Abstract 3826: A reversible DNA methylation signature precedes sporadic mutations in the colorectal adenoma-dysplasia-cancer development
Authors: Molnar Bela , Peterfia Balint , Kalmar Alexandra , Wichmann Barnabas , Patai Arpad V. , Tulassay Zsolt 
Published: August 2015

Abstract
Background and aims: New generation sequencing and array technologies now allow the systematic investigation and comparison of genetic or epigenetic alterations in AD-CRC. The colorectal adenoma-dysplasia-cancer (AD-CRC) development is characterised by sporadic (&lt;45% frequency) mutations in a limited number of genes. Recent epigenetic investigations showed that methylation of selected genes occurs in high frequency (&gt;95%) in early stages of cancers already.
Aims: Evaluation of the role of epigenetic and genetic alterations in the AD-CRC development using new generation sequencing and DNA methylation arrays.
Materials and methods: DNA was isolated from fresh frozen biopsy specimens (20 normal; 33 adenomas; 17 CRCs). First, a multiplex PCR panel was designed to amplify mutation hot spots of 12 selected genes (APC, BRAF, CTNNB1, EGFR, FBXW7, KRAS, MSH6, NRAS, PIK3CA, SMAD2, SMAD4, TP53). Further DNA methylation analysis of 94 genes was performed on Human Colon Cancer EpiTect Methyl II Signature PCR Array (Qiagen) from the same DNA specimen. After RNA isolation whole genome expression analysis was performed with HGU133plus2 microarrays (Affymetrix) from 49 normal, 49 adenoma and 49 CRC specimens and on HT-29 cells with and without 5-aza-deoxycytidin treatment. Immunohistochemistry confirmation of expression changes on protein level was performed using tissue microarrays.
Results: Mutations were found in 76% of adenomas and 78% of the cancer cases. The average number of mutations found in mutated samples was 1; 1,8; 1,9 and 2,3 in low grade adenomas, high grade adenomas, carcinomas and serrated adenomas, respectively. The APC suppressor gene was mutated in adenomas more frequently than in carcinomas (36% vs. 24%). The most frequently mutated genes were APC, TP53 and KRAS with 36%, 18% and 26% frequencies in adenomas and 24%, 47% and 45% frequencies in carcinomas.
DNA methylation was found in 100% of the investigated colorectal adenoma and cancer specimens. Eight genes were found to be methylated in all of the cases. This gen set included SFRP1, MAL, SLIT2, SST, VIM; another set of genes (DKK1, SLI3, TMEFF2) was found to be hypermethylated in adenomas and cancers in &gt;75% of the cases. In adenomas 56 genes, in dysplasias 40 in cancer 37 genes were methylated (&gt;50% of the cases). The effect of methylation could be confirmed by decreased mRNA and protein expression. Demethylation treatment successfully restored the expression profile of the top methylated genes.
Conclusion: Methylation of gene sets occurred in early premalignant stages followed by somatic mutations in increasing number through the AD-CRC development. Demethylation treatment could reverse the systematic, robust methylation alterations. Epigenetic alterations precede the somatic mutations of the AD-CRC and show higher significance in CRC development than genetic.
Citation Format: Bela Molnar, Balint Peterfia, Alexandra Kalmar, Barnabas Wichmann, Arpad V. Patai, Zsolt Tulassay. A reversible DNA methylation signature precedes sporadic mutations in the colorectal adenoma-dysplasia-cancer development. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3826. doi:10.1158/1538-7445.AM2015-3826