| Abstract |
Colon cancer is one of the most common human tumors. There are approximately 1 million new cases of colorectal cancer annually world-wide, and about 500.000 deaths every year by the disease. Racial and ethnic differences as well as environmental influences are associated with differences in the incidence rates of the disease. Colorectal cancer is the result of a multistage prossess. Inactivation of the APC gene (Adenomatous Polyposis Colί), DNA methylation disturbances, activation of RAS oncogene, loss of DCC gene (deleted ίn colon cancer), inactivation through deletion of p53, the role of MMR mutagenesis genes as well as additional mutations are steps which induce tumorgenesis and contribute to malignant behaviour. RAS family genes (ΚRAS, NRAS, HRAS), in normal cells, are involved in many cellular prossesses, controlling cell proliferation and differentiation. The protein kinase Ras/Raf/MEΚ/MAPK cascade, through phosphorylation, catalyses the induction of mitogene signals in the cell nucleus, resulting in control of the cell cycle. Point mutations of KRAS codon 12 are strongly associated with its mutagenic activation. The RAF group of genes includes three protoongogenes, the ARAF1, RAF1 and BRAF. BRAF gene is located in chromosomal position 7q34 and is a functional gene. The encoded protein is a member of serine/threonine kinase proteins and is activated by the link GΤΡ – Ras. It is involved in the induction of mitogen signals from the cellular membrane to the nucleus. Point mutations with inefficient mismatch repair may promote its oncogenic activation. It is involved in the cell cycle and in antiapoptotic activity. BRAF might be favorable mutation target scince the encoded protein has constitutively higher kinase activity comparative to RAF1. The most common RAF mutation in colorectal cancer is V600E (exon 15) which converts a valine residue to glutamic acid at amino acid position 600 (previously reported as V599E) of the encoded protein and it represents more than 50% of the observed mutations whereas no coexistence of KRAS mutations was observed. The aim of the present study is to investigate the contribution of ongogenic BRAF mutations to colon cancer, the potential relation with KRAS mutations in the same group of cancer specimens and the unveiling of possible correlations with clinical and histopathological features of the examined tumors. In this study, direct sequencing was performed of exon 15 of the BRAF gene in 61 sporadic colorectal cancers, surgically excised. Every specimen was examined pathologically for conformation of the histology (adenocarcinomas). Also, personal and family medical history of the patients was recorded as well as demographic and clinical data. Major criterion was the sporadic nature of the tumors. For the detection of point mutations in BRAF and KRAS genes, polymerase chain reaction was used to amplify exon 15 and codon 12 respectively, using specific oligonucleotide primers. Direct sequencing was performed for exon 15 BRAF analysis, in all specimens, using automated analyzer [BigDyeTM Tenninator Cycle Sequencing Ready Reaction Κit (Applied Biosystems), ΑΒΙ PRISM 3100 Avant Genetic Analyzer]. For the detection of point mutations in KRAS codon 12, restriction fragment length polymorphism analysis was performed (RFLP) by using restriction endonuclease MvaI (Roche Diagnostics GmbH, Germany). DNA from the cancer cell line SW480 which harbored the KRAS codon 12 mutation in homozygous status was used as a positive control. Subsequently, randomly selected samples, positive for KRAS mutations after RFLP analysis, were sequenced and the initial results were confirmed. It was found that the coding sequence of exon 15 was perfectly concerved in all
samples. No nucleotide exchange that could interfere by modifying the aminoacid sequence of the BRAF protein was detected. Molecular analysis for the detection of KRAS mutations was possible for 58 samples. Seventeen samples were positive for KRAS mutations. All KRAS mutations were detected in heterozygous state. Among the 17 tumors with KRAS codon 12 mutations, 12 (34,3%) were male cases (among 35 male cases examined) and 5 (21.7%) were women cases (among 23 women cases examined). Patients with age 70 years or younger were more commonly carriers of KRAS codon 12 mutations than older patients (>70 years), (P= 0.028, Pearson chi square test). Males between 61 and 70 years were more commonly carriers of KRAS mutations compared to younger or older patients, (Ρ= 0.040, Pearson chi square test). The 38.1% of the tumors located in the proximal colon (cecum, ascending colon, and transverse colon) and 24.3% of the tumors located in the distal colon (descending or sigmoid colon and rectum) harbored KRAS mutations respectively. The 20.0% of Dukes’ stage A and 28.6% of Dukes’ stage B tumors harbored KRAS mutations, while Dukes’ stage C and D tumors carried these mutations in rates of 34.6% and 27.3% respectively. Tumor stage was uncertain in two cases. The descriptive approach of these results reveals that Dukes stage C and D tumors harbored most of the observed mutations (over 60%). From 43 tumors of good and moderate differentiation, 27.9% were carriers of KRAS mutations, while from 8 tumors of poor differentiation, 37.5% were carriers of the same mutations (data available for 51 tumor samples). Finally, KRAS mutations were found in 25.6% of non mucinous tumors examined and in 40% of mucinous tumors (data available for 44 tumor samples). The absence of BRAF mutations results perhaps, from environmental, ethnic differences or/and other molecular events. Sequencing of exon 15 and subsequent results make the possibility of methodological discrepancies unlikely. On the other hand, several studies report differences of BRAF mutation frequencies of wide range (5%-20%) from line to line, which presumably correlates, at least in part, with population differences due to environmental, ethnic or racial factors. KRAS mutations seem to remain “ the common suspects” since their detection frequency, in the present study, is approximately 30%, which is in alignment with the international reports.
|
Search
