Methylation status of the APC and RASSF1A promoter in cell-free circulating DNA and its prognostic role in patients with colorectal cancer

Abstract

DNA methylation is the most frequent epigenetic alteration. Using methylation-specific polymerase chain reaction (MSP), the methylation status of the adenomatous polyposis coli (<i>APC</i>) and Ras association domain family 1 isoform A (<i>RASSF1A</i>) genes was examined in cell-free circulating DNA from 155 plasma samples obtained from patients with early and advanced colorectal cancer (CRC). <i>APC</i> and <i>RASSF1A</i> hypermethylation was frequently observed in both early and advanced disease, and was significantly associated with a poorer disease outcome. The methylation status of the <i>APC</i> and <i>RASSF1A</i> promoters was investigated in cell-free DNA of patients with CRC. Using MSP, the promoter methylation status of <i>APC</i> and <i>RASSF1A</i> was examined in 155 blood samples obtained from patients with CRC, 88 of whom had operable CRC (oCRC) and 67 had metastatic CRC (mCRC). The frequency of <i>APC</i> methylation in patients with oCRC was 33%. Methylated <i>APC</i> promoter was significantly associated with older age (P=0.012), higher stage (P=0.014) and methylated <i>RASSF1A</i> status (P=0.050). The frequency of <i>APC</i> methylation in patients with mCRC was 53.7%. In these patients, <i>APC</i> methylation was significantly associated with methylated <i>RASSF1A</i> status (P=0.016). The frequency of <i>RASSF1A</i> methylation in patients with oCRC was 25%. Methylated <i>RASSF1A</i> in oCRC was significantly associated with higher stage (P=0.021). The frequency of <i>RASSF1A</i> methylation in mCRC was 44.8%. Methylated <i>RASSF1A</i> in mCRC was associated with moderate differentiation (P=0.012), high levels of carcinoembryonic antigen (P=0.023) and methylated <i>APC</i> status (P=0.016). Patients with an unmethylated <i>APC</i> gene had better survival in both early (81±5 vs. 27±4 months, P<0.001) and advanced disease (37±7 vs. 15±3 months, P<0.001), compared with patients with methylated <i>APC</i>. Patients with an unmethylated <i>RASSF1A</i> gene had better survival in both early (71±6 vs. 46±8 months, P<0.001) and advanced disease (28±4 vs. 16±3 months, P<0.001) than patients with methylated <i>RASSF1A</i>. The observed significant correlations between <i>APC</i> and <i>RASSF1A</i> promoter methylation status and survival may be indicative of a prognostic role for these genes in CRC, which requires additional testing in larger studies.