CpG Island Methylation in PTEN, MGMT, and E/H Cadherin Promoters, Mutations of K-ras and B-raf May Indicate How Progression in the Colorectal Adenocarcinoma Sequence Occurs.

3608 Background: The colorectal adenocarcinoma sequence summarizes the epigenetic and genetic modifications, which arise during cancer progression. These modifications have effect on the methylation status of different gene promoters and mutations in the K-ras and B-raf genes. Included in the sequence during cancer progression may be, as we have previously shown (ESMO 2010, abstr.:646P): methylation of the gene promoters of H-cadherin then of MGMT deregulating removal toxic methyl adducts on guanine bases and inducing the activation of K-ras through G to A point mutations, activation of B-raf gene by a V600E mutation and finally methylation of E-cadherin, linked to emergence of metastasis. Methods: DNA extraction was obtained by standard methods from resected tumour samples. In order to add PTEN methylation status in this sequence, shown to co-occur with K-ras and B-raf mutations in colorectal cancer (CRC), we screened for PTEN inactivation by promoter hypermethylation over 200 CRC patients from which MGMT, E- and H-cadherin methylation, K-ras and B-raf status we determined. PTEN methylation was analyzed by methylation-specific PCR, gel electrophoresis after Sybr green staining and UV-photography. From each individual patient we examined germline DNA from white blood cells as described above. Results: Of the 95 out of 222 tumors (43%) with a PTEN hypermethylation, 77 (81%) were also methylated in CDH13, 52 (55%) were MGMT methylated, 35 (37%) had a K-ras gene mutation and were B-raf wild type as expected, their mutations being mutually exclusive. We are analysing the 127 non PTEN hypermethylated patients especially in regards to B-raf mutations, Smad and others. All results were tumor specific as all the sequenced blood controls were unmethylated respectively wild type. Conclusions: PTEN methylation occurs after CDH13 and MGMT methylation and induces mutations in K-ras or B-raf genes. These results further add on and sustain the colorectal adenocarcinoma sequence.