Analytical performance of a real-time PCR-based assay for V600 mutations in the BRAF gene, used as the companion diagnostic test for the novel BRAF inhibitor vemurafenib in metastatic melanoma
Melanomas frequently harbor BRAFV600 mutations. Vemurafenib (RG7204/PLX4032), a small-molecule inhibitor of mutant BRAF, has shown striking clinical efficacy in BRAFV600 mutant melanoma, creating the need for a well-validated companion diagnostic to select patients for treatment. We describe analyti...
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Published in | Diagnostic molecular pathology Vol. 21; no. 1; p. 1 |
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Main Authors | , , , , , , , , |
Format | Journal Article |
Language | English |
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United States
01.03.2012
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Abstract | Melanomas frequently harbor BRAFV600 mutations. Vemurafenib (RG7204/PLX4032), a small-molecule inhibitor of mutant BRAF, has shown striking clinical efficacy in BRAFV600 mutant melanoma, creating the need for a well-validated companion diagnostic to select patients for treatment. We describe analytic performance characteristics of the cobas 4800 BRAF V600 Mutation Test, the test used to select patients for the pivotal vemurafenib trials. This real-time polymerase chain reaction assay was designed to detect the V600E (1799T>A) mutation DNA from formalin-fixed paraffin-embedded tissue samples. Sensitivity was assessed using blends of cell lines or tumor DNA, and tumor specimens with low levels of mutant alleles, as determined by 454 sequencing (a quantitative next-generation pyrosequencing method). A >96% hit rate was obtained across all specimen types with 5% mutant alleles at a DNA input of 125 ng, an amount readily obtained from one 5-μm section. The cobas test showed a higher sensitivity and specificity than direct bidirectional sequencing in a panel of 219 melanoma specimens. Cross reactivity with V600K and V600D was observed. Repeated testing of 5 specimens by 2 operators, using different instruments and reagent lots, yielded correct calls in 158/160 tests (98.8%). A set of 26 highly pigmented samples were identified that gave invalid test results. A simple 1:2 dilution resulted in a valid test result of 76% in such cases. The cobas test is a reproducible assay that detects some non-V600E mutations and is more accurate than direct sequencing in detecting BRAFV600E. |
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AbstractList | Melanomas frequently harbor BRAFV600 mutations. Vemurafenib (RG7204/PLX4032), a small-molecule inhibitor of mutant BRAF, has shown striking clinical efficacy in BRAFV600 mutant melanoma, creating the need for a well-validated companion diagnostic to select patients for treatment. We describe analytic performance characteristics of the cobas 4800 BRAF V600 Mutation Test, the test used to select patients for the pivotal vemurafenib trials. This real-time polymerase chain reaction assay was designed to detect the V600E (1799T>A) mutation DNA from formalin-fixed paraffin-embedded tissue samples. Sensitivity was assessed using blends of cell lines or tumor DNA, and tumor specimens with low levels of mutant alleles, as determined by 454 sequencing (a quantitative next-generation pyrosequencing method). A >96% hit rate was obtained across all specimen types with 5% mutant alleles at a DNA input of 125 ng, an amount readily obtained from one 5-μm section. The cobas test showed a higher sensitivity and specificity than direct bidirectional sequencing in a panel of 219 melanoma specimens. Cross reactivity with V600K and V600D was observed. Repeated testing of 5 specimens by 2 operators, using different instruments and reagent lots, yielded correct calls in 158/160 tests (98.8%). A set of 26 highly pigmented samples were identified that gave invalid test results. A simple 1:2 dilution resulted in a valid test result of 76% in such cases. The cobas test is a reproducible assay that detects some non-V600E mutations and is more accurate than direct sequencing in detecting BRAFV600E. |
Author | Hillman, Grantland Shah, Sweta Cheng, Suzanne Halait, Harkanwal Demartin, Kelli Soviero, Stephen Langland, Rachel Wu, Lin Lawrence, H Jeffrey |
Author_xml | – sequence: 1 givenname: Harkanwal surname: Halait fullname: Halait, Harkanwal organization: Roche Molecular Diagnostics, Pleasanton, CA 94588, USA – sequence: 2 givenname: Kelli surname: Demartin fullname: Demartin, Kelli – sequence: 3 givenname: Sweta surname: Shah fullname: Shah, Sweta – sequence: 4 givenname: Stephen surname: Soviero fullname: Soviero, Stephen – sequence: 5 givenname: Rachel surname: Langland fullname: Langland, Rachel – sequence: 6 givenname: Suzanne surname: Cheng fullname: Cheng, Suzanne – sequence: 7 givenname: Grantland surname: Hillman fullname: Hillman, Grantland – sequence: 8 givenname: Lin surname: Wu fullname: Wu, Lin – sequence: 9 givenname: H Jeffrey surname: Lawrence fullname: Lawrence, H Jeffrey |
BackLink | https://www.ncbi.nlm.nih.gov/pubmed/22306669$$D View this record in MEDLINE/PubMed |
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Snippet | Melanomas frequently harbor BRAFV600 mutations. Vemurafenib (RG7204/PLX4032), a small-molecule inhibitor of mutant BRAF, has shown striking clinical efficacy... |
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SubjectTerms | Cell Line, Tumor DNA Mutational Analysis - methods DNA, Neoplasm - analysis Formaldehyde Humans Indoles - therapeutic use Melanoma - drug therapy Melanoma - genetics Melanoma - secondary Mutation Paraffin Embedding Patient Selection Predictive Value of Tests Proto-Oncogene Proteins B-raf - antagonists & inhibitors Proto-Oncogene Proteins B-raf - genetics Proto-Oncogene Proteins B-raf - metabolism Real-Time Polymerase Chain Reaction - methods Reproducibility of Results Skin Neoplasms - drug therapy Skin Neoplasms - genetics Skin Neoplasms - pathology Sulfonamides - therapeutic use Tissue Fixation |
Title | Analytical performance of a real-time PCR-based assay for V600 mutations in the BRAF gene, used as the companion diagnostic test for the novel BRAF inhibitor vemurafenib in metastatic melanoma |
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