Performance Characteristics of a PCR Assay for the Detection of KRAS Mutations in Formalin-Fixed Paraffin-Embedded Tissue Samples of Non-Small Cell Lung Cancer

Abstract

Abstract Introduction: Approximately 25% of non-small cell lung cancer (NSCLC) tumors contain mutations in KRAS. These tumors are insensitive to therapy directed against the epidermal growth factor receptor and appear to be resistant to adjuvant chemotherapy. The current study demonstrates the performance of the cobas® KRAS Mutation Test (cobas test), a TaqMelt polymerase chain reaction (PCR) assay designed to detect 19 mutations in codons 12, 13, and 61. Methods: To reflect real-world testing conditions, the study used formalin-fixed, paraffin-embedded tissue (FFPET) NSCLC samples for the predominant mutations. DNA blends of cell lines and plasmids were used where FFPET samples were not available. Results: In the limit of detection study, a correct mutation call rate of ≥95% was obtained with approximately 5% mutant sequences using 3.1-50.0 ng DNA per PCR reaction. Mutation levels as low as 2.4% consistently yielded correct mutation calls when 50.0 ng DNA was used for testing. The cobas test performance was compared to Sanger sequencing in a method correlation using two cobas reagent lots and 194 specimens. After resolution of discordant results using 454 sequencing, the positive, negative, and overall percent agreement for mutations in codons 12/13 and 61 between the cobas test and sequencing ranged from 97.0% to 100%. Mutation detection was 100% reproducible and showed greater specificity than Sanger sequencing. Test performance was not impaired by the presence of interfering substances or clinically relevant microbes, and inclusivity testing demonstrated the kit’s ability to detect rare mutations. Conclusions: The cobas test is a robust, sensitive, and reproducible method for detecting KRAS mutations in FFPET tumor samples from NSCLC patients.