title: KRAS mutation analysis by PCR: a comparison of two methods.
abstract: BACKGROUND
  KRAS mutation assays are important companion diagnostic tests to guide anti-EGFR antibody treatment of metastatic colorectal cancer. Direct comparison of newer diagnostic methods with existing methods is an important part of validation of any new technique. In this this study, we have compared the Therascreen (Qiagen) ARMS assay with Competitive Allele-Specific TaqMan PCR (castPCR, Life Technologies) to determine equivalence for KRAS mutation analysis.
  
  
  METHODS
  DNA was extracted by Maxwell (Promega) from 99 colorectal cancers. The ARMS-based Therascreen and a customized castPCR assay were performed according to the manufacturer's instructions. All assays were performed on either an Applied Biosystems 7500 Fast Dx or a ViiA7 real-time PCR machine (both from Life Technologies). The data were collected and discrepant results re-tested with newly extracted DNA from the same blocks in both assay types.
  
  
  RESULTS
  Of the 99 tumors included, Therascreen showed 62 tumors to be wild-type (WT) for KRAS, while 37 had KRAS mutations on initial testing. CastPCR showed 61 tumors to be wild-type (WT) for KRAS, while 38 had KRAS mutations. Thirteen tumors showed BRAF mutation in castPCR and in one of these there was also a KRAS mutation. The custom castPCR plate included several other KRAS mutations and BRAF V600E, not included in Therascreen, explaining the higher number of mutations detected by castPCR. Re-testing of discrepant results was required in three tumors, all of which then achieved concordance for KRAS. CastPCR assay Ct values were on average 2 cycles lower than Therascreen.
  
  
  CONCLUSIONS
  There was excellent correlation between the two methods. Although castPCR assay shows lower Ct values than Therascreen, this is unlikely to be clinically significant.
authors.affiliation: Department of Pathology, Queen Alexandra Hospital, Portsmouth, United Kingdom.
authors.forename: Louise
authors.initials: L
authors.lastname: Bolton
authors.affiliation: Department of Pathology and Warwick Medical School, University Hospitals Coventry and Warwickshire, Coventry, United Kingdom.
authors.forename: Anne
authors.initials: A
authors.lastname: Reiman
authors.affiliation: Department of Pathology and Warwick Medical School, University Hospitals Coventry and Warwickshire, Coventry, United Kingdom.
authors.forename: Katie
authors.initials: K
authors.lastname: Lucas
authors.affiliation: Department of Pathology and Warwick Medical School, University Hospitals Coventry and Warwickshire, Coventry, United Kingdom.
authors.forename: Judith
authors.initials: J
authors.lastname: Timms
authors.affiliation: Department of Pathology and Warwick Medical School, University Hospitals Coventry and Warwickshire, Coventry, United Kingdom.
authors.forename: Ian A
authors.initials: IA
authors.lastname: Cree
mesh_terms: D000483:Alleles; D015179:Colorectal Neoplasms / Q000235:genetics / Q000473:pathology; D004247:DNA / Q000032:analysis*; D004252:DNA Mutational Analysis / Q000379:methods*; D006801:Humans; D009154:Mutation; D048493:Proto-Oncogene Proteins B-raf / Q000235:genetics; D011933:Reagent Kits, Diagnostic; D060888:Real-Time Polymerase Chain Reaction; D018631:ras Proteins / Q000235:genetics*
chemical_list: D011933:Reagent Kits, Diagnostic; D004247:DNA; C482119:BRAF protein, human; D048493:Proto-Oncogene Proteins B-raf; D018631:ras Proteins