The model describing the accumulation of genetic alterations of the APC, K-ras, TP53 and SMAD4 genes that drive the development of a carcinoma, has become generally accepted as a paradigm for the genetic basis of colorectal carcinogenesis [3,32]. The relatively low frequency of simultaneous occurrence of mutations in both APC and K-ras observed in this study seems to argue against this synergy. This contention is in accordance with observations from another cohort study, in which APC, K-ras and TP53 gene mutations were studied in 109 tumours and these mutations were found to rarely occur together in the same tumour [9]. However, the simultaneous occurrence of APC and K-ras mutations observed in our study occurs more frequently than expected based on chance alone and therefore mutations in the APC and K-ras genes do not seem to occur independently. When data on APC and K-ras mutations are derived from the study by Smith et al. [9], similar results, although not statistically significant, could be obtained.

The K-ras mutation frequency of 37% is in accordance with reported frequencies of 30 to 60% [33-43]. The frequency of 37% of truncating mutations in the mutation cluster region of APC in this study, however, seems low in comparison to the general assumption that most colorectal tumours harbour a mutation in the APC gene. When only reports from studies on sporadic rather than familial colorectal cancer or colorectal cancer cell lines are considered, the mutation frequencies are lower and vary between 30 and 70% [17,44-49], and a population-based case-control study in the Netherlands reported a 32% mutation frequency [50].

The method for mutation analysis of the APC mutation cluster region and exon 1 of K-ras is based on nested amplification and direct sequencing of purified PCR fragments, a highly sensitive method. Since no screening step was performed prior to the sequencing of the gene fragments, it is unlikely that mutations would have escaped detection. The reproducibility of the applied assays was good, with a reproducibility of 85% and 88% for APC and K-ras, respectively. Arguably, this indicates the extent of heterogeneity present in the tumour samples.

In 103 sporadic colorectal cancers no alterations were found in the K-ras, APC or hMLH1 genes. It is plausible that these tumours have harboured mutations in other components of the Wnt signalling pathway, e.g. mutations in the Axin genes, which are essential for the degradation of β-catenin, and were observed in 11% of patient samples [51]. In addition, an epi-genetic change, i.e. promotor hypermethylation of the APC gene that leads to impaired APC function has been observed in 18% of sporadic colorectal adenomas and carcinomas [52].

