BACKGROUND: Despite a myriad of attempts in the last three decades to diagnose ovarian cancer (OC) earlier, this clinical aim still remains a significant challenge. Aberrant methylation patterns of linked CpGs analyzed in DNA fragments shed by cancers into the bloodstream (i.e. cell-free DNA) can provide highly specific signals indicating cancer presence. METHODS: We analyzed 699 cancerous and non-cancerous tissues using a methylation array or reduced representation bisulfite sequencing to discover the most specific OC methylation patterns. A three-DNA-methylation-serum-marker panel was developed using targeted ultra-high coverage bisulfite sequencing in 151 women and validated in 250 women with various conditions, particularly in those associated with high CA125 levels (endometriosis and other benign pelvic masses), serial samples from 25 patients undergoing neoadjuvant chemotherapy, and a nested case control study of 172 UKCTOCS control arm participants which included serum samples up to two years before OC diagnosis. RESULTS: The cell-free DNA amount and average fragment size in the serum samples was up to ten times higher than average published values (based on samples that were immediately processed) due to leakage of DNA from white blood cells owing to delayed time to serum separation. Despite this, the marker panel discriminated high grade serous OC patients from healthy women or patients with a benign pelvic mass with specificity/sensitivity of 90.7% (95% confidence interval [CI] = 84.3-94.8%) and 41.4% (95% CI = 24.1-60.9%), respectively. Levels of all three markers plummeted after exposure to chemotherapy and correctly identified 78% and 86% responders and non-responders (Fisher's exact test, p = 0.04), respectively, which was superior to a CA125 cut-off of 35 IU/mL (20% and 75%). 57.9% (95% CI 34.0-78.9%) of women who developed OC within two years of sample collection were identified with a specificity of 88.1% (95% CI = 77.3-94.3%). Sensitivity and specificity improved further when specifically analyzing CA125 negative samples only (63.6% and 87.5%, respectively). CONCLUSIONS: Our data suggest that DNA methylation patterns in cell-free DNA have the potential to detect a proportion of OCs up to two years in advance of diagnosis and may potentially guide personalized treatment. The prospective use of novel collection vials, which stabilize blood cells and reduce background DNA contamination in serum/plasma samples, will facilitate clinical implementation of liquid biopsy analyses.

CAS Max Planck Partner Institute for Computational Biology Shanghai Institute of Biological Sciences 320 Yue Yang Road Shanghai 200031 China

Department of Women's Cancer UCL Elizabeth Garrett Anderson Institute for Women's Health University College London Medical School Building Room 340 74 Huntley Street London WC1E 6AU UK

Department of Women's Cancer UCL Elizabeth Garrett Anderson Institute for Women's Health University College London Medical School Building Room 340 74 Huntley Street London WC1E 6AU UK CAS Max Planck Partner Institute for Computational Biology Shanghai Institute of Biological Sciences 320 Yue Yang Road Shanghai 200031 China

GATC Biotech AG Jakob Stadler Platz 7 78467 Konstanz Germany

Genedata AG Margarethenstrasse 38 4053 Basel Switzerland

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

References provided by Crossref.org