Analysis of cell-free DNA methylation (cfDNAme), alone or combined with CA125, could help to detect ovarian cancers earlier and may reduce mortality. We assessed cfDNAme in regions of ZNF154, C2CD4D and WNT6 via targeted bisulfite sequencing in diagnostic and early detection (preceding diagnosis) settings. Diagnostic samples were obtained via prospective blood collection in cell-free DNA tubes in a convenience series of patients with a pelvic mass. Early detection samples were matched case-control samples derived from the UK Familial Ovarian Cancer Screening Study (UKFOCSS). In the diagnostic set (ncases = 27, ncontrols = 41), the specificity of cfDNAme was 97.6% (95% CI: 87.1%-99.9%). High-risk cancers were detected with a sensitivity of 80% (56.3%-94.3%). Combination of cfDNAme and CA125 resulted in a sensitivity of 94.4% (72.7%-99.9%) for high-risk cancers. Despite technical issues in the early detection set (ncases = 29, ncontrols = 29), the specificity of cfDNAme was 100% (88.1%-100.0%). We detected 27.3% (6.0%-61.0%) of high-risk cases with relatively lower genomic DNA (gDNA) contamination. The sensitivity rose to 33.3% (7.5%-70.1%) in samples taken <1 year before diagnosis. We detected ovarian cancer in several patients up to 1 year before diagnosis despite technical limitations associated with archival samples (UKFOCSS). Combined cfDNAme and CA125 assessment may improve ovarian cancer screening in high-risk populations, but future large-scale prospective studies will be required to validate current findings.

Department of Gynaecology Obstetrics and Neonatology 1st Faculty of Medicine Charles University Prague and General University Hospital Prague Czech Republic

Department of Gynecology and Obstetrics Charles University Prague 1st Faculty of Medicine and Hospital Na Bulovce Czech Republic

Department of Women's and Children's Health Division of Obstetrics and Gynecology Karolinska Institutet and Karolinska University Hospital Stockholm Sweden

Department of Women's Cancer UCL EGA Institute for Women's Health University College London London UK

Eurofins Genomics Europe Sequencing GmbH Konstanz Germany

European Translational Oncology Prevention and Screening Institute Hall in Tirol Austria

GENEWIZ Germany GmbH Bahnhofstraße Germany

MRC Clinical Trials Unit at UCL Institute of Clinical Trials and Methodology University College London London UK

Research Institute for Biomedical Aging Research Universität Innsbruck Innsbruck Austria

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