Figure 1 Mutations of BRAF and KRAS genes in stomach cancers. Genomic DNA each from normal cells or tumor cells was amplified with two primer pairs covering exons 11 and 15 of BRAF gene. KRAS gene was amplified with two primer pairs covering exons 2 and 4. Radioisotope was incorporated into the PCR products for detection by autoradiogram.
The PCR reaction
mixture was denatured for 1 min at 94°C and incubated for 30
procedures of
analysis were
performed as described previously (Shin et al., 1999). After SSCP, DNAs showing mobility shifts were cut out from the dried gel, and reamplified for 30 cycles using the same primer sets. Sequencing of the PCR products was carried out using a capillary automatic sequencer (ABI Prism Genetic Analyzer, Applied Foster City, CA, USA) according to the manufacturer's recom-mendation. SSCP (a-d) and DNA sequencing analyses (e-h) of DNA from tumors (lane T) and normal tissues (lane N). Exon 15 (a and b) of BRAF, and exon 2 (c) and exon 4 (d) of KRAS were amplified. SSCPs of DNA from the tumors show wild-type bands and additional aberrant bands (arrows) as compared to SSCP from corresponding normal cells.
(e) Sequencing analysis from the
aberrant band in (a). There is a G to A transition at nucleotide 1795 of BRAF (arrow) in tumor tissue as compared to normal tissue. (f) Sequencing analysis from the aberrant band in (b). There is an A to G transition at nucleotide 1778 of BRAF (arrow) in tumor tissue as compared to normal tissue. (g) Sequencing analysis from the aberrant band in (e). There is a G to A transition at nucleotide 34 (arrow) of KRAS in tumor tissue as compared to normal tissue. (h) Sequencing analysis from the aberrant band in (d). There is a G to A transition at nucleotide 175 (arrow) of KRAS in tumor tissue as compared to normal tissue. Numbering of cDNA of BRAF and KRAS was done with respect to the ATG start codon
Oncogene
mutation at the amino acid 599, raising the possibility that the V599 M mutation might be a stomach cancer-specific BRAF mutation. In two stomach cancers, we also found D593G BRAF mutation, which has also been detected in colon tumors previously. In BRAF protein, several amino acids at the activation segment are conserved among the species, and these amino acids (D593, F594, G595, L596, T598, V599 and K600) were reported to be mutated in tumors (Brose et al., 2002;
Davies et al., 2002; Naoki et al., 2002; Rajagopalan et al., 2002; Yuen et al., 2002; Pollock et al., 2003;
Satyamoorthy et al., 2003). Currently, it is not known how the V599 M and D593G mutants alter the function of BRAF, and how they contribute to the development of stomach cancer. Clearly, further studies are required to characterize the functional consequences of these mutants in gastric carcinogenesis.
Previous documents on BRAF mutations revealed that the coincidence of BRAF and RAS mutations in the same tumor is not a rare event (Brose et al., 2002; Davies et al., 2002; Naoki et al., 2002; Rajagopalan et al., 2002;
Yuen et al., 2002; Pollock et al., 2003; Satyamoorthy et al., 2003). In agreement with these reports, one of the seven stomach cancers with BRAF mutation had a RAS mutation. Cells both with activating mutations of KRAS BRAF had a substantially higher BRAF kinase activity and ERK1/2 phosphorylation activity than those with BRAF mutation alone (Davies et al., 2002).
It is possible that the tumors with both BRAF and RAS mutations might have an accelerated course in the development or progression of the tumors. BRAF mutations have been detected in early stages of colon cancer and melanoma development (Yuen et al., 2002;
Pollock et al., 2003). By contrast, we detected BRAF mutations in AGC, but not in EGC. Although it was not statistically significant, this result suggested the role of mutant BRAF proteins in the relatively late stage of stomach cancer development. However, since number of stomach cancers with BRAF mutations is small, the common clinical features of the tumors with BRAF mutation remain unknown at this stage.
In summary, we have found seven BRAF mutations in 319 primary stomach cancers. Despite the low frequency of BRAF mutation in stomach cancer compared with that of malignant melanoma, our data suggest that alteration of RAS RAF kinase pathway by BRAF mutation together with RAS mutation may play an important role in gastric carcinogenesis.