K-ras gene mutation status in colorectal cancer: comparative analysis of pyrosequencing and PCR-RFLP

In patients with high-stage colorectal carcinomas (CRC), anti-EGFR therapy is known to be effective only in cases with a wild-type K-ras gene status. Different procedures have been proposed for such evaluation.

The mutation status of K-ras gene, codons 12, 13 and 61 was determined in 250 CRC cases using the pyrosequencing assay. In addition, we compared the performance of the pyrosequencing procedure with that of PCR-RFLP in a subset (n=100) of the CRC samples the latter only in codons 12 and 13.

Using pyrosequencing, 46.4% of the 250 CRC cases were found mutated. Most mutations were located in codon 12 (36.4% from all cases) and several were located in codon 61 (3.2%). All mutation identified by PCR-RFLP were confirmed by pyrosequencing and, in addition, one more mutated sample was identified in the subset of 100 samples.

Both methods are highly specific and can profitably be used in the molecular diagnosis of colorectal cancer in order to establish the adequate therapy.