Z Gastroenterol 2012; 50 - A64
DOI: 10.1055/s-0032-1312418

Epigenetic alterations in colorectal adenomas outnumber those in cancer. Is adenoma-dysplasia-carcinoma sequence a non-sequential process at the molecular level?

VÁ Patai 1, O Galamb 2, G Valcz 2, A Kalmár 1, Á Patai 3, B Péterfia 4, B Wichmann 1, K Leiszter 1, K Tóth 1, A Schöller 1, I Fűri 1, B Barták 1, Z Nagy 1, B Molnár 2, Z Tulassay 2
  • 12nd Department of Internal Medicine, Semmelweis University, Budapest, Hungary
  • 2Molecular Medicine Research Unit, Hungarian Academy of Sciences, Budapest, Hungary
  • 31st Department of Internal Medicine, Sopron County Elisabeth Hospital, Sopron, Hungary
  • 41st Department of Pathology and Experimental Cancer Research, Semmelweis University, Budapest, Hungary

Background: The adenoma-dysplasia-carcinoma sequence (ADCS) driven by sequential accumulation of genetic mutations is a widely accepted concept in colorectal carcinogenesis. In some proximal cancers arising from different precursor lesions DNA methylation has been shown to be an important mechanism, however in the distal colon DNA methylation has scarcely been studied.

Aims: Our primary aim was to study DNA methylation alterations in 96 genes during ADCS, to identify characteristic methylation pattern for the distal colon and to compare the different stages. Our further aim was to correlate our DNA methylation profiles with whole-genome mRNA expression microarray results and also to analyze potential markers at protein level.

Methods: For DNA methylation analysis 15 healthy colonic, 7 low-grade dysplasia (LGD), 5 high-grade dysplasia (HGD), 12 CRC (UICC stage I-III), 4 normal adjacent and 5 ulcerative colitis (UC) samples were endoscopically removed from the left side of the colon. DNA methylation percentages of 96 genes were determined using Methyl-Profiler PCR array system based on methylation-sensitive restriction enzyme digestion followed by fluorescence real-time PCR. For mRNA expression analysis 49 healthy colonic, 25 LGD, 24 HGD and 24 CRC samples were applied for Affymetrix Whole-Genome Expression Microarray. To examine the underlying mechanisms, immunohistochemistry for DNA methyltransferases (DNMT) 1, 3a, 3b and SFRP1 were also performed.

Results: Analysis of 96 genes revealed hypermethylation of 8 genes in all 48 samples, additionally 34 genes were hypermethylated in LGD, 50 genes in HGD and 30 genes in CRC. A characteristic panel of 10 hypermethylated genes, including MAL, SFRP1, SLIT2 and ALDH1A3 (latter a novel finding in CRC) significantly distinguished LGD, HGD and CRC from UC and normal tissue (p<0.05). SFRP1, SLIT2 and MAL methylation levels inversely correlated with mRNA expression, and SFRP1 protein level correlated with mRNA expression levels. DNMT3a protein level correlated with overall methylation, showing strongest expression in LGD and HGD.

Conclusions: Distal CRC has a characteristic methylation pattern. Precancerous lesions have more epigenetic alterations as compared to CRC and this might indicate that ADCS is a non-sequential process at the molecular level.