EGFR was assessed by immunohistochemistry as previously described [21]. Briefly, after deparaffinization of the sections, endogenous peroxidase was blocked in 0.3% H2O2 in PBS for 20 min. For antigen retrieval, the sections were submitted to high temperature and pressure with Tris-EDTA buffer (pH 9) for 5 min. The slides were preincubated in PBS for 10 min. The primary mouse monoclonal antibody GSK126 mouse directed against EGF receptor (clone 31G7, Zymed labs, South San Francisco, CA, USA) receptor was diluted 1:100, and incubated overnight at 4°C. The secondary biotinylated antibodies (goat anti-mouse from Dako, Glostrup, Denmark) and the peroxidase-labelled streptavidin-biotin

complex (Dako) were diluted 1:200 and incubated for 30 min at room temperature. All slides were developed in 0.05% diamino benzidine (Sigma, St Louis, MO, USA) for 5 min and counterstained in Harris haematoxylin (Sigma). Finally, the slides were dehydrated through graded alcohol to xylene and mounted in organic mounting medium. EGFR-scores EGFR stainings were mainly in the cell membranes and the expression pattern

of EGFR was quite similar to BGJ398 that of HER2. Thus EGFR expression was therefore evaluated using the HercepTest scoring criterion as reported in previous studies [21–23]. Sections were considered as positive when at least 10% of the tumor cells to be stained. Cytoplasmic staining without associated membrane staining was considered non-specific and was reported as negative. The score was based on a scale where 0 corresponded to tumor cells that were completely negative, 1+ corresponded to faint perceptible staining of the tumor cell membranes, 2+ corresponded to moderate staining of the entire tumor cell membranes and 3+ was strong circumferential staining of the entire tumor cell membranes creating a fishnet Adenosine pattern. As positive controls we used in house positive control tissue sections. As negative controls we used normal tissues, which are expected not to express EGFR such as connective tissue seen in the same sections as the tumor cells. In the metastases sections we used lymphocytes and the surrounding capsule of the lymph nodes as negative internal

controls. Excluded cases In 3 cases, no tumor cells could be found in the sections of lymph nodes. In another case, there were no tumor cells in the sections supposed to be primary lung cancer. Thus, we started from 51 patient cases and ended up with 47 cases with high quality material of both primary tumors and the corresponding metastases. Results EGFR expression of primary tumors and metastases The EGFR-scores for the analyzed 47 primary NSCLC and the corresponding 47 lymph node metastases are shown in Table 2. In 36 of 47 (76.6%) analysed primary tumors, immunostaining for EGFR was evident. Among these, 11 (23.4%) had EGFR expression scored as 1+, 10 (21.3%) had EGFR expression scored as 2+, and 15 (31.9%) had EGFR expression scored as 3+.