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

Sandrine Dufort,Marie-Jeanne Richard,Florence De Fraipont,Sylvie Lantuejoul

doi:10.1186/1756-9966-30-57

Abstract

BackgroundEpidermal 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.MethodsWe designed a pyrosequencing assay based on nested PCR for the characterization of theses mutations on formalin-fixed and paraffin-embedded tumor tissue.ResultsThis 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.Conclusionspyrosequencing 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.

Highlights

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 nonsmall-cell lung cancer (NSCLC) [1]
Clinical samples Between 1st January and 30 June 2010, 213 tumor samples were collected from consecutive patients with an advanced lung adenocarcinoma, DNA extracted and their Epidermal Growth Factor Receptor (EGFR) mutation status determined for selection for anti EGFR treatments by clinicians
Pyrosequencing assay of exon 19 deletions In order to test the pyrosequencing method for the analysis of exon 19 deletions, we used DNA from the NCIH1650 cell line as positive control and DNA extracted from human peripheral blood lymphocytes (PBL) as wild-type control

Summary

Introduction

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 nonsmall-cell lung cancer (NSCLC) [1]. 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 aminoacid residues leucine 747 to glutamic acid 749 (ΔLRE). The second mutation is a single-point mutation in exon. 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

Results

Conclusion