Smoking Signature in Lung 4.7 5.5 6.7 5.9 5.4 5.9 5.0 3.4 0.8 0.8 0.8 0.8 NT NT NT NT NT NT T T NT NT NT T Probe ID Increased risk 204641_at 204822_at 201292_at 219306_at 218542_at 209642_at 201637_s_at 213189_at Decreased risk 202068_s_at 214894_x_at 218804_at 201651_s_at NEK2 TTK TOP2A KIF15 C10orf3 BUB1 FXR1 LDLR MACF1 TMEM16A PACSIN2 DKFZp667G2110 0.0008 0.0011 0.0041 0.0048 0.0068 0.0084 0.0007 0.0088 0.0068 0.0091 0.0095 0.0046 RRa 2.6 2.9 3.1 2.8 2.7 2.8 2.8 2.0 0.5 0.4 0.4 0.4 1.5 1.5 1.4 1.4 1.3 1.3 1.5 1.2 0.3 0.2 0.2 0.2 Table 3. Mortality risk in smokers for gene expression differentiating current from never smokers (C/N) in lung tumor and non- tumor tissue samples (p,0.01) Gene Symbol p-value 95% CIb Lowest 95% CIb Highest Tissue typec aRelative Risk of death. Analysis based on 54 current and former smokers using 262 probes from the Current/Never smoking comparisons (98 down- and 64 up- regulated probes from T and 75 down- and 28 up-regulated probes from NT, minus 3 overlapping probes in T and NT); models adjusted for tumor stage, sex, and smoking status b95% Confidence Interval cT = Lung adenocarcinoma samples of any stage (N = 42); NT = Non-Tumor tissue samples (N = 34) doi:10.1371/journal.pone.0001651.t003 risk. The latter finding was most evident in non-tumor tissue, likely reflecting the widely recognized field-cancerization effect by smoking,[22] while in the tumor tissue, smoking-related genes’ effects on survival may be masked by extensive molecular alterations occurring during tumorigenesis. In the non-tumor tissue, current smoking strongly altered immune response genes, consistent with the defense mechanisms of the lung tissue against the acute toxic effects of smoking. Among the gene most strongly down-regulated in NT was CX3CR1, located on chromosome 3p21.3, an area known to be often deleted in lung cancer,[23] particularly in smokers.[24] Current knowledge of gene expression altered by cigarette smoking is based on bronchoscopy-obtained airway epithelial cells or macrophages[9,25–27] or peripheral leukocytes[10] from healthy smokers rather than directly on lung tissue. The few studies with lung tissue samples are very small[28] or used RNA amplification[29] or RNA pooling[30] methods. Our results are consistent with some previous findings, such as smoking-related alteration of CYP1B1[9,10] or of the mitotic pathway in cancer survival.[29] However, earlier studies were often limited by the small sample size, or lacked information on potential confounders, or availability of paired tumor and non-tumor lung tissue samples for the distinction of gene changes involved in lung carcinogenesis from those representing a transient smoking effect. We overcame these pitfalls with a relatively large sample size of fresh tumor and non-tumor lung tissues, detailed covariate information (e.g., sex, age, stage, previous lung diseases or chemotherapy), biochemical validation of the smoking status, and confirmation of the main findings in independent tissue samples. In conclusion, our study provides clues on how cigarette smoking affects lung cancer development and survival. Functional assays to confirm these findings are warranted. If confirmed, these genes could become important targets for chemoprevention and treatment for lung cancer in smokers. Supporting Information Appendix S1 Quality Assurance. 1A Description of analysis of sample quality assurance 1B Samples’ description 1C Surfactant genes in Tumor (T) and Non-Tumor (NT) lung tissues by smoking Found at: doi:10.1371/journal.pone.0001651.s001 (0.07 MB DOC) Appendix S2 Current/Never (C/N) and Former/Never (F/N) smoking comparisons in early stage Tumor (T) tissue. 2A Current/ Never (C/N) comparison, early stage Tumor (T) tissues: up- regulated probes. 2B Current/Never (C/N) comparison, early stage Tumor (T) tissues: down-regulated probes. 2C Current/ Never (C/N) comparison, late stage Tumor tissues: up+down- regulated probes. 2D Gene Ontology (GO) functional categories for the Current/Never (C/N) smoker comparison. 2E Current/ Never (C/N) and Former/Never (F/N) comparisons: overlapping probe list. 2F Gene list from GSEA comparison of up-regulated C/N genes and F/N genes in early stage Tumor (T) tissues. 2G Gene list from GSEA comparison of down-regulated C/N genes and F/N genes in early stage Tumor (T) tissues. Found at: doi:10.1371/journal.pone.0001651.s002 (0.62 MB DOC) Appendix S3 Current/Never (C/N) smoking comparisons in Non-Tumor (NT) lung tissue. 3A Current/Never (C/N) compar- ison in Non-Tumor (NT) lung tissues: up-regulated probes. 3B Current/Never (C/N) comparison in Non-Tumor (NT) lung tissues: down-regulated probes . 3C Gene Ontology (GO) functional categories for the Current/Never (C/N) comparison (up and down-regulated genes) in Non-Tumor (NT) lung tissues. Found at: doi:10.1371/journal.pone.0001651.s003 (0.21 MB DOC) Appendix S4 Comparison between Tumor (T) and Non-Tumor (NT) lung tissue for the genes whose expression significantly PLoS ONE | www.plosone.org 6 February 2008 | Volume 3 | Issue 2 | e1651