Purpose: DNA damage repair can lead to epigenetic changes. DNA mismatch repair proteins bind to platinum DNA adducts and at sites of DNA damage can recruit the DNA methylating enzyme DNMT1, resulting in aberrant methylation. We hypothesised that DNA damage repair during platinum-based chemotherapy may cause aberrant DNA methylation in normal tissues of patients such as blood.Experimental Design: We used Illumina 450k methylation arrays and bisulphite pyrosequencing to investigate methylation at presentation and relapse in blood DNA from ovarian cancer patients enrolled in the SCOTROC1 trial (n=247) and in a cohort of ovarian tumour DNA samples collected at first relapse (n=46). We used an ovarian cancer cell line model to investigate the role of the DNA mismatch repair gene MLH1 in platinum induced methylation changes.Results: Specific CpG methylation changes in blood at relapse are observed following platinumbased chemotherapy and are associated with patient survival, independent of other clinical factors (HR=3.7; 95%CI 1.8-7.6, p=2.8x10 3 Translational RelevancePlatinum-based chemotherapy is the cornerstone of treatment for a wide variety of cancers. Greater than 80% of ovarian cancer patients respond to platinum-based chemotherapy at first line treatment and is therefore standard of care. However, over 50% of women will not respond to platinum-based chemotherapy at second-line treatment. There are a range of other treatment options at second-line, mainly guided by patient fitness and the platinum free interval. However, there are no biomarkers that guide this choice. We show for the first time that novel DNA methylation biomarkers, measured in blood DNA at the time of first relapse following platinum-based chemotherapy, are prognostic for overall survival of ovarian cancer patients. This opens the potential of a relatively non-invasive prognostic test that could help guide second line treatment options for ovarian cancer patients.4

Purpose: We aimed to identify DNA methylation biomarkers of progression-free survival (PFS) to platinum-based chemotherapy in high-grade serous ovarian cancer (HGSOC) within biologically relevant ovarian cancer-associated pathways.Experimental Design: Association with PFS of CpG island (CGI) promoter DNA methylation at genes in the pathways Akt/mTOR, p53, redox, and homologous recombination DNA repair was sought with PFS as the primary objective in a prospectively collected ovarian cancer cohort (n ¼ 150). Significant loci were validated for associations between PFS, methylation, and gene expression in an independent The Cancer Genome Atlas (TCGA) data set of HGSOC (n ¼ 311).Results: DNA methylation at 29 CGI loci linked to 28 genes was significantly associated with PFS, independent from conventional clinical prognostic factors (adjusted P < 0.05). Of 17 out of the 28 genes represented in the TCGA data set, methylation of VEGFB, VEGFA, HDAC11, FANCA, E2F1, GPX4, PRDX2, RAD54L, and RECQL4 was prognostic in this independent patient cohort (one-sided P < 0.05, false discovery rate < 10%). A multivariate Cox model was constructed, with clinical parameters (age, stage, grade, and histologic type) and significant loci. The final model included NKD1, VEGFB, and PRDX2 as the three best predictors of PFS (P ¼ 6.62 Â 10 À6, permutation test P < 0.05). Focussing only on known VEGFs in the TCGA cohort showed that methylation at promoters of VEGFA, VEGFB, and VEGFC was significantly associated with PFS.Conclusions: A three loci model of DNA methylation could identify two distinct prognostic groups of patients with ovarian cancer (PFS: HR ¼ 2.29, P ¼ 3.34 Â 10 À5