title: Competitive allele specific TaqMan PCR for KRAS, BRAF and EGFR mutation detection in clinical formalin fixed paraffin embedded samples.
abstract: BACKGROUND
  The development of targeted therapies has created a need for robust molecular characterization of cancer and it has become a challenge to validate methods to ensure accuracy in tumor mutation testing.
  
  
  METHODS
  The current study was designed to evaluate KRAS, BRAF and EGFR genotyping by Competitive Allele Specific hydrolysis probes (TaqMan) PCR technology (CAST), on suboptimal formalin fixed paraffin embedded (FFPE) tumor samples. Assays were calibrated on FFPE samples and a minimal quantification cycle (Cq) cut-off was determined to standardize analyses and avoid over-interpretation of degraded material. Sensibility, specificity and blinded clinical sample screenings (n=63) were evaluated.
  
  
  RESULTS
  CAST PCR allowed efficient amplification of FFPE samples, probes were highly specific and all assays had a sensibility inferior to 1% except for the EGFR p.T790M assay. 60/63 samples were correctly typed. The three missed mutations were EGFR exon 19 deletions that were not recognized by the DEL19 assays that were used.
  
  
  CONCLUSIONS
  This technology is less laborious and prevent crossover of PCR products as compared to multistep methods. TaqMan® Mutation Detection assay is an important technology to consider in the field of mutation detection for KRAS, BRAF and EGFR point mutation screening. Assay calibration on FFPE samples may prevent erroneous interpretations that will ultimately harm clinical oncology practice.
authors.affiliation: UMR-S775, INSERM, Paris, F-75006, France. audrey.didelot@parisdescartes.fr
authors.forename: Audrey
authors.initials: A
authors.lastname: Didelot
authors.forename: Delphine
authors.initials: D
authors.lastname: Le Corre
authors.forename: Armelle
authors.initials: A
authors.lastname: Luscan
authors.forename: Aurélie
authors.initials: A
authors.lastname: Cazes
authors.forename: Karine
authors.initials: K
authors.lastname: Pallier
authors.forename: Jean-François
authors.initials: JF
authors.lastname: Emile
authors.forename: Pierre
authors.initials: P
authors.lastname: Laurent-Puig
authors.forename: Hélène
authors.initials: H
authors.lastname: Blons
mesh_terms: D045744:Cell Line, Tumor; D015342:DNA Probes / Q000235:genetics; D066246:ErbB Receptors / Q000235:genetics*; D005557:Formaldehyde; D005838:Genotype; D060005:Genotyping Techniques / Q000379:methods; D019073:HT29 Cells; D006801:Humans; D009154:Mutation; D009369:Neoplasms / Q000235:genetics* / Q000473:pathology; D016612:Paraffin Embedding; D016133:Polymerase Chain Reaction / Q000379:methods*; D048493:Proto-Oncogene Proteins B-raf / Q000235:genetics*; D015203:Reproducibility of Results; D016707:Tissue Fixation; D018631:ras Proteins / Q000235:genetics*
chemical_list: D015342:DNA Probes; D005557:Formaldehyde; C512478:EGFR protein, human; D066246:ErbB Receptors; C482119:BRAF protein, human; D048493:Proto-Oncogene Proteins B-raf; D018631:ras Proteins