Identification and Validation of a 3-Gene Methylation Classifier for HPV-Based Cervical Screening on Self-Samples.

Abstract

<b>Purpose:</b> 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.<b>Experimental Design:</b> 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 (<i>n</i> = 245) and brush (<i>n</i> = 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 (<i>n</i> = 199) and brush (<i>n</i> = 287) self-samples was analyzed.<b>Results:</b> 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 (<i>ASCL1, LHX8,</i> and <i>ST6GALNAC5</i>). 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.<b>Conclusions:</b> 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. <i>Clin Cancer Res; 24(14); 3456-64. ©2018 AACR</i>.