Specific and sensitive detection of BRAF and KRAS mutations in clinical samples

• Published in: Journal of clinical oncology Vol. 30; no. 15_suppl; p. 10587
• Main Authors: Kelchner, Vanessa, Chupreta, Sergey, Mishra, Anjali, Beaubien, Ron, Kurihara, Laurie, Laliberte, Julie, Mikheykin, Andrey, Beer, David G., Makarov, Vladimir
• Format: Journal Article
• Language: English
• Published: 20.05.2012
• ISSN: 0732-183X; 1527-7755
• DOI: 10.1200/jco.2012.30.15_suppl.10587

Abstract only 10587 Background: The development of highly sensitive and specific genotyping assays that are suitable for clinical research and molecular diagnostics opens new opportunities for the detection, assessment, and research of cancer. Swift Biosciences has developed myT Primer qPCR technology which has unique structural and thermodynamic properties that enables highly sensitive mismatch discrimination. The myT Primer reagents can detect 1% mutant BRAF or KRAS present in a background of 10 3 wild-type genomic DNA copies with no background signal from wild-type. A separate ultrasensitive BRAF format has been developed which enables the detection of a single mutant BRAF copy in > 10 4 wild-type genomic DNA copies, representing 0.01% detection. Methods: To evaluate the performance on clinical samples, DNA was prepared from 26 melanoma, 40 colorectal, and 49 lung tumor fresh frozen and FFPE samples, and their corresponding normal adjacent samples. qPCR was performed using standard cycling conditions in single tube format with allele-specific myT primers to assess two BRAF alleles or seven KRAS alleles. Performance of the assay was evaluated on the ABI 7500, CFX96, and Roche LightCycler. A subset of samples were selected for validation by Sanger sequencing. Results: Mutations of BRAF in melanoma, colorectal, and lung tumors were observed to be 46%, 5%, and 2%, in concordance with published data for the frequency of BRAF mutations. Mutations of KRAS in colorectal cancer were observed to be in 17/40, or 42% of samples, consistent with published reports of KRAS in colorectal cancer. 34% of lung cancer samples had a KRAS mutation, also consistent with available statistics. Mutation status was confirmed with Sanger sequencing in the subset of selected samples. Conclusions: The extreme selectivity of the myT BRAF Primers makes them well suited for genotyping of conventional FFPE and frozen tissue samples, whereas the ultrasensitive format is ideal for use with difficult samples such as needle biopsies, CTCs, and serum. The extreme selectivity of these primers results in a definitive Yes/No answer and eliminates the need to use a delta Ct method.