Novel genetic and epigenetic alterations in ... - Ous-research.no

pseudogene, leading to a high rate of falsepositives.[38] In the present study, we used MSPprimers specifically designed to amplify the proteinencodingPTEN gene,[39] and showed that PTENwas not subject to promoter hypermethylation incolorectal carcinomas. A novel study confirms thatmethylation of PTEN is an unusual event incolorectal cancer as a whole.[40]Figure 5. Distribution of colorectal carcinomasaccording to site associates with sex, age, MSIstatusand methylation frequencies. The circlesindicate 52 carcinomas placed according to site, the redcircle=female, the blue=male. Top right section of thecircle: blue=MSS, red=MSI. The lowest section:green=patient 68years. Widespread methylation is given in the top leftsection: white= methylation in < 5 genes,black=widespread methylation > 5 genes.polarization of epithelial cells caused by apicaltransport of lipids and proteins. Loss of cell polarityis often seen in neoplastic transformation.[31] ForMGMT the early involvement is further supported bythe fact that promoter methylation has previouslybeen identified in aberrant crypt foci.[32]Our data do not suggest that any of the markersincluded here were methylated in an age dependentmanner. Of the 11 analyzed genes, six wereunmethylated in all normal samples from nonaffectedindividuals, excluding them as age-specificmethylation targets. For two genes (SCGB3A1 andMAL) only one of 21 samples was methylated.Although the sample in question was from an olderindividual (75 years), the resulting overallmethylation frequency was only 5%. This is instrong contrast to the frequent reported age-specificmethylation of the N33 gene, which showsapproximately 46% methylation among normalsamples in general and 58% methylation in normalsamples from individuals over 60 years.[33] HOXA9is the only gene in the present study harboring“frequent” promoter methylation in normal samples(19% overall, and 43% for individuals of 60 years orolder). Binary regression analysis resulted in asignificant P value, however, when using the samestatistical analysis in the tumor sample series agedependence could not be confirmed. Both technicaland biological aspects influence the interpretation ofDNA promoter methylation analyses.The importance of primer design is emphasized inthe PTEN assay. Promoter hypermethylation ofPTEN has been frequently reported in various tumortypes, including CRC.[34-37] However, the majorityof MSP primer sets used have failed to discriminatebetween PTEN and its frequently methylatedInterdependence among hypermethylated genesand wide spread methylationThe hierarchical clustering analysis of genepromoter methylation status in normal, benign, andmalignant samples confirmed that the distribution ofHOXA9 and MGMT methylation frequencies acrosssample groups differed from the other genes.Overall, methylation of NR3C1 and RUNX3 had thehighest correlation (figure 3 and Additional file 7), inaddition to MLH1, which was also closely related toNR3C1 and RUNX3. Furthermore, the present studyconfirmed that hypermethylation of MLH1 wascharacteristic of right-sided sporadic colon tumorswith MSI.[41] The lack of MLH1 hypermethylation inadenomas analyzed in the present study supportsthe theory that CIMP and MSI-tumors arise fromsessile serrated polyps rather than fromadenomas.[42] NR3C1, RUNX3, CRABP1, andSCGB3A1 were also shown to have the samecharacteristics as MLH1, supporting the hypothesisthat DNA methylation plays a more prominent role inproximal than in distal carcinogenesis. CRABP1,MLH1, NR3C1, and RUNX3 have recently beenshown to belong to a panel of epigeneticallyregulated genes which best discriminate betweenCIMP-positive and CIMP-negative tumors, aphenotype strongly related with MSI status.[43]We found that the MSI positive samples with V600EBRAF mutations were accompanied by promoterhypermethylation of several genes, in agreementwith the CIMP phenotype (Figure 4). Furthermore,we also confirmed that MSS tumors with TP53mutations had less overall methylation, and thus inagreement with a CIMP negative phenotype. KRASmutations were evenly distributed between MSI andMSS samples but seemingly the KRAS/MSIsamples had more methylation than KRAS/MSSsamples. Interestingly, three MSS samples hadBRAF mutations, and all differed from the V600Emutation found among the MSI tumors.Methylation markers suitable for early tumordetectionFor genes previously analyzed for promotermethylation in normal colon samples, our results arewithin the expected range (CDKN2A, 0-33% (rangeof samples 9-100, total methylation frequency~4%)[44-57]; MGMT, 0-39% (range of samples 12-220, total methylation frequency~7%)[14,15,44,49,50,53,56-61]; and MLH1, 0-50%(range of samples 8-100, total methylationfrequency ~5%)).[44,46,49,50,52,53,55-57,62-67]SCGB3A1 and RUNX3 have previously beenanalyzed in only one study, and both wereunmethylated in 57 normal samples.[48] The studyshowing the highest methylation frequency ofCDKN2A and MLH1 were biased towards normal6