title: Multisite analytic performance studies of a real-time polymerase chain reaction assay for the detection of BRAF V600E mutations in formalin-fixed, paraffin-embedded tissue specimens of malignant melanoma.
abstract: BACKGROUND
  A polymerase chain reaction-based companion diagnostic (cobas 4800 BRAF V600 Mutation Test) was recently approved by the US Food and Drug Administration to select patients with BRAF-mutant metastatic melanoma for treatment with the BRAF inhibitor vemurafenib.
  
  
  OBJECTIVE
  (1) To compare the analytic performance of the cobas test to Sanger sequencing by using screening specimens from phase II and phase III trials of vemurafenib, and (2) to assess the reproducibility of the cobas test at different testing sites.
  
  
  METHODS
  Specimens from 477 patients were used to determine positive and negative percent agreements between the cobas test and Sanger sequencing for detecting V600E (1799T>A) mutations. Specimens were evaluated with a massively parallel pyrosequencing method (454) to resolve discordances between polymerase chain reaction and Sanger results. Reproducibility of the cobas test was assessed at 3 sites by using 3 reagent lots and an 8-member panel of melanoma samples.
  
  
  RESULTS
  A valid cobas result was obtained for all eligible patients. Sanger sequencing had a failure rate of 9.2% (44 of 477). For the remaining 433 specimens, positive percent agreement was 96.4% (215 of 223) and negative percent agreement, 80% (168 of 210). Among 42 cobas mutation-positive/Sanger V600E-negative specimens, 17 were V600E positive and 24 were V600K positive by 454. The cobas test detected 70% of V600K mutations. In the reproducibility study, a correct interpretation was made for 100% of wild-type specimens and specimens with greater than 5% mutant alleles; V600E mutations were detected in 90% of specimens with less than 5% mutant alleles.
  
  
  CONCLUSIONS
  The cobas test (1) had a lower assay failure rate than that of Sanger, (2) was more sensitive in detecting V600E mutations, (3) detected most V600K mutations, and (4) was highly reproducible.
authors.affiliation: Medical and Scientific Affairs, Roche Molecular Systems, Inc, 4300 Hacienda Dr, Pleasanton, CA 94588, USA.
authors.forename: Steven
authors.initials: S
authors.lastname: Anderson
authors.forename: Kenneth J
authors.initials: KJ
authors.lastname: Bloom
authors.forename: Dino U
authors.initials: DU
authors.lastname: Vallera
authors.forename: Josef
authors.initials: J
authors.lastname: Rueschoff
authors.forename: Cliff
authors.initials: C
authors.lastname: Meldrum
authors.forename: Robert
authors.initials: R
authors.lastname: Schilling
authors.forename: Barbara
authors.initials: B
authors.lastname: Kovach
authors.forename: Ju Ruey-Jiuan
authors.initials: JR
authors.lastname: Lee
authors.forename: Pam
authors.initials: P
authors.lastname: Ochoa
authors.forename: Rachel
authors.initials: R
authors.lastname: Langland
authors.forename: Harkanwal
authors.initials: H
authors.lastname: Halait
authors.forename: H Jeffrey
authors.initials: HJ
authors.lastname: Lawrence
authors.forename: Michael C
authors.initials: MC
authors.lastname: Dugan
mesh_terms: D000293:Adolescent; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D019943:Amino Acid Substitution; D004252:DNA Mutational Analysis / Q000379:methods*; D005260:Female; D005557:Formaldehyde; D006801:Humans; D007211:Indoles / Q000627:therapeutic use; D008297:Male; D008545:Melanoma / Q000188:drug therapy / Q000235:genetics* / Q000473:pathology / Q000556:secondary; D008875:Middle Aged; D020125:Mutation, Missense; D016612:Paraffin Embedding; D047428:Protein Kinase Inhibitors / Q000627:therapeutic use; D048493:Proto-Oncogene Proteins B-raf / Q000037:antagonists & inhibitors / Q000235:genetics*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D013449:Sulfonamides / Q000627:therapeutic use; D016707:Tissue Fixation; D000077484:Vemurafenib; D055815:Young Adult
chemical_list: D007211:Indoles; D047428:Protein Kinase Inhibitors; D013449:Sulfonamides; D005557:Formaldehyde; D000077484:Vemurafenib; C482119:BRAF protein, human; D048493:Proto-Oncogene Proteins B-raf