title: Analytic performance studies and clinical reproducibility of a real-time PCR assay for the detection of epidermal growth factor receptor gene mutations in formalin-fixed paraffin-embedded tissue specimens of non-small cell lung cancer.
abstract: BACKGROUND
  Epidermal growth factor receptor (EGFR) gene mutations identify patients with non-small cell lung cancer (NSCLC) who have a high likelihood of benefiting from treatment with anti-EGFR tyrosine kinase inhibitors. Sanger sequencing is widely used for mutation detection but can be technically challenging, resulting in longer turn-around-time, with limited sensitivity for low levels of mutations. This manuscript details the technical performance verification studies and external clinical reproducibility studies of the cobas EGFR Mutation Test, a rapid multiplex real-time PCR assay designed to detect 41 mutations in exons 18, 19, 20 and 21.
  
  
  METHODS
  The assay's limit of detection was determined using 25 formalin-fixed paraffin-embedded tissue (FFPET)-derived and plasmid DNA blends. Assay performance for a panel of 201 specimens was compared against Sanger sequencing with resolution of discordant specimens by quantitative massively parallel pyrosequencing (MPP). Internal and external reproducibility was assessed using specimens tested in duplicate by different operators, using different reagent lots, instruments and at different sites. The effects on the performance of the cobas EGFR test of endogenous substances and nine therapeutic drugs were evaluated in ten FFPET specimens. Other tests included an evaluation of the effects of necrosis, micro-organisms and homologous DNA sequences on assay performance, and the inclusivity of the assay for less frequent mutations.
  
  
  RESULTS
  A >95% hit rate was obtained in blends with >5% mutant alleles, as determined by MPP analysis, at a total DNA input of 150 ng. The overall percent agreement between Sanger sequencing and the cobas test was 96.7% (negative percent agreement 97.5%; positive percent agreement 95.8%). Assay repeatability was 98% when tested with two operators, instruments, and reagent lots. In the external reproducibility study, the agreement was > 99% across all sites, all operators and all reagent lots for 11/12 tumors tested. Test performance was not compromised by endogenous substances, therapeutic drugs, necrosis up to 85%, and common micro-organisms. All of the assessed less common mutations except one (exon 19 deletion mutation 2236_2248 > AGAC) were detected at a similar DNA input level as that for the corresponding predominant mutation.
  
  
  CONCLUSIONS
  The cobas EGFR Mutation Test is a sensitive, accurate, rapid, and reproducible assay.
authors.affiliation: Roche Molecular Systems, Inc, Pleasanton, CA 94588, USA. patrick.odonnell@roche.com
authors.forename: Patrick
authors.initials: P
authors.lastname: O'Donnell
authors.forename: Jane
authors.initials: J
authors.lastname: Ferguson
authors.forename: Johnny
authors.initials: J
authors.lastname: Shyu
authors.forename: Robert
authors.initials: R
authors.lastname: Current
authors.forename: Taraneh
authors.initials: T
authors.lastname: Rehage
authors.forename: Julie
authors.initials: J
authors.lastname: Tsai
authors.forename: Mari
authors.initials: M
authors.lastname: Christensen
authors.forename: Ha Bich
authors.initials: HB
authors.lastname: Tran
authors.forename: Sean Shih-Chang
authors.initials: SS
authors.lastname: Chien
authors.forename: Felice
authors.initials: F
authors.lastname: Shieh
authors.forename: Wen
authors.initials: W
authors.lastname: Wei
authors.forename: H Jeffrey
authors.initials: HJ
authors.lastname: Lawrence
authors.forename: Lin
authors.initials: L
authors.lastname: Wu
authors.forename: Robert
authors.initials: R
authors.lastname: Schilling
authors.forename: Kenneth
authors.initials: K
authors.lastname: Bloom
authors.forename: Warren
authors.initials: W
authors.lastname: Maltzman
authors.forename: Steven
authors.initials: S
authors.lastname: Anderson
authors.forename: Stephen
authors.initials: S
authors.lastname: Soviero
mesh_terms: D002289:Carcinoma, Non-Small-Cell Lung / Q000235:genetics*; D004252:DNA Mutational Analysis / Q000379:methods*; D066246:ErbB Receptors / Q000235:genetics*; D005091:Exons; D006801:Humans; D057230:Limit of Detection; D008175:Lung Neoplasms / Q000235:genetics*; D025202:Molecular Diagnostic Techniques; D060885:Multiplex Polymerase Chain Reaction; D009154:Mutation; D016612:Paraffin Embedding; D060888:Real-Time Polymerase Chain Reaction; D015203:Reproducibility of Results
chemical_list: D066246:ErbB Receptors