Automated Universal BRAF State Detection within the Activation Segment in Skin Metastases by Pyrosequencing-Based Assay U-BRAF

Malignant melanoma is a highly-aggressive type of malignancy with considerable metastatic potential and frequent resistance to cytotoxic agents. BRAF mutant protein was recently recognized as therapeutic target in metastatic melanoma. We present a newly-developed U-BRAF approach – a universal pyrosequencing-based assay for mutation detection within activation segment in exon 15 of human braf. We identified 5 different BRAF mutations in a single assay analyzing 75 different formalin-fixed paraffin-embedded (FFPE) samples of cutaneous melanoma metastases from 29 patients. We found BRAF mutations in 21 of 29 metastases. All mutant variants were quantitatively detectable by the newly-developed UBRAF assay. These results were confirmed by ultra-deep-sequencing validation (60,000-fold coverage). In contrast to all other BRAF state detection methods, the U-BRAF assay is capable of automated quantitative identification of at least 36 previously-published BRAF mutations. Under the precaution of a minimum of 3% mutated cells in front of a background of wild type cells, U-BRAFV600 assay design completely excludes false wild-type results. The corresponding algorithm for classification of BRAF-mutated variants is provided. The single-reaction assay and data analysis automation makes our approach suitable for the assessment of large clinical sample sizes. Therefore, we suggest U-BRAF assay as a most powerful sequencing-based diagnostic tool to automatically identify BRAF state as a prerequisite to targeted therapy. Citation: Skorokhod A, Helmbold P, Brors B, Schirmacher P, Enk A, et al. (2013) Automated Universal BRAF State Detection within the Activation Segment in Skin Metastases by Pyrosequencing-Based Assay U-BRAF. PLoS ONE 8(3): e59221. doi:10.1371/journal.pone.0059221 Editor: H. Peter Soyer, The University of Queensland, Australia Received November 28, 2012; Accepted February 12, 2013; Published March 26, 2013 Copyright: 2013 Skorokhod et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This research was funded by Deutsche Krebshilfe [DKH108856]. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: AS declares an inventorship of U-BRAF-V600 assay, a patent for diagnostic application of U-BRAF-V600 is pending. Application Number: EP 12 153 477.0. Filling date: 1st February 2012. Title: Method for variant differentiation utilizing a pyrosequencing dispensation order. State: Pending application. Herewith AS confirms that this patent does not alter his adherence to all the PLOS ONE policies on sharing data and materials. * E-mail: [email protected]