Additional file 1: Table S1. of Airway wall thickness on HRCT scans decreases with age and increases with smoking

Cumulative APF at the different airway diameters per lung lobe. Table S2. Airway wall thickness per lobe. Table S3. Airway wall area per lobe. Table S4. Airway wall thickness according to smoking status. Table S5. Airway wall area according to smoking status. Table S6: Associations between airway wall thickness and age, gender and smoking status. Table S7. Associations between airway wall area and age, gender and smoking status. Table S8. Association between pulmonary function parameters and airway wall thickness, independent of age, gender, smoking status and height. Table S9. Association between pulmonary function parameters and airway wall area, independent of age, gender, smoking status and height. (DOCX 51 kb

Additional file 1: Table S1. of Genetic evaluation of the effect of GLCCI1 rs37973 on corticosteroid response in chronic obstructive pulmonary disease

Clinical characteristics of patients with COPD by rs37973 genotype (Studies 1 and 2). Table S2. Clinical characteristics of patients with COPD by rs37973 genotype (GLUCOLD study). Appendix 1. Sensitivity analyses exploring the effect of transformation and covariate adjustment in Study 2. Table S3. The influence of transformation and covariate adjustment on the analysis of change in FEV1 versus rs37973 genotype in patients with COPD (Study 2). Appendix 2. List of Institutional Review Boards/Independent Ethics Committees and the Chairperson (s) for each site. (DOCX 68 kb

NSCLC patient characteristics and<i>KRAS/EGFR</i> mutation^*.

<p>NSCLC patient characteristics and<i>KRAS/EGFR</i> mutation^{<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0152317#t003fn001" target="_blank">*</a>}.</p

Distribution of different <i>KRAS</i> amino acid changes in advanced NSCLC patients^*.

<p>Distribution of different <i>KRAS</i> amino acid changes in advanced NSCLC patients^{<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0152317#t004fn001" target="_blank">*</a>}.</p

Chronic Obstructive Pulmonary Disease Is Not Associated with <i>KRAS</i> Mutations in Non-Small Cell Lung Cancer

<div><p>Mutations in epithelial growth factor receptor <i>(EGFR)</i>, as well as in the EGFR downstream target <i>KRAS</i> are frequently observed in non-small cell lung cancer (NSCLC). Chronic obstructive pulmonary disease (COPD), an independent risk factor for developing NSCLC, is associated with an increased activation of EGFR. In this study we determined presence of <i>EGFR</i> and <i>KRAS</i> hotspot mutations in 325 consecutive NSCLC patients subjected to <i>EGFR</i> and <i>KRAS</i> mutation analysis in the diagnostic setting and for whom the pulmonary function has been determined at time of NSCLC diagnosis. Information about age at diagnosis, sex, smoking status, forced vital capacity (FVC) and forced expiratory volume in 1 sec (FEV₁) was collected. Chronic obstructive pulmonary disease(COPD) was defined according to 2013 GOLD criteria. Chi-Square, student t-test and multivariate logistic regression were used to analyze the data. A total of 325 NSCLC patients were included, 193 with COPD and 132 without COPD. COPD was not associated with presence of <i>KRAS</i> hotspot mutations, while <i>EGFR</i> mutations were significantly higher in non-COPD NSCLC patients. Both female gender (HR 2.61; 95% CI: 1.56–4.39; p<0.001) and smoking (HR 4.10; 95% CI: 1.14–14.79; p = 0.03) were associated with <i>KRAS</i> mutational status. In contrast, only smoking (HR 0.11; 95% CI: 0.04–0.32; p<0.001) was inversely associated with <i>EGFR</i> mutational status. Smoking related G>T and G>C transversions were significantly more frequent in females (86.2%) than in males (61.5%) (p = 0.008). The exon 19del mutation was more frequent in non-smokers (90%) compared to current or past smokers (36.8%). In conclusion, <i>KRAS</i> mutations are more common in females and smokers, but are not associated with COPD-status in NSCLC patients. <i>EGFR</i> mutations are more common in non-smoking NSCLC patients.</p></div

Logistic regression analysis of patient characteristics associated with COPD, <i>KRAS</i> and <i>EGFR</i>.

<p>Logistic regression analysis of patient characteristics associated with COPD, <i>KRAS</i> and <i>EGFR</i>.</p

Distribution of different <i>EGFR</i> mutations in advanced NSCLC patients.

<p>Distribution of different <i>EGFR</i> mutations in advanced NSCLC patients.</p

Distribution of different <i>KRAS</i> nucleotide changes in advanced NSCLC patients^*.

<p>Distribution of different <i>KRAS</i> nucleotide changes in advanced NSCLC patients^{<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0152317#t005fn001" target="_blank">*</a>}.</p

Differentially expressed miRNAs after TGF-β1 stimulation in control lung fibroblasts in the discovery group.

<p>(A) The miRNA expression in primary parenchymal lung fibroblasts of control subjects with and without TGF-β1 stimulation was determined by microarray. Unsupervised hierarchical clustering was used to generate the heatmap and pearson correlation was used as the distance metric. Twenty-nine miRNAs were differentially expressed after TGF-β1 stimulation (FDR<0.05). The heatmap shows the median-centered expression of the 29 miRNAs of which 8 miRNAs were downregulated and 21 miRNAs were upregulated after TGF-β1 stimulation. (B) Validation of differentially expressed miRNAs after TGF-β1 stimulation in the discovery group using qRT-PCR. Data are presented as relative expression (2^-ΔCp) normalized to RNU48. *p<0.05, **p<0.01.</p

Upregulation of ECM genes and α-SMA after TGF-β1 stimulation in primary parenchymal lung fibroblasts.

<p>(A) Effective TGF-β1 stimulation of control fibroblasts in the discovery group was confirmed by the upregulation of <i>FN1</i> (fibronectin 1), <i>COL1A1</i> (collagen type I alpha I) and <i>α-SMA</i> (alpha-smooth muscle actin), genes that are well-known to be affected by TGF-β. (B) These genes were also upregulated in the control and COPD fibroblasts in the replication group after 2.5 ng/ml TGF-β1 and (C) after 7.5 ng/ml TGF-β1 stimulation. Data are presented as relative expression (2^-ΔCp). **p<0.01, ***p<0.001, ****p<0.0001.</p