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.

In various cell types certain stresses can stimulate p38 mitogen-activated protein kinase (p38 MAPK), leading to the transcriptional activation of genes that contribute to appropriate compensatory responses. In this report the mechanism of p38-activated transcription was studied in cardiac myocytes where this MAPK is a key regulator of the cell growth and the cardiac-specific gene induction that occurs in response to potentially stressful stimuli. In the cardiac atrial natriuretic factor (ANF) gene, a promoter-proximal serum response element (SRE), which binds serum response factor (SRF), was shown to be critical for ANF induction in primary cardiac myocytes transfected with the selective p38 MAPK activator, MKK6 (Glu). This ANF SRE does not possess sequences typically required for the binding of the Ets-related ternary complex factors (TCFs), such as Elk-1, indicating that p38-mediated induction through this element may take place independently of such TCFs. Although p38 did not phosphorylate SRF in vitro, it efficiently phosphorylated ATF6, a newly discovered SRF-binding protein that is believed to serve as a coactivator of SRF-inducible transcription at SREs. Expression of an ATF6 antisense RNA blocked p38-mediated ANF induction through the ANF SRE. Moreover, when fused to the Gal4 DNA-binding domain, an N-terminal 273-amino acid fragment of ATF6 was sufficient to support trans-activation of Gal4/luciferase expression in response to p38 but not the other stress kinase, Nterminal Jun kinase (JNK); p38-activating cardiac growth promoters also stimulated ATF6 trans-activation. These results indicate that through ATF6, p38 can augment SRE-mediated transcription independently of Ets-related TCFs, representing a novel mechanism of SRF-dependent transcription by MAP kinases.

Approximately 50% of human melanomas harbor activating mutations in the BRAF oncogene, and a large majority are of the V600E (1799T>A) type. RG7204 (PLX4032), a highly selective small molecule that targets V600E-mutated BRAF, has shown striking efficacy in Phase 1 and Phase 2 clinical trials in metastatic melanoma. These findings create a critical need for a robust well-validated companion diagnostic test that is both sensitive and specific for the mutation. Here we describe analytic performance characteristics of the cobas BRAF V600 Mutation Test, a real-time PCR assay, which was designed to detect the V600E (1799T>A) mutation in formalin-fixed paraffin-embedded (FFPE) samples of malignant melanoma, and has been used to select patients in clinical trials of RG7204. Analytic sensitivity was assessed using DNA blends from melanoma cell lines, DNA blends from FFPE tumor sections of melanoma, and individual FFPE tumor specimens with low levels of BRAF mutant alleles, as determined by 454 sequencing (a quantitative “deep” sequencing method). A > 96% hit rate was obtained across all specimen types with 5% mutant sequences at a DNA input of 125 ng per PCR reaction, a DNA amount readily obtained from a single 5-micron FFPE tissue section. The calculated PROBIT values (95% probability) for the cobas test ranged from 0.6 to 30 ng per PCR genomic DNA input for specimens with 5% mutant sequences. The cobas test was compared to bidirectional Sanger sequencing using a set of 219 FFPE melanoma samples. The cobas test detected 14 V600E mutations among the 120 samples (11.7%) that were called mutation-negative by Sanger sequencing, and in all 14 cases a V600E mutation was confirmed by 454 sequencing. Conversely the cobas test detected no V600E mutation in 5 of 99 samples (5%) that were called mutant-positive by Sanger, and in all 5 cases 454 sequencing detected no mutation. The cobas test showed no cross reactivity with BRAF homologs, including RAF1, ARAF and the BRAF pseudogene; however, some cross-reactivity with V600K and V600D was observed with the cobas test. When a panel of tumor samples (including samples with low levels of mutant alleles and low tumor content) was repeatedly tested by two operators, using different instruments and reagents lots, a correct mutation call was obtained in 98.75% of tests. These analytic studies demonstrate that the cobas BRAF V600 Mutation Test is a reproducible assay that is more sensitive and more specific than conventional Sanger sequencing for the detection of the V600E (1799T>A) mutation and detects some non-V600E mutations. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2212. doi:10.1158/1538-7445.AM2011-2212

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