the date of diagnosing stage IV disease until 14192894 censorship or death. Only patients with available clinical data who had progressed to stage IV disease and subsequently were treated were included for survival analysis. All patients treated with an EGFR TKI irrespective of their mutational status were evaluated for overall survival. Univariate Cox regression analysis was performed with the covariates age, gender, histology, KRAS and EGFR Methods Patients This study concerns all the NSCLC tumor samples from eight regional Dutch hospitals during the period of November 2008 until April 2011 that were tested for mutational status by a central pathology department. Data on gender, smoking status, age at diagnosis, stage at diagnosis, localization of metastases, start date and lines of treatment received were collected. Tumor samples were obtained by either bronchoscopy, transthoracic lung biopsies and/or from pulmonary resections and were sent to the respective pathology department for histological examination. Histology was according to 2004 WHO criteria. Response to treatment was performed according to RECIST criteria. Sample collection procedure and DNA extraction From each formalin-fixed and paraffin embedded tumor tissue block that was sent to the pathology department 4 mm sections were cut. After hematoxylin and eosin staining, slides were evaluated by an experienced lung pathologist for the presence of sufficient tumor tissue and estimating the percentage of tumor cells. Samples with clearly less than 50% tumor cells were defined as inadequate for EGFR/KRAS SU6668 Mutation testing. Areas with.50% tumor cells marked by the pathologist on the slide. This area was scraped from the slide using a scalpel and dissolved in TE-4 and 20 mg/ml Proteinase K. DNA was extracted by incubation overnight at 55uC, followed by heating to 100uC for 5 minutes to inactivate proteinase K and centrifuged at room temperature at 13,000 rpm. The aqueous solution was directly used for PCR analysis or stored at 220uC. DNA concentration was measured on a ND1000 spectrophotometer. All DNA isolates were set to 10 ng/ml in TE-4 prior 
to use. For quality control, genomic DNA was amplified in a multiplex PCR containing a control gene primer set resulting in products of 100, 200, 300, 400 and 600 bp according to the BIOMED-2 protocol. Only DNA samples with PCR products of 300 bp and larger were used for mutation analysis. All samples were tested on DNA extracted from two independent slides. All standard 22880633 precautions were taken to avoid contamination of amplification products using separate laboratories for pre- and N Number of patients Number of biopsies Histology Adenocarcinoma SCC Large cell undifferentiated Adenosquamous Carcinoid Salivary gland NSCLC-NOS 353 27 42 7 3 2 8 442 474 Percentage 100 80 6 9 1 1 1 2 SCC is squamous cell lung carcinoma. NSCLC-NOS is non-small cell lung cancer not otherwise specified. doi:10.1371/journal.pone.0070346.t001 EGFR/KRAS Mutation Status in Dutch NSCLC Patients mutation status, metastatic site were also analyzed. Variables with p-value less than 0.20 were used for the multivariate analysis. All statistical analysis was performed using SPSS version 18.0. Nominal P-values less than 0.05 were considered significant. Results EGFR and KRAS mutations From November 2008 until April 2011 474 samples from 442 patients were sent to the central pathology department for mutation analysis. The most common histological classification was adenocarcinoma, 8%
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