Automated universal BRAF state detection within the activation segment in skin metastases by pyrosequencing-based assay U-BRAF(V600).
A. Skorokhod, P. Helmbold, B. Brors, P. Schirmacher, A. Enk, and R. Penzel.
PLoS One 8 (3): e59221 (2013)

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.

URL: http://dx.doi.org/10.1371/journal.pone.0059221
DOI: 10.1371/journal.pone.0059221
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@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
}

%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.

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Automated universal BRAF state detection within the activation segment in skin metastases by pyrosequencing-based assay U-BRAF(V600).
A. Skorokhod, P. Helmbold, B. Brors, P. Schirmacher, A. Enk, and R. Penzel.
PLoS One 8 (3): e59221 (2013)

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Automated universal BRAF state detection within the activation segment in skin metastases by pyrosequencing-based assay U-BRAF(V600).A. Skorokhod, P. Helmbold, B. Brors, P. Schirmacher, A. Enk, and R. Penzel. PLoS One 8 (3): e59221 (2013)

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Automated universal BRAF state detection within the activation segment in skin metastases by pyrosequencing-based assay U-BRAF(V600).
A. Skorokhod, P. Helmbold, B. Brors, P. Schirmacher, A. Enk, and R. Penzel.
PLoS One 8 (3): e59221 (2013)