Comparative Evaluation of the FDA Approved THxIDTM BRAF Test in a Hospital-Based Molecular Laboratory

摘要

Background: Since patients diagnosed with BRAF V600E and V600K mutated advanced melanoma show response to treatment with MAP kinase inhibitors, several sensitive methods have been developed to determine the V600 allele status of melanoma patients. The new FDA-approved THxID? BRAF companion diagnostic test was compared to two other testing methods designed and used for routine diagnostic testing in the Montpellier CHU hospital. Patients and methods: A series of 82 formalin-fixed paraffin-embedded (FFPE) tissue specimens of malignant melanoma were analyzed. DNA was extracted from whole 5 urn tissue sections (n=18) and punches (n=64). Samples were tested for BRAF V600 mutations using the THxID? BRAF test, High Resolution Melting (HRM), and bidirectional Sanger sequencing, on 7500 Fast Dx Real-Time PCR (Applied Biosystems), cobas? 4800 System (Roche Molecular Diagnostics), and ABI PRISM? 3100 Genetic Analyzer (Applied Biosystems), respectively. Positive (PPA) and negative (NPA) percent agreements were determined between the THxID? BRAF test and the other assays. Results: Invalid results were observed in 1/82 specimens (1.2%) with Sanger (not included in percent agreement calculations), 0/82 with HRM, and 1/82 (1.2%) with the THxID? BRAF test. PPA was 35/36 (97.2%) for V600E and V600K mutations combined for the THxID? BRAF test and HRM, and NPA was 44/46 (95.7%). For the THxID? BRAF test and Sanger, PPA was 36/37 (97.3%) and NPA 44/44 (100%). One V600E sample identified by THxID? BRAF test was detected as wild-type by HRM and uninterpretable by Sanger. Interestingly, all V600K (n=3) were detected using the 3 different approaches. Percent agreement values were not lower when using punches (n=64) versus slides (n=18) suggesting this new format is suitable for the THxID? BRAF assay. Conclusion: This study demonstrated the high agreement between the FDA approved THxID? BRAF assay and two technically different testing methods: Sanger sequencing and HRM. It has also highlighted the potential of THxID? BRAF to be applied to a broader range of sample types than claimed in the current "instructions for use", an extension that would require the ad hoc validation and approval.