Genome-wide cell-free DNA methylation profiling in advanced stage ovarian cancer. Are we looking at the tumor or the patient's immune response to the tumor?

The aim of this study was to identify differentially methylated regions in cell-free DNA (cfDNA) between healthy persons and patients with advanced stage ovarian cancer (ASOC) and to identify differences in cfDNA methylation before and after cytoreductive surgery. Plasma-derived cfDNA was analyzed by a high-throughput genome wide DNA methylation sequencing technique: MeD-seq. A training set of therapy naïve cfDNA samples of patients with ASOC (≥FIGO stage IIIB, n=10) was compared with cfDNA of healthy controls (n=10) to define a ASOC specific cfDNA methylation signature. A cumulative hypermethylation score was constructed and a validation set of pre- and postoperative samples of 39 patients were compared using this score. MeD-seq results of tumor tissue samples were correlated with cfDNA results. Patients with ASOC showed a clear distinct cfDNA methylation signature from healthy controls (p<0.0001). This cfDNA-methylation signature resulted in preoperative hypermethylation scores (135; interquartile range 110-163) that were significantly higher than postoperative hypermethylation scores (91; interquartile range 76-101) (p<0.001). The cfDNA methylation signature at baseline differed from tumor tissue and was more closely related to DNA methylation of immune-related cells (T-lymphocytes, neutrophil granulocytes, monocytes, and B-lymphocytes) than to ASOC tissue. MeD-seq provides a promising method for genome wide methylation profiling of cfDNA. Patients with ASOC could clearly be distinguished from healthy controls and differed pre- and postoperatively.