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

Results in Briefinstability, and served to classify MSI-tumors. Furthermore, and in agreement with the CIMPconcept, the samples with MSI and hypermethylation of several genes also carried BRAFmutations. The promoters of ADAMTS1, MAL, and MGMT were frequently methylatedboth in benign and malignant tumors, independent of microsatellite instability. In addition,MGMT was the gene with most frequent promoter hypermethylation among the normalsamples taken in distance from primary tumors, which may indicate that it is involved in thecreation of a “field effect”. From these data we conclude that methylated ADAMTS1,MGMT, and MAL are suitable as markers for early tumor detection.Paper III. “Dysregulation of RAS signaling through alterations of RAS, RAF, NF1 and/orRASSF1A in colorectal carcinomas with known microsatellite instability status”In this article, four components involved in MAP-kinase signaling were analyzed in a seriesof colorectal carcinomas. The MAP-kinase pathway is shown to be hyperactive in a largefraction of CRC due to mutations in KRAS and BRAF, but the possible role of NF1, anegative regulator of KRAS signaling, had never been examined in a series of CRC. This ismost likely due to the large size of the gene (61 exons) and the fact that no mutation hotspotsor mutation cluster region have been identified. A total of 65 colorectal carcinomaswere included in the study, in which mutations in BRAF and KRAS was assessed by directsequencing, and promoter hypermethylation of the RASSF1A was analyzed by MSP. Arepresentative subset of these tumors (n =24) was included in the NF1-analysis, which wasperformed with denaturing high-performance liquid chromatography (dHPLC), sequencing,multiple ligation-dependent probe amplification (MLPA) and real-time polymerase chainreaction (PCR). Forty percent of the carcinomas (26/65) carried a KRAS mutation, and allbut two (c.184-189delGAG and c.49insTTG) occurred in codon 12, 13 or 61 which areknown to produce a constitutively active protein. BRAF was mutated in 22% (14/64) of thecarcinomas, and all but three of the mutations were the V600E mutation which also yields aconstitutively active protein. Mutations in KRAS and BRAF were mutually exclusive, andwhile BRAF mutations were strongly associated with MSI (P = 0.006), KRAS mutationswere more common among MSS-samples (P = 0.08). We found that 31% (18/59) sampleswere hypermethylated in the promoter of RASSF1A, but there were no covariance betweenRASSF1A methylation and mutation status of either of the analyzed genes. One of the 24carcinomas analyzed for NF1 mutations contained two missense mutations and additional40