Comparison of Testing Methods for the Detection of BRAF V600E Mutations in Malignant Melanoma: Pre-Approval Validation Study of the Companion Diagnostic Test for Vemurafenib

Fernando Lopez-Rios,Debbie Mair,Rebeca Martinez,Belen Gomez,Barbara Angulo,Rachel Langland,Esther Conde,David Gonzalez De Castro,Felice Shieh,Jeffrey Vaks,H Jeffrey Lawrence,Keiran Smalley

doi:10.1371/journal.pone.0053733

Abstract

BackgroundThe cobas 4800 BRAF V600 Mutation Test is a CE-marked and FDA-approved in vitro diagnostic assay used to select patients with metastatic melanoma for treatment with the selective BRAF inhibitor vemurafenib. We describe the pre-approval validation of this test in two external laboratories.MethodsMelanoma specimens were tested for BRAF V600 mutations at two laboratories with the: cobas BRAF Mutation Test; ABI BRAF test; and bidirectional direct sequencing. Positive (PPA) and negative (NPA) percent agreements were determined between the cobas test and the other assays. Specimens with discordant results were tested with massively parallel pyrosequencing (454). DNA blends with 5% mutant alleles were tested to assess detection rates.ResultsInvalid results were observed in 8/116 specimens (6·9%) with Sanger, 10/116 (8·6%) with ABI BRAF, and 0/232 (0%) with the cobas BRAF test. PPA was 97·7% for V600E mutation for the cobas BRAF test and Sanger, and NPA was 95·3%. For the cobas BRAF test and ABI BRAF, PPA was 71·9% and NPA 83·7%. For 16 cobas BRAF test-negative/ABI BRAF-positive specimens, 454 sequencing detected no codon 600 mutations in 12 and variant codon 600 mutations in four. For eight cobas BRAF test-positive/ABI BRAF-negative specimens, four were V600E and four V600K by 454 sequencing. Detection rates for 5% mutation blends were 100% for the cobas BRAF test, 33% for Sanger, and 21% for the ABI BRAF. Reproducibility of the cobas BRAF test was 111/116 (96%) between the two sites.ConclusionsIt is feasible to evaluate potential companion diagnostic tests in external laboratories simultaneously to the pivotal clinical trial validation. The health authority approved assay had substantially better performance characteristics than the two other methods. The overall success of the cobas BRAF test is a proof of concept for future biomarker development.

Highlights

The new paradigm of targeted drug development in cancer medicine is to design agents that inhibit specific recurring genetic lesions in tumors
A successful example of this strategy is the focused and integrated co-development of the novel BRAF inhibitor vemurafenib and its companion in vitro diagnostic (IVD), the cobas 4800 BRAF V600 Mutation Test (‘‘RT-PCR test’’), which resulted in Food and Drug Administration (FDA) approval of vemurafenib in 2011, followed soon thereafter with CE-IVD marking in Europe - indicator that allows the free distribution of products within the European Union to meet essential requirements regarding safety, health and environmental protection, less than 5 years after the Investigational New Drug Application (Figure 1) [2,3]
Approval of vemurafenib was granted for the treatment of BRAF V600E mutation positive metastatic melanoma based on the results of a pivotal randomized phase 3 trial of vemurafenib vs. dacarbazine, which demonstrated that vemurafenib treatment results in significant improvements in overall survival, progression-free survival, and objective response rate [4]

Summary

Introduction

The new paradigm of targeted drug development in cancer medicine is to design agents that inhibit specific recurring genetic lesions in tumors. A critical component of this model is the codevelopment of robust and accurate companion in vitro diagnostic (IVD) assays to detect these specific genetic lesions and to identify patients likely to benefit from a given targeted treatment [1]. A successful example of this strategy is the focused and integrated co-development of the novel BRAF inhibitor vemurafenib and its companion IVD, the cobas 4800 BRAF V600 Mutation Test (‘‘RT-PCR test’’), which resulted in Food and Drug Administration (FDA) approval of vemurafenib in 2011, followed soon thereafter with CE-IVD marking in Europe - indicator that allows the free distribution of products within the European Union to meet essential requirements regarding safety, health and environmental protection-, less than 5 years after the Investigational New Drug Application (Figure 1) [2,3]. The cobas 4800 BRAF V600 Mutation Test is a CE-marked and FDA-approved in vitro diagnostic assay used to select patients with metastatic melanoma for treatment with the selective BRAF inhibitor vemurafenib. We describe the preapproval validation of this test in two external laboratories

Methods

Results

Discussion

Conclusion