​ncbi.​nlm.​nci). The EGFR exon 21 L858R mutation [21] was also analyzed by PCR-RFLP based on the presence of a new Sau96I restriction site. Codons 12/13 of KRAS were also

analyzed by PCR-RFLP [22, 23]. BRAF exons 14 & 15 were analyzed as previously described [20], and the 3’ and 5’ intron-exon splice sites of MET exon 14 were also screened. Immunohistochemistry Following H&E review by an experienced pathologist, only the cases with adequate material were selected for further analysis (Figure  1). Tissue microarrays (5x 0.6 mm diameter cores per case), were created. Cases not included on TMAs were further handled as whole BMS-907351 in vivo tissue sections. Immunohistochemical staining was performed according this website to standard protocols, with slight modifications, on serial

2.5 μm thick sections using the Bond MaxTM (Leica Microsystems, Germany/Menarini Diagnostics, Athens, Hellas) and i6000 (Biogenex, USA) auto-stainers. The conditions of staining for the antibodies against EGFR (clone 31 G7, Invitrogen, CA, USA) and c-MET/HGFR (8 F1, NovocastraTM, Leica Biosystems, UK) were as previously described [24]. For the detection of phosphorylated EGF Receptor at Tyr1173 monoclonal rabbit antibody (clone 53A5 CST, MA, USA) at a dilution of 1:150 was used, staining was performed using a Bond Max autostainer. Figure 1 Expression of proteins in NSCLC tumors studied by immunohistochemistry in tissue microarrays. A) EGFR strong membrane positivity; B) EGFR absence; C) pEGFRTyr1173 membrane and cytoplasmic positivity; D) pEGFRTyr1173 lack of immunoreaction; E) c-MET strong cytoplasmic staining; F) Absence of c-MET staining. (Full size images X100). Immunohistochemical scoring EGFR protein staining was evaluated, using a previously proposed semi-quantitative approach based on staining intensity (0–4) and percentage of stained

tumor cells (0–100) [25]. Diffuse cytoplasmic or granular staining was diagnosed as negative. Scores of 0–200 were considered as negative/low expression, scores of 201–400 Resveratrol were considered as positive/high expression. For evaluation of phospho-EGFRTyr1173 and c-MET expression we used a semi-quantitative scoring system based on intensity and staining pattern. Intensity was scored as follows: 0 = no staining, 1 = weakly, 2 = moderately, and 3 = strongly positive. The scoring of the staining pattern was based on the percentage of positive tumor cells: 0 = 0 to 5%, 1 = 6 to 25%, 2 = 26% to 50%, 3 = 51% to 100%. The localization of staining for each protein was also indicated, as cytoplasmic and cytoplasmic/membranous for MET and nuclear, cytoplasmic and cytoplasmic/nuclear for phospho-EGFRTyr1173. The total score was calculated as the sum of the intensity score and the staining pattern score.