Pyrosequencing, a method approved to detect the two major EGFR mutations for anti EGFR therapy in NSCLC

We designed a pyrosequencing assay based on nested PCR for the characterization of theses mutations on formalin-fixed and paraffin-embedded tumor tissue.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.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.Detection of mutations of the epidermal growth factor receptor (EGFR) gene is critical for predicting the response to therapy with tyrosine kinase inhibitors (TKIs, e.g.: gefitinib and erlotinib) in patients with non-small-cell lung cancer (NSCLC) [1]. Practically all mutations are on exons 18 through 21 where they affect the ATP-binding cleft of EGFR [2]. In vitro studies have shown that EGFR mutants have constitutive TK activity and, therefore, a greater sensitivity to anti-EGFR inhibition. Two classes of mutation account for approximately 90% of EGFR mutations reported to date in lung adenocarcinoma [3]. The class I mutations are in-frame deletions in exon 19, which almost always include amino-acid residues leucine 747 to glutamic acid 749 (ΔLRE). The second mutation is a single-point mutation in exon 21, which substitutes an arginine for a leucine at codon 858 (L858R).Thus far, the direct DNA sequencing method is the most common and conventional method used for the detection and identification of mutations in tumor cells. However, its sensitivity is suboptimal for clinical tumor samples. Mutant DNA needs to comprise ≥25