modified the standard GSEA method by substituting an ANOVA test for the standard two-sample t-test to adjust for sex. Furthermore, we changed the permutation test for calculating the p-values by permuting residuals and using as weights the observed ANOVA coefficients divided by the standard error values. Up- and down- regulated genes were included in different gene-sets for the analyses. Molecular function classification of smoking-altered genes Gene Ontology was used to assign the genes to functional categories.[3] GoMiner[8] was utilized to rank-order the GO categories for the genes identified in the smoking comparisons. Quantitative PCR validation and confirmation in independent samples We used quantitative real-time PCR (QRTPCR) to confirm the differential expression of 19 C/N selected genes (20 probes), including 14 genes from T and 5 from NT analyses. Primer and probe sets for the selected genes as well as control probes for GUSB and S18 (ABI) were run on 7500 Taqman under the manufacturer’s standard protocol. Ct values were normalized based on GUSB expression. Validation assays were performed in 68 samples used in the original microarray analyses, including 43 T (27 C and 16 N smokers), and 25 NT (18 C and 7 N smokers). Confirmation assays were performed in 40 independent samples, including 19 T (12 C and 7 N smokers) and 21 NT samples (12 C and 9 N smokers). These samples were collected in EAGLE (10 T samples from 7 C and 3 N smokers, and 12 NT samples from 7 C and 5 N smokers-these samples were not used for the microarray analyses), and from the Mayo Clinic, Rochester, MN (9 T and 9 NT paired samples from 5 C and 4 N smokers). Results The molecular signature of cigarette smoking in lung adenocarcinoma To investigate the molecular changes associated with smoking in the tumor tissue, we compared gene expression changes between current and never (C/N) smokers (Table 1). To avoid potential alteration of gene expression due to advanced tumor status, we limited smoking comparisons in tumor tissue to the early stages (stages I and II). Unless specified differently, ‘‘T’’ samples represent Smoking Signature in Lung early stage adenocarcinomas. Results from the advanced tumor stage tissues are reported for completeness in Appendix S2C. Using stringent selection criteria (fold-change .1.5 and p- value,0.001), we identified 64 up- and 98 down-regulated probe- sets, representing 54 up- and 81 down-regulated genes (Appendix S2A, S2B). Most of the significantly up-regulated genes were involved in cell cycle/mitosis/cell division (e.g., TTK, CENPF, NEK2), while many of those down-regulated were involved in cell adhesion/cell cycle arrest (e.g., ADRB2, APLP2, MACF1), consistent with a role of these genes in neoplasia development. The GoMiner results (Appendix S2D) confirmed that the mitosis genes (12 altered genes among the 127 mitotic genes on the HG- U133A chip, p,0.001), and more generally those involved in cell cycle were the most commonly altered in the tumor tissue (Table 2). Lung cancer gene expression is similar in current and former smokers To verify whether the C/N smoking signature in the tumor was present also in former smokers, we compared the C/N and F/N signatures in T and found 26 probes (22 down- and 4 up- regulated, representing 21 genes) that differentiated both C/N and F/N using stringent selection criteria (Appendix S2E). Some of these genes, e.g., STOM, SSX2IP, TRPC6, APLP2 (2 probes), and DHRS7, exhibited a persistent alteration even in subjects (n = 6) who quit smoking more than 20 years before the study. The GSEA analysis showed that among the 64 up- and 98 down- regulated probes found in the C/N comparison in T, 58 and 90 probes, representing 50 up- and 73 down-regulated genes, were also up- and down-regulated, respectively in the F/N smoking comparison (p,0.001, Fig. 1, and Appendix S2F, S2G). All cell cycle genes that differentiated C/N were also altered in F/N, although less prominently (Table 2), indicating that alterations of these genes persist following smoking cessation. Importantly, the mitosis/cell cycle genes identified in C/N and F/N also differentiated the early stage tumor from the non-tumor tissue samples (T/NT, paired analysis) (Table 2), while pack years of cigarette smoking, a composite index of intensity and duration that does not consider the time when smoking occurred, were not associated with gene expression in either T or NT. Smoking signature in the noninvolved lung tissue The C/N comparison in NT revealed 28 up- and 75 down- regulated probes, representing 25 up- and 73 down-regulated Table 1. Number of probes and genes differentiating current from never smokers (C/N) and former from never smokers (F/N) in all tumor samples, early stage tumor samples (T), and all non-tumor (NT) tissue samples. All stages Tumor Stages I and II Tumor (‘‘T’’) Non-Tumor (‘‘NT’’) Comparison 24 Current vs. 18 Former vs. between smokers 16 Never 16 Never 20 Current vs. 10 Never 13 Former vs. 10 Never 16 Current vs. 15 Never 18 Former vs. 15 Never Criteria for significance p-value,0.001 p-value,0.001+ Fold change.1.5 aFDR = False Discovery Rate [2] doi:10.1371/journal.pone.0001651.t001 PLoS ONE | www.plosone.org FDRa Direction Probes Genes Probes Genes 8.5% Down 142 119 61 48 Up 119 104 63 56 17.0% Down 25 22 17 15 Up 105 97 3 3 3 9.5% Down 126 104 98 81 Up 106 89 64 54 27.8% Down 31 25 26 21 Up 40 35 4 4 7.8% Down 211 191 75 73 Up 71 64 28 25 78.3% Down 7 7 1 1 Up 2 2 0 0 February 2008 | Volume 3 | Issue 2 | e1651