A sensitive and accurate test for simultaneous detection of 36 BRAF and NRAS mutations.

Abstract

e24153 Background: Activating mutations in the mitogen-activated protein kinase pathway are among the most common mutations found in cancer cells. Because of the interaction between BRAF and NRAS, there is interest in simultaneous testing for mutation status of these genes. We have developed a multiplexed, allele-specific PCR kit for broad and sensitive detection of 36 BRAF and NRAS mutations. The BRAF/NRAS Mutation Test kit has been verified for use with FFPE tissue and plasma (Research Use Only).The sample to result can be performed in eight hours or less. A web-based analysis tool enables automated and rapid interpretation of results. We describe the studies done to verify the test performance and to evaluate its correlation with an Illumina MiSeq method. Methods: We tested contrived specimens as well as FFPE tissue and plasma specimens from melanoma, non-small cell lung, and colorectal cancer patients. Results for analytical sensitivity, exclusivity, repeatability, and correlation with the MiSeq method are reported. Results: The BRAF/NRAS Mutation Test demonstrated high sensitivity, specificity and broad mutation coverage. Repeatability was 100% across all samples, operators, and instruments. Analytical sensitivity was high for tissue and plasma samples. 1% sensitivity was achieved for 34 mutations using mutant plasmid DNA in 50ng genomic DNA. 2.5% mutant allele frequency in 50ng FFPE DNA and as low as 0.03% from 2mL plasma were detected. Plasmids containing highly similar HRAS, KRAS, and KRAS pseudogene plus A-RAF, C-RAF, and BRAF pseudogene sequences were not detected, showing specificity for NRAS and BRAF. The test showed high concordance for 164 tumor FFPE tissue from melanoma, CRC, and NSCLC (98.2%) and 186 plasma specimens from melanoma and CRC (98.9%) when compared to MiSeq sequencing. Conclusions: The BRAF/NRAS Mutation Test is a sensitive and reproducible kit for the detection of 11 BRAF mutations in exons 11 and 15 and 25 NRAS mutations in exons 2, 3, and 4. The simplified lab workflow and assay reliability make it suitable for the accurate detection of mutations from tissue and plasma biopsies.