These samples were also analyzed for KRAS mutations because (i) EGFR and KRAS mutations are mutually exclusive in NSCLC and (ii) emerging data suggest that KRAS mutations see more are negative predictors of benefit from both adjuvant chemotherapy and anti-EGFR-directed therapies [12, 14, 15]. We found 26.7% of the samples with a KRAS mutation (data not shown). This is also in accordance with the literature [14] and validated

our cohort as being well representative. We found 8 exon 19 deletions and 10 exon 21 mutations. These results were in accordance with those described by Tanaka et al. [16]. They noticed that exon 19 deletions were significantly associated with a male gender. In our cohort, 15 of

the 18 patients with EGFR mutations were female. We observed a deficit in mutation detection when the samples were very poor in tumor cells whereas the others could be accurately analyzed. As only bronchial or trans-thoracic Selleck Dibutyryl-cAMP fine needle biopsies are usually available in the medical setting of patients with advanced stage NSCLC (around 90% of the samples analyzed here, with only 10% being surgical specimens), these results demonstrate the need for a pathologist’s expertise to qualify the samples and perform microdissection if samples contain less than 20% of tumor cells. Indeed, Masago et al. [17] have demonstrated that results could be obtained from biopsy specimens only if the quantity of the specimen is sufficient to make a pathological diagnosis and if cancer cells were carefully buy LY2874455 selected. However, microdissection is very time-consuming and it is not always possible. Alternatively, methods such as peptide nucleic acid-locked nucleic acid PCR clamp [18, 19] or real-time PCR based on scorpion primers coupled with the

Amplified Refractory Mutation System (ARMS) [20] have a sensitivity around 1% of cancer cells. However, they could be difficult to use in routine clinical assay because they require special equipments and expensive reagents. Conclusions The present pyrosequencing method is sufficiently sensitive and specific to enable the detection of the to two major TKI-sensitive mutations in a large majority of the DNA extracted from paraffin-embedded clinical samples. Acknowledgements and funding Excellent technical support was provided by Emilie Bonin, Monique Delon, Valérie Konik-Mathevet, Maryse Samuel and Odile Vermeulen. We also acknowledge the Department of Cytology and Pathology for tumor sample preparations. We thank Dr Alison Foote for correcting our English usage. This project was supported by the clinical research direction of the Grenoble’s hospital, INCa (the French National Cancer Institute) and the French ministry of health initiated the ERMETIC project. References 1.