title: Automated universal BRAF state detection within the activation segment in skin metastases by pyrosequencing-based assay U-BRAF(V600).
abstract: 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(V600) 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 U-BRAF(V600) 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(V600) 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(V600) assay as a most powerful sequencing-based diagnostic tool to automatically identify BRAF state as a prerequisite to targeted therapy.
authors.affiliation: Department of Dermatology, Heidelberg University Hospital, Heidelberg, Germany. Alexander.Skorokhod@med.uni-heidelberg.de
authors.forename: Alexander
authors.initials: A
authors.lastname: Skorokhod
authors.forename: Peter
authors.initials: P
authors.lastname: Helmbold
authors.forename: Benedikt
authors.initials: B
authors.lastname: Brors
authors.forename: Peter
authors.initials: P
authors.lastname: Schirmacher
authors.forename: Alexander
authors.initials: A
authors.lastname: Enk
authors.forename: Roland
authors.initials: R
authors.lastname: Penzel
mesh_terms: D000368:Aged; D000369:Aged, 80 and over; D005091:Exons; D005260:Female; D005557:Formaldehyde; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D008297:Male; D008545:Melanoma / Q000175:diagnosis* / Q000235:genetics; D008875:Middle Aged; D009362:Neoplasm Metastasis; D009363:Neoplasm Proteins / Q000235:genetics*; D016612:Paraffin Embedding; D048493:Proto-Oncogene Proteins B-raf / Q000235:genetics*; D017422:Sequence Analysis, DNA / Q000379:methods*; D012878:Skin Neoplasms / Q000175:diagnosis* / Q000235:genetics; D016707:Tissue Fixation
chemical_list: D009363:Neoplasm Proteins; D005557:Formaldehyde; C482119:BRAF protein, human; D048493:Proto-Oncogene Proteins B-raf
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