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

Sandrine Dufort12, Marie-Jeanne Richard12, Sylvie Lantuejoul23 and Florence de Fraipont12*

Author Affiliations

1 UM Biochimie des Cancers et Biothérapies, CHU Grenoble, Institut de Biologie et Pathologie, parvis Belledonne, 38 043 Grenoble, France

2 Centre de recherche INSERM/UJF U823, Institut Albert Bonniot, Rond-point de la Chantourne, 38 709 La Tronche cedex 9, France

3 Département d'Anatomie et Cytologie Pathologiques, CHU Grenoble, Institut de Biologie et Pathologie, parvis Belledonne, 38 043 Grenoble, France

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Journal of Experimental & Clinical Cancer Research 2011, 30:57  doi:10.1186/1756-9966-30-57

Published: 16 May 2011



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.


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.