%0 Journal Article %1 Skorokhod2013 %A Skorokhod, Alexander %A Helmbold, Peter %A Brors, Benedikt %A Schirmacher, Peter %A Enk, Alexander %A Penzel, Roland %D 2013 %J PLoS One %K 80 Aged, Aged; Analysis, B-raf, DNA, Embedding; Exons; Female; Fixation Formaldehyde; High-Throughput Humans; Male; Melanoma, Metastasis; Middle Neoplasm Neoplasms, Nucleotide Paraffin Proteins Proteins, Proto-Oncogene Sequence Sequencing; Skin Tissue and diagnosis/genetics; genetics; methods; over; %N 3 %P e59221 %R 10.1371/journal.pone.0059221 %T Automated universal BRAF state detection within the activation segment in skin metastases by pyrosequencing-based assay U-BRAF(V600). %U http://dx.doi.org/10.1371/journal.pone.0059221 %V 8 %X 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.

@article{Skorokhod2013, __markedentry = {[bbrors:6]}, 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.}, added-at = {2015-04-09T12:36:21.000+0200}, author = {Skorokhod, Alexander and Helmbold, Peter and Brors, Benedikt and Schirmacher, Peter and Enk, Alexander and Penzel, Roland}, biburl = {https://www.bibsonomy.org/bibtex/2aa3cea99bd9ee6e0a2b2dc2d60875770/bbrors}, doi = {10.1371/journal.pone.0059221}, institution = {Department of Dermatology, Heidelberg University Hospital, Heidelberg, Germany. Alexander.Skorokhod@med.uni-heidelberg.de}, interhash = {97eb72de3fbddf7d02bd09c995a483f5}, intrahash = {aa3cea99bd9ee6e0a2b2dc2d60875770}, journal = {PLoS One}, keywords = {80 Aged, Aged; Analysis, B-raf, DNA, Embedding; Exons; Female; Fixation Formaldehyde; High-Throughput Humans; Male; Melanoma, Metastasis; Middle Neoplasm Neoplasms, Nucleotide Paraffin Proteins Proteins, Proto-Oncogene Sequence Sequencing; Skin Tissue and diagnosis/genetics; genetics; methods; over;}, language = {eng}, medline-pst = {ppublish}, number = 3, owner = {bbrors}, pages = {e59221}, pii = {PONE-D-12-37414}, pmid = {23555633}, timestamp = {2015-04-09T12:36:21.000+0200}, title = {Automated universal BRAF state detection within the activation segment in skin metastases by pyrosequencing-based assay U-BRAF(V600).}, url = {http://dx.doi.org/10.1371/journal.pone.0059221}, volume = 8, year = 2013 }