Automated universal BRAF state detection within the activation segment in skin metastases by pyrosequencing-based assay U-BRAFV600
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-BRAFV600 approach - a universal py...
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| Main Authors: | , , , , , |
|---|---|
| Format: | Article (Journal) |
| Language: | English |
| Published: |
2013
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| In: |
PLOS ONE
Year: 2013, Volume: 8, Issue: 3, Pages: 1-10 |
| ISSN: | 1932-6203 |
| DOI: | 10.1371/journal.pone.0059221 |
| Online Access: | Verlag, kostenfrei, Volltext: https://doi.org/10.1371/journal.pone.0059221 Verlag, kostenfrei, Volltext: https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0059221 
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| Author Notes: | Alexander Skorokhod, Peter Helmbold, Benedikt Brors, Peter Schirmacher, Alexander Enk, Roland Penzel |
MARC
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| 245 | 1 | 0 | |a Automated universal BRAF state detection within the activation segment in skin metastases by pyrosequencing-based assay U-BRAFV600 |c Alexander Skorokhod, Peter Helmbold, Benedikt Brors, Peter Schirmacher, Alexander Enk, Roland Penzel |
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| 520 | |a 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-BRAFV600 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-BRAFV600 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-BRAFV600 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-BRAFV600 assay as a most powerful sequencing-based diagnostic tool to automatically identify BRAF state as a prerequisite to targeted therapy. | ||
| 650 | 4 | |a Amplification-refractory mutation system analysis | |
| 650 | 4 | |a Colorectal cancer | |
| 650 | 4 | |a Cutaneous melanoma | |
| 650 | 4 | |a Dideoxy DNA sequencing | |
| 650 | 4 | |a Metastasis | |
| 650 | 4 | |a Mutation databases | |
| 650 | 4 | |a Mutation detection | |
| 650 | 4 | |a Polymerase chain reaction | |
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