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Pyrosequencing, a method approved to detect the two major EGFR mutations for anti EGFR therapy in NSCLC.
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Edité par CCSD ; BioMed Central -
International audience. ABSTRACT: BACKGROUND: Epidermal Growth Factor Receptor (EGFR) mutations, especially in-frame deletions in exon 19 (delta LRE) 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 delta LRE and L858R EGFR mutations in patients with NSCLC when the samples contain at least 20% of tumor cells.
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