title: Pyrosequencing, a method approved to detect the two major EGFR mutations for anti EGFR therapy in NSCLC.
abstract: BACKGROUND
  Epidermal Growth Factor Receptor (EGFR) mutations, especially in-frame deletions in exon 19 (ΔLRE) and a point mutation in exon 21 (L858R) predict gefitinib sensitivity in patients with non-small cell lung cancer. Several methods are currently described for their detection but the gold standard for tissue samples remains direct DNA sequencing, which requires samples containing at least 50% of tumor cells.
  
  
  METHODS
  We designed a pyrosequencing assay based on nested PCR for the characterization of theses mutations on formalin-fixed and paraffin-embedded tumor tissue.
  
  
  RESULTS
  This method is highly specific and permits precise characterization of all the exon 19 deletions. Its sensitivity is higher than that of "BigDye terminator" sequencing and enabled detection of 3 additional mutations in the 58 NSCLC tested. The concordance between the two methods was very good (97.4%). In the prospective analysis of 213 samples, 7 (3.3%) samples were not analyzed and EGFR mutations were detected in 18 (8.7%) patients. However, we observed a deficit of mutation detection when the samples were very poor in tumor cells.
  
  
  CONCLUSIONS
  pyrosequencing is then a highly accurate method for detecting ΔLRE and L858R EGFR mutations in patients with NSCLC when the samples contain at least 20% of tumor cells.
authors.affiliation: UM Biochimie des Cancers et Biothérapies, CHU Grenoble, Institut de Biologie et Pathologie, Grenoble, France.
authors.forename: Sandrine
authors.initials: S
authors.lastname: Dufort
authors.forename: Marie-Jeanne
authors.initials: MJ
authors.lastname: Richard
authors.forename: Sylvie
authors.initials: S
authors.lastname: Lantuejoul
authors.forename: Florence
authors.initials: F
authors.lastname: de Fraipont
mesh_terms: D000483:Alleles; D002289:Carcinoma, Non-Small-Cell Lung / Q000188:drug therapy / Q000235:genetics*; D045744:Cell Line, Tumor; D066246:ErbB Receptors / Q000235:genetics*; D005091:Exons / Q000235:genetics; D005260:Female; D006801:Humans; D008175:Lung Neoplasms / Q000188:drug therapy / Q000235:genetics*; D008297:Male; D009154:Mutation / Q000235:genetics*; D017422:Sequence Analysis, DNA / Q000592:standards; D017384:Sequence Deletion
chemical_list: D066246:ErbB Receptors