Identification and validation of a novel 3-gene methylation classifier for HPV-based cervical screening on self-samples

Background: Offering self-sampling of cervico-vaginal material for human papillomavirus (HPV) testing is an effective method to increase the coverage in cervical screening programmes. Molecular triage directly on HPV-positive self-samples for colposcopy referral opens the way to full molecular cervical screening. Here, we set out to identify a methylation classifier for detection of precancer (CIN3) or cancer, applicable to self-samples obtained by different devices. Findings: Genome-wide DNA methylation profiling of HPV-positive self-samples revealed 12 methylation targets for CIN3 detection. Multiplex quantitative methylation-specific PCR of these targets yielded a 3-gene methylation classifier (ASCL1, LHX8 and ST6GALNAC5), showing 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. All self-samples from women with cervical cancer scored methylation-positive. Conclusion: By genome-wide DNA methylation profiling on self-samples, we identified a highly effective 3-gene methylation classifier for direct triage on self-samples from HPV-positive women. 68 self-sampled cervico-vaginal specimens were analysed by Illumina Infinium HumanMethylation450 BeadChip. This series comprised 28 HPV-positive controls, 36 from women with a CIN3 (precancer) and 4 from women with cervical squamous cell carcinoma.