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Diagnostic Technologies and Molecular and Cellular Profiling: Biomarker Assay Development, Validation, and Qualification

Development of a companion diagnostic test for inhibitors of V600E BRAF

Roche Molecular Systems, Pleasanton, CA and Plexxikon, Berkeley, CA

Abstract

A13

Mutations of the human BRAF gene are detected in approximately 8% of cancer samples, primarily in cutaneous melanomas (70%). The most common mutation (90%) is a valine-to-glutamic acid mutation at residue 600 (V600E), which mimics the phosphorylation of the kinase domain activation segment that is thought to destabilize a hydrophobic interaction of V600 in the inactive state. PLX4032 is a novel small molecule selected for its specific inhibitory activity to the mutant B-Raf protein. This compound showed strong specificity for and potency against melanoma and colorectal cell lines bearing the V600E mutation. In addition, it demonstrated robust efficacy in xenograft models using a once-daily low oral dose, with evidence of tumor shrinkage and prolonged delay of tumor growth for a significant post-dosing period. The development of PLX4032 as a cancer therapy will be expedited by a companion diagnostic test to detect the BRAF^V600E mutation as a biomarker for patient selection. For this important biomarker, we have initiated development of a real-time PCR assay on the Roche Molecular Diagnostics COBAS® TaqMan® 48. The assay, which can be used with fresh frozen or paraffin-embedded tissue, is based on the amplification of a short DNA fragment. We evaluated the sensitivity and specificity of the test with cell line DNA samples containing the V600E mutation, showing that it can detect the V600E mutation when it is present in only 10% of the mixture with the wild-type BRAF gene. We also validated the analytical performance of this test with clinical samples of melanomas. The development of our assay as a companion diagnostic test to select patients with the BRAF^V600E mutation for treatment with PLX4032 is a compelling example of personalized medicine in cancer management.