KRAS/BRAF Mutations in Colorectal Carcinoma. A Comparative Study between Real-Time PCR, Sanger Sequencing and Chip-Hybridization PCR Based Technology
Javier Hernandez-Losa, Rosa Somoza, Roso Mares, Esmeralda Lindo, Ana Solsona, Teresa Moline, Stefania Landolfi, Santiago Ramon y Cajal. Hospital University Vall d´Hebron, Barcelona, Spain; Vall d´Hebron Research Institute, Barcelona, Spain
Background: KRAS mutation testing is mandatory before prescribing anti-EGFR mAbs in the treatment of advanced colorectal cancer. Other studies suggest that BRAF mutation could also be relevant in the management of those patients. Sanger sequencing have been established as a Gold Standard method, nevertheless recently several methods have been described to determine those mutations. We compare a new Cancer Mutation Array kit with two well established methods in a series of 200 human carcinoma samples.
Design: We have compared three methods for mutation detections in KRAS and BRAF genes in 200 human colorectal carcinoma samples.We used two different sample sets.To assess the KRAS mutation status we used a kit based on real-time PCR (Therascreen® KRAS mutation kit,Qiagen), and a kit based on PCR amplification and array-hybridization with different probes (CLART® CMA KRAS-BRAF kit,Genomica SAU) in 159 samples. In the BRAF set we used Sanger Sequencing with specific primers of BRAF and the above mentioned CMA kit in 41 samples. The CMA kit detects 9 KRAS mutations (all G12 variants, G13D, Q61H and Q61L) and 2 BRAF mutations (V600E and V600K). DNAs were extracted from FFPE tissue samples. We compare the sensitivity of each method with several plasmids, and cell lines.
Results: The CMA kit detected KRAS and BRAF mutations in less than 5% of DNA mutant in a background of wild type DNA. Therascreen kit detected 67% (108/159) KRAS mutants whereas CMA kit detected 66% (105/159). In the BRAF set, both Sequencing and CMA kit detected 34% (14/41) of BRAF mutants.Discordant cases were analyzed in a second extraction. One G12V detected by Therascreen kit was demonstrated being a false positive in the second round by all methods. G12A and G12V sample mutants detected in both extractions by Therascreen kit were unable to be reproduced by other technologies, probably due to a lower sensitivity in these assays.Furthermore using CMA kit we detected 3 samples with KRAS and BRAF mutations that had been missed in the first analysis. In the KRAS set we detected two V600E samples and we also detected a G12C in the BRAF set.
Conclusions: The new CMA kit detects simultaneously the most prevalent mutations of KRAS and BRAF with high sensitivity and reproducibility in 2 steps. The concordant results obtained by CMA kit compared to a well established methods supports the use of this technology in different screenings to testing KRAS and BRAF mutations in human colorectal cancer samples.
Wednesday, March 6, 2013 9:30 AM
Poster Session V # 259, Wednesday Morning