RASSF1A promoter methylation in high-grade serous ovarian cancer: A direct comparison study in primary tumors, adjacent morphologically tumor cell-free tissues and paired circulating tumor DNA

// Lydia Giannopoulou 1 , Issam Chebouti 2 , Kitty Pavlakis 3 , Sabine Kasimir-Bauer 2 , Evi S. Lianidou 1 1 Analysis of Circulating Tumor Cells Laboratory, Laboratory of Analytical Chemistry, Department of Chemistry, University of Athens, University Campus, Athens, 15771, Greece 2 Department of Gynecology and Obstetrics, University Hospital of Essen, University of Duisburg-Essen, Essen, D-45122, Germany 3 Pathology Department, IASO Women’s Hospital, Marousi, Athens, 15123, Greece Correspondence to: Evi S. Lianidou, email: lianidou@chem.uoa.gr Keywords: circulating tumor DNA, RASSF1A, high-grade serous ovarian cancer, methylation specific PCR, high resolution melting analysis Received: April 10, 2016 Accepted: November 11, 2016 Published: February 10, 2017 ABSTRACT The RASSF1A promoter is frequently methylated in high-grade serous ovarian cancer (HGSC). We examined RASSF1A promoter methylation in primary tumors, adjacent morphologically tumor cell-free tissues and corresponding circulating tumor DNA (ctDNA) samples of patients with HGSC, using a real-time methylation specific PCR (real-time MSP) and a methylation-sensitive high-resolution melting analysis (MS-HRMA) assay for the detection and semi-quantitative estimation of methylation, respectively. Two groups of primary HGSC tumor FFPE samples were recruited (Group A n=67 and Group B n=61), along with matched adjacent morphologically tumor cell-free tissues (n=58) and corresponding plasma samples (n=59) for group B. Using both assays, RASSF1A promoter was found highly methylated in primary tumors of both groups, and at lower percentages in the adjacent morphologically tumor cell-free tissues. Interestingly, RASSF1A promoter methylation was also observed in ctDNA by real-time MSP. Overall survival (OS) was significantly associated with RASSF1A promoter methylation in primary tumor samples using MS-HRMA (P=0.023). Our results clearly indicate that RASSF1A promoter is methylated in adjacent tissue surrounding the tumor in HGSC patients. We report for the first time that RASSF1A promoter methylation provides significant prognostic information in HGSC patients.