Prognostic Role of Kras Mutations Detected by High-Sensitivity Taqmelt PCR Assay in Metastatic Colorectal Cancer

ABSTRACT Background KRAS mutation is present in approximately 35-40% of patients with metastatic colorectal cancer (mCRC) and has been established as a predictive marker of resistance to anti-EGFR therapy, but its role as prognostic factor is not yet clear. This study is aimed to analyze the prevalence and the impact in prognosis of expanded number of KRAS mutations in caucasian mCRC population and the sensitivity of the TaqMelt PCR assay cobas KRAS Mutation Test. Methods A single institution retrospective cohort of 669 consecutive mCRC patients between 2000-9 with clinical follow-up was studied for frequent 7 mutations in codons 12/13 by ARMS-scorpion real-time PCR (Therascreen, Qiagen) and TaqMelt PCR assay cobas KRAS Mutation Test (Roche), which are designed to detect 19 mutations in KRAS codons 12, 13 and 61. DNA was obtained by cobas DNA preparation kit (Roche) from one single 5um FFPE tissue section and by QIAamp DNA FFPE Tissue Kit (Qiagen) from 50um of tissue. Results KRAS mutation was detected by the cobas KRAS Mutation Test in 41.2% of mCRC patients and was correlated with poor overall survival (OS) (p = 0.013), OS from the metastatic diagnosis for metachronous disease (p = 0.025), disease-free survival for metastatic disease (p = 0.012) and time to progression (p = 0.046). KRAS mutation was significantly associated with tumor grade (p = 0.025) and liver as first site of dissemination (p = 0.047). However, KRAS mutations detected by Therascreen did not impact on prognosis. The median of DNA concentration obtained by cobas and QIAamp was 120ng/ul and 80ng/ul, respectively. The frequency of invalid cases was 5.7% (cobas) and 17.9% (Therascreen). Finally, cobas identified 19.2% additional KRAS mutations not detected by Therascreen. Next Generation Sequencing by 454 GS FLX+ System analysis is ongoing to validate discrepant mutations. Conclusions The cobas KRAS Mutation Test is a robust and reproducible assay that, 1) obtains superior DNA recovery from very small amount of FFPE tissue; 2) detects up to 19.2% of mutations not detected by other methods; and, 3) KRAS mutations detected by cobas correlate with poor outcome in mCRC. Disclosure All authors have declared no conflicts of interest.