Optimization of routine KRAS genotyping procedure for rational selection of first-line targeted therapy in metastatic colorectal cancer (mCRC).

Abstract

e14008 Background: KRAS genotyping represents a first step towards individualized therapy and has become a crucial issue in mCRC. The optimization of KRAS genotyping delay enabling rational prescription of first-line treatment in mCRC including anti-EGFR targeted therapy, requires robust and rapid molecular biology techniques. Methods: Between June 2008 and December 2009, 844 mCRC have been analyzed for KRAS mutations. Following prescription of KRAS genotyping by medical oncologists, the tumor specimens were collected from private and academic pathologists, as paraffin-embedded tissue specimens, and macrodissected after hematoxylin-eosin slide analysis by a pathologist to ensure a minimum of 50% tumor tissue content. DNA was then extracted and controlled. Routine KRAS genotyping, restricted to exon 2 mutations in codon 12 and 13, was performed using three molecular biology techniques i.e. high resolution melting (HRM), PCR-RFLP and allelic discrimination PCR (TaqMan). Results: Among the 844 tumor specimens, 3% (25/844) had excessive DNA degradation and could not be analyzed. KRAS mutations were detected in 41% (334/819) of the analysable specimens with 81% (274/334) of the mutations being located in codon 12 and 19% (60/334) in codon 13. Among 549 specimens in which all three techniques were used, 15 (2.7%) cases of discordance between the three techniques were observed. In these discordant cases, the whole procedure (from DNA extraction) was systematically repeated. 53% of the discordances was due to PCR-RFLP, as revealed by a second analysis of the same sample, 33% to TaqMan, 13% to HRM. From these results, in order to reduce the duration of KRAS genotyping, HRM and TaqMan are used as genotyping techniques and PCR-RFLP only used in cases of discordance, helping to optimize the genotyping delay that is a crucial point for the prescription of first-line treatment in mCRC. Conclusions: The results obtained show an excellent correlation between the three techniques used. Using HRM warrants high quality and rapid routine KRAS genotyping in paraffin-embedded tumor specimens enabling rational prescription of first-line targeted therapy in mCRC. No significant financial relationships to disclose.