 methylation markers. Results on native urine and sediment were highly comparable. A strong agreement was found between hrHPV testing on urine and scrapes (kappa = 0.79). Also, methylation levels in urine were moderately to strongly correlated to those detected in scrapes (
r
= 0.508–0.717). All markers were significantly increased in urine from cervical cancer patients compared to controls and showed a good discriminatory power for cervical cancer (AUC = 0.744–0.887). Our results show a good agreement of urine-based molecular analysis with reference cervical samples, and suggest that urine-based DNA methylation testing may provide a promising strategy for cervical cancer detection.
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