Abstract 1477: The topography of DNA methylation-mediated field defects in colorectal carcinogenesis
Authors: Subramaniam Manish Mani , Loh Marie , Chan Jason Yongsheng , Liem Natalia , Lim Pei Li , Yeoh Khay Guan , Soong Richie , Salto-Tellez Manuel 
Published: February 2011

Purpose: High-throughput DNA methylation analysis of the entire non-neoplastic colonic mucosa (NNCM) at different distances from tumor was performed to explore the extent and distribution (“topography”) of methylation-mediated field defects in colorectal cancer (CRC). Methylation in NNCM of CRC was compared with that of colonic mucosa from non-CRC patients.

Materials and Methods: Seventy-one samples (NNCM and tumor) were obtained from rectosigmoid specimens of 3 CRCs (case 1, 2 stage IV; case 3, stage II). Entire NNCM from resection specimens was completely stripped from underlying layers, divided into equally-sized quadrants and numbered with first and last being proximal and distal to tumor respectively. Distances from tumor center to center of each NNCM quadrant were recorded. Eight unequivocally normal colonic mucosal samples from diverticulosis (n=3) and solitary rectal ulcer (n=1) were also analyzed. NNCM and tumor DNA were subjected to Illumina's Golden Gate DNA methylation analysis for 1506 CpG sites. Probes were defined as methylated, if beta-value &gt; 0.297 and unmethylated, if ≤ 0.297, based on methylated (M) and unmethylated (HSD) controls at 5% false discovery rate. Unsupervised hierarchical cluster analysis was performed with methylation as both continuous and binarized variable. Average levels and frequency of methylation for all probes was used for correlating methylation with distance of NNCM from tumor at 5% significance level.

Results: Tumor of case-1 was significantly methylated followed by that of case-2 and case-3 (average β-values: 0.415, 0.181 and 0.144). In case-1, NNCM samples approximately 4-5cm from tumor were highly methylated and clustered with tumor samples. Beyond 4-5 cms, levels (correlation coefficient: −0.418 and p=0.017) and frequency of methylation (correlation coefficient:-0.493 and p=0.004) in NNCM significantly decreased with increasing distance away from tumor. In case-2, the NNCM samples within 4-5 cms from tumor were less methylated and clustered with the tumor, which was equally methylated and although not significant, levels and frequency of methylation tend to increase with increasing distance away from tumor. In contrast, in case-3, neither the tumor clustered with the immediately adjacent NNCM nor was any correlation detected between methylation and distance from the tumor. Overall, NNCM of CRCs was significantly methylated than that of non-CRC patients. Significant methylation of HDAC9, ACTG2 and HRASLS (all p&lt; 0.001) were detected in NNCM of CRCs than non-CRC patients.

Conclusions: Methylation-mediated field cancerization was detected in NNCM of advanced CRCs usually within 4-5cms radius of the tumor. NNCM of CRCs was significantly methylated compared to colonic mucosa of non-CRC patients. Our data supports the notion that methylation-mediated field defects may be due to presence of a frankly invasive malignant tumor rather than a pre-existing event.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1477.