Abstract 5361: Characteristic DNA methylation signature in sporadic colorectal cancer and in premalignant lesions.

Abstract

Abstract BACKGROUND: DNA methylation plays a crucial role in the carcinogenesis of proximal colon, however distal colorectal cancer and its premalignant lesions have scarcely been studied. AIMS: Our aim was to study DNA methylation in multiple genes during adenoma-dysplasia-carcinoma sequence in distal colon and to identify a characteristic methylation pattern and to confirm it on FFPE samples. Our further aim was to correlate DNA methylation levels with mRNA levels. METHODS: For DNA methylation analysis 20 healthy colonic, 12 low-grade dysplasia (LGD), 10 high-grade dysplasia (HGD), 20 colorectal cancer (CRC) samples were endoscopically removed from the distal colon, 14 samples were matched CRC and LGD samples. DNA methylation percentages of 96 genes were determined using methylation-sensitive restriction enzyme-based quantitative array PCR (EpiTect Methyl qPCR Array System (Qiagen). For validation, methylation-specific high resolution melting analysis (MS-HRM) analysis was performed for SFRP1, SFRP2 and MAL genes in fresh frozen and FFPE tissue specimen after automatic DNA isolation by the MagNA Pure 96 system (Roche). For mRNA expression analysis 49 healthy colonic, 25 LGD, 24 HGD and 24 CRC samples were applied for Whole Genome Expression Profile Microarray (Affymetrix). RESULTS: Analysis of 96 genes revealed hypermethylation of 8 genes in all 62 samples, additionally 34 genes were hypermethylated in LGD, 50 genes in HGD and 30 genes in CRC. A characteristic panel of 10 hypermethylated genes (e.g. ALDH1A3, MAL, SFRP1, SFRP2, SLIT2) significantly distinguished LGD, HGD and CRC from normal tissue (p<0.05) and was detected in 95% of the analyzed premalignant and malignant lesions. Hypermethylation of SFRP1, SFRP2 and MAL genes was confirmed by MS-HRM on fresh frozen and FFPE specimens and their methylation levels inversely correlated with mRNA expression. CONCLUSIONS: A characteristic methylation signature was identified in sporadic distal CRC and premalignant lesions. Our results suggest that some epigenetic alterations accumulate in precursor lesions. Hypermethylation was detected with DNA methylation analysis based on restriction enzyme digestion and MS-HRM in biopsy, fresh frozen and FFPE tissue samples. Citation Format: Arpad V. Patai, Alexandra Kalmar, Orsolya Galamb, Gabor Valcz, Arpad Patai, Balint Peterfia, Peter Hollosi, Barnabás Wichmann, Katalin Leiszter, Kinga Tóth, Andrea Schöller, Sandor Spisak, Ferenc Sipos, Zsolt Tulassay, Bela Molnar. Characteristic DNA methylation signature in sporadic colorectal cancer and in premalignant lesions. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5361. doi:10.1158/1538-7445.AM2013-5361