title: 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.
abstract: BACKGROUND
  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 pre-approval validation of this test in two external laboratories.
  
  
  METHODS
  Melanoma 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.
  
  
  RESULTS
  Invalid 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.
  
  
  CONCLUSIONS
  It 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.
authors: Laboratorio de Dianas Terapéuticas, Hospital Universitario Sanchinarro, Madrid, Spain. flopezrios@hospitaldemadrid.com
authors: Fernando
authors: F
authors: Lopez-Rios
authors: Barbara
authors: B
authors: Angulo
authors: Belen
authors: B
authors: Gomez
authors: Debbie
authors: D
authors: Mair
authors: Rebeca
authors: R
authors: Martinez
authors: Esther
authors: E
authors: Conde
authors: Felice
authors: F
authors: Shieh
authors: Jeffrey
authors: J
authors: Vaks
authors: Rachel
authors: R
authors: Langland
authors: H Jeffrey
authors: HJ
authors: Lawrence
authors: David Gonzalez
authors: DG
authors: de Castro
mesh_terms: D004252:DNA Mutational Analysis / Q000379:methods*; D003955:Diagnostic Tests, Routine / Q000379:methods*; D006801:Humans; D007211:Indoles / Q000494:pharmacology / Q000627:therapeutic use*; D008545:Melanoma / Q000188:drug therapy* / Q000235:genetics* / Q000473:pathology; D009154:Mutation / Q000235:genetics*; D048493:Proto-Oncogene Proteins B-raf / Q000235:genetics*; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013449:Sulfonamides / Q000494:pharmacology / Q000627:therapeutic use*; D000077484:Vemurafenib
chemical_list: D007211:Indoles; D013449:Sulfonamides; D000077484:Vemurafenib; C482119:BRAF protein, human; D048493:Proto-Oncogene Proteins B-raf