BRAF mutation and gene methylation frequencies of colorectal tumours with microsatellite instability increase markedly with patient age

The microsatellite instability phenotype (MSI⁺) is observed in approximately 25% of colon cancers and 2% of rectal cancers. MSI⁺ is a hallmark of almost all hereditary non‐polyposis colorectal cancers (HNPCC) where it is associated with germline mutations in one of the DNA mismatch repair genes.¹ However, the large majority of MSI⁺ cancers occur as sporadic cases that arise following methylation induced silencing of the hMLH1 gene promoter. Sporadic MSI⁺ tumours are believed to originate in serrated polyps and display frequent and concurrent methylation of gene promoter regions but low frequencies of KRAS and TP53 mutations.² In contrast, HNPCC associated MSI⁺ tumours originate in conventional adenomas and show frequent APC and KRAS mutations but infrequent methylation. Another striking difference is that BRAF mutations occur in most sporadic MSI⁺ tumours but have never been observed in HNPCC‐MSI⁺ tumours.³,⁴ While there are clearly major differences between sporadic and familial MSI⁺ tumours, we investigated here whether patient age was also an important factor that could influence MSI⁺ tumour phenotype.

The frequency of the BRAF mutation was evaluated in MSI⁺ tumours from a consecutive series of colorectal cancer (CRC) patients aged <60 years (n = 828) who were enrolled in a population based screening programme for HNPCC in the state of Western Australia. A total of 66 MSI⁺ cases (8%) were identified using the BAT26 mononucleotide marker, of which only five (7.6%) contained a BRAF mutation. Family cancer history and germline mutation status for all 66 MSI⁺ cases have yet to be determined but preliminary data suggest that less than one third will be HNPCC. For comparison, BRAF mutations were also investigated in non‐selected MSI⁺ patients aged ⩾60 years. Of 45 MSI⁺ cases, 27 (60%) showed a BRAF mutation, with a highly significant difference in frequency between young and old MSI⁺ patients (p<1×10⁻⁵). These results are almost identical to those of another recent population based study which reported a BRAF mutation frequency of 7% in MSI⁺ tumours from patients aged <55 years and 61% in those aged 55–79 years (p = 0.0002).⁵

The frequency of methylation in five gene promoter regions (hMLH1, p16, p14, TIMP3, and MINT2) was also compared between MSI⁺ tumours from young and old patients derived from a non‐selected CRC series. In six MSI⁺ tumours from patients aged <60 years, only 2/30 (7%) of the CpG islands investigated by MethyLight assay were methylated. A much higher frequency (61/100, 61%) was observed in 20 MSI⁺ tumours from patients aged ⩾60 years (p<1×10⁻⁵). These results concur with an earlier study showing that patients with hMLH1 methylated MSI⁺ tumours were, on average, 18 years older than those without hMLH1 methylation.⁶

The above results on the frequencies of BRAF mutation and promoter methylation demonstrate the existence of striking age related differences in MSI⁺ phenotype. In view of the very low incidence of HNPCC (0.5–1.5% of all CRC), these differences are likely to involve a much greater proportion of MSI⁺ tumours than simply the rare cases with germline mutations in mismatch repair genes. We estimate that 30–40% of MSI⁺ tumours in population based CRC cohorts belong to a subgroup characterised by the absence of both BRAF mutation and promoter methylation.

The clinical significance of these findings relates to the potential prognostic and predictive values of MSI⁺. Current disagreement in the literature concerning the predictive value of MSI⁺ for response to 5‐fluorouracil (5FU) chemotherapy⁷,⁸,⁹,¹⁰ could be due to the relative proportion of HNPCC and younger MSI⁺ cases included within these studies. BRAF mutation and gene promoter methylation, or other factors closely associated with these features, may be strong determinants of the response to 5FU. We therefore recommend consideration of this issue in future studies aimed at evaluating the predictive significance of MSI⁺ in colon cancer.