Optimization of a Molecular Genetic Assay for the Sensitive Detection of KRAS Mutations in Colorectal Carcinoma

Abstract

Activating mutations of codons 12 and 13 of the KRAS gene occur frequently in colorectal carcinomas. Clinical trialshave shown patients with metastatic colorectal cancer benefit from treatment with tyrosine kinase inhibitors, eg.Vectibix (panitumumab), used to block EGFR overexpression if point mutations in codons 12 or 13 of the KRAS geneare absent. The DxS KRAS mutation kit, mandated by Health Canada as a companion diagnostic, doubled in price to$200-$500 per patient tested, making it less attractive for use by clinical laboratories. Using 25 cases of colorectalcancer previously analyzed by an external laboratory, we validated and compared DxS (Amplification Refractory)and a less costly method, SNaPshot (Primer Extension), for KRAS mutation analysis as well as two extractionmethods, Qiagen FFPE and PicoPure. We demonstrated that DNA purified using the Qiagen FFPE kit produced moreconsistent results. KRAS mutation status was obtained for all samples assayed and were compared with results fromthe external laboratory. Both DxS and SNaPshot met the required analytic sensitivity of 1% mutant to wild-typebackground. One sample was discordant by both assays with those previously reported due to either the assaysor sampling from a different location in the tumour tissue. SNaPshot detected a Gly13Asp variant as an artefactin addition to the correct genotype in four samples. DNA quality from the fixed tissues and PCR amplificationconditions were investigated as the cause. After further validation and refinement of the amplification conditions,the SNaPshot method has now been adopted for clinical use.