Keigo Kimura

Colorectal Cancer With Mutation in BRAF, KRAS, and Wild-Type With Respect to Both Oncogenes Showing Different Patterns of DNA Methylation

PURPOSE BRAF mutations are common in sporadic colorectal cancers (CRCs) with a DNA mismatch repair (MMR) deficiency that results from promoter methylation of hMLH1, whereas KRAS mutations are common in MMR proficient CRCs associated with promoter methylation of MGMT. The aim of this study was to further investigate the link between genetic alterations in the RAS/RAF/ERK pathway and an underlying epigenetic disorder. PATIENTS AND METHODS Activating mutations of BRAF and KRAS were identified and correlated with promoter methylation of 11 loci, including MINT1, MINT2, MINT31, CACNA1G, p16(INK4a), p14(ARF), COX2, DAPK, MGMT, and the two regions in hMLH1 in 468 CRCs and matched normal mucosa. RESULTS BRAF V599E mutations were identified in 21 (9%) of 234 CRCs, and KRAS mutations were identified in 72 (31%) of 234 CRCs. Mutations in BRAF and KRAS were never found in the same tumor. CRCs with BRAF mutations showed high-level promoter methylation in multiple loci, with a mean number of methylated loci of 7.2 (95% CI, 6.6 to 7.9) among 11 loci examined (P < .0001). Tumors with KRAS mutations showed low-level promoter methylation, and CRCs with neither mutation showed a weak association with promoter methylation, with an average number of methylated loci of 1.8 (95% CI, 1.5 to 2.1) and 1.0 (95% CI, 0.79 to 1.3), respectively. CONCLUSION In CRC, the methylation status of multiple promoters can be predicted through knowledge of BRAF and, to a lesser extent, KRAS activating mutations, indicating that these mutations are closely associated with different patterns of DNA hypermethylation. These changes may be important events in colorectal tumorigenesis.

No Duplicate KRAS Mutation is Identified on the Same Allele in Gastric or Colorectal Cancer Cells with Multiple KRAS Mutations

Codon 12 and 13 mutations in 170 colorectal cancer (CRC) and 66 gastric cancer (GC) specimens were analysed by an ‘enriched’ polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) method. All identified mutations were verified by direct sequencing of the second PCR products. Among the 170 CRC specimens, mutations were identified in 47 (28%) and 13 (7.6%) cases in codons 12 and 13, respectively. In the 66 GC specimens examined, however, mutations in codons 12 and 13 were only detected in two (3.0%) and one (1.5%) cases, respectively. Mutations in both codon 12 and 13 were found in 3/170 (1.8%) CRCs and 1/66 (1.5%) GCs. Duplicate mutations were never identified in the same allele, which was confirmed by direct sequencing of the second amplified products. The majority of colorectal and gastric cancer cells with KRAS mutations are homogeneous because they have the same KRAS mutation. A few colorectal or gastric cancers, however, showed heterogeneity, as verified by the fact that single mutations were identified in the same allele.