To ensure the appropriate assignment of vemurafenib to patients with unresectable or metastatic melanoma, accurate detection of activating BRAF mutations is now a clinical imperative. However, the performance of commercially available test kits on challenging samples is unknown. Methods: 126 formalin-fixed, paraffin-embedded melanoma samples were selected for challenging attributes, such as small sample size, that might affect test kit performance. The Qiagen BRAF RGQ PCR version 2 (RGQv2) test

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... kit, intended for research use only, and the FDA-approved companion diagnostic cobas 4800 BRAF V600 Mutation Test were compared for their ability to detect the V600E mutation in challenging samples, using a single 5 μm unstained slide. Results: of the 126 specimens, three samples were invalid by the RGQv2 test, three other samples were invalid by the cobas test, and an additional two samples were found invalid by both tests. For the 118 samples that yielded valid results with both tests, concordant results were observed for 105 (89.0% [95% CI, 82.1%-93.5%]) samples. Of the 12 discordant samples with sufficient material for further testing, next generation sequencing confirmed the cobas test result for 6 (50.0%) and confirmed the RGQv2 test result for the other 6 (50.0%) samples. Five (4.0%) of the RGQv2 test results yielded multiple positive mutation calls and two results had a sample control assay PCR cycle threshold (CT) >33, indicating insufficient amounts of DNA template, but gave accurate mutation calls. Workflow analysis showed that the total time to result was 5.65 hours for the cobas test to process 24 samples and 7.84 hours for RGQv2 assay to process 7 samples. Conclusions: The two commercially available, PCR-based methods demonstrated similar abilities to detect BRAF V600 mutations in challenging melanoma samples. However, the total time-to-result, assay hands-on time, and diagnostic interpretation were more efficient and rapid with the cobas test.