Novel, Simplex And Multiplex Assays For Braf Mutation Detection.

e23257 Background: BRAF gene mutations play key roles in colorectal cancer (CRC) and in malignant melanoma (MM). In CRC, BRAF mutation is associated with poorer prognosis and may be a marker for new targeted therapies. In MM, BRAF mutations are predictive of the efficacy of BRAF-targeted therapies. In CRC, only the V600E change is observed regularly, while in MM different BRAF mutations may occur. To improve the identification of BRAF mutations, we developed novel simplex and multiplex real-time PCR-based assays (SensiScreen) and compared them with Direct Sequencing (DS), Mutant-Enriched PCR (ME-PCR) and Cobas. Methods: SensiScreen assays (PentaBase, Denmark) comprise SuPrimers (DNA primers with increased specificity), BaseBlockers (suppressing amplification of wild-type genes) and HydrolEasy probes (hydrolysis probe with increased S/N ratio and sensitivity). Sensitivity studies on plasmids and extracted cell line DNA were performed. Two cohorts of patients were analysed: 100 CRC patients used for comparison of the V600E SensiScreen assay, DS and ME-PCR; and 100 MM patients used for comparison between SensiScreen assays (simplex and multiplex) and Cobas. Results: Our sensitivity studies gave superimposable results for both simplex and multiplex assays. In plasmids and cell lines experiments, DS had a limit of detection of 10%, ME-PCR of up to 0,5% and SensiScreen of < 0,1%. In 100 CRC cases, we found 7 (7%) BRAF mutated patients by DS, 9 (9%) by ME-PCR and 10 (10%) by SensiScreen. In 100 MM patients, we found 51 (51%) BRAF mutations by Cobas and 55 (55%) by SensiScreen. Simplex and multiplex SensiScreen assays identified the same mutations. Conclusions: SensiScreen simplex and multiplex assays are novel, rapid, easy-to-use and highly sensitive methods for BRAF mutation analysis in CRC and in MM. Indeed, it enables the detection of additional BRAF alterations in CRC and MM patients compared with widely used methodologies.