Table 4. Quantitative Real-Time PCR validation of microarray results of genes differentiating current from never smokers (C/N) in tumor (T) and non-tumor (NT) tissue samples and confirmation in independent samples Gene Name ABI Assay ID Average expression in validation samplesa Average expression in confirmation samplesb Early Stage Adenocarcinoma Current Smokers N = 27 Never Smokers N = 16 Fold Change C/Nc Smoking Signature in Lung Never Smokers Fold Change C/Nc Current Smokers N = 12 19.13 12.31 13.13 15.16 12.89 13.72 11.19 12.25 14.79 15.26 14.64 13.95 15.91 12.57 13.12 13.30 8.20 14.16 11.03 17.42 N = 12 N = 7 20.21 13.42 14.24 15.93 13.00 14.74 11.69 12.92 15.70 15.76 15.82 14.57 18.01 13.30 13.28 N = 9 13.51 9.30 14.09 15.36 18.47 2.10 2.16 2.16 1.71 1.08 2.03 1.41 1.60 1.88 1.41 2.27 1.54 4.27 1.66 1.11 1.16 2.15 0.96 20.13 2.07 C10orf23 AURKA BIRC5 BIRC5 CCNA2 CENPF CKS1B FOXM1 GGH KIF20A KIF4A MKI67 NEK2 TPX2 TTK CEACAM5 CYBB CYTL1 FGG TM7SF4 Non-Tumor Lung Tissue Hs00269212_m1 Hs00153353_m1 Hs00977611_g1 Hs00153138_m1 Hs00193201_m1 Hs00216688_m1 Hs01029137_g1 Hs00153543_m1 Hs00608257_m1 Hs00194882_m1 Hs00602211_g1 Hs00267195_m1 Hs00601227_mH Hs00201616_m1 Hs00177412_m1 Hs00237075_m1 Hs00166163_m1 Hs00184064_m1 Hs00241037_m1 Hs00229255_m1 16.57 10.48 10.84 13.13 11.23 11.78 9.36 10.43 11.92 12.40 12.42 12.53 12.61 10.60 11.49 10.98 5.86 11.54 9.05 14.99 N = 18 17.37 11.46 12.45 13.42 12.87 12.58 9.91 11.65 13.75 13.65 13.66 12.71 14.52 11.73 12.80 N = 7 14.85 8.19 14.61 11.99 18.20 aValidation in 68 samples used also for microarray analysis; bConfirmation in 40 independent samples from EAGLE and Mayo Clinic cC/N = Current/Never smoking comparison doi:10.1371/journal.pone.0001651.t004 differentiates Current from Never smokers (C/N) in early stage lung Tumor (T). 4A C16orf30 and UBE21 transcription sites. 4B Comparison of C/N results in early stage Tumor (T) tissues vs. C/ N results in Non-Tumor (NT) lung tissues by GSEA analysis. 4C Gene list from GSEA comparison of up-regulated C/N genes between early stage Tumor (T) tissues and Non-Tumor (NT) tissues. 4D Gene list from GSEA comparison of down-regulated C/N genes between early stage Tumor (T) tissues and Non-Tumor (NT) tissues. Found at: doi:10.1371/journal.pone.0001651.s004 (0.51 MB DOC) Appendix S5 Mortality risk in smokers associated with the expression of genes differentiating Current from Never smokers (C/N) in Tumor and Non-Tumor tissue samples. 5A Current/ Never (C/N) genes and related mortality risk in Tumor and Non- Tumor lung tissues (all stages) from Current and Former smokers. Found at: doi:10.1371/journal.pone.0001651.s005 (0.55 MB DOC) References 1.74 1.97 3.07 1.22 3.13 1.74 1.46 2.33 3.55 2.38 2.37 1.14 3.74 2.19 2.47 14.58 5.03 8.36 7.71 8.15 Acknowledgments We thank Ms. Juliet Joly for her technical help with the manuscript preparation, and Drs. Subhashree Madhavan and Wei Lin for their help with the CEL files on the GEO site, Drs. Abbas Shakoori and Konstantin Shilo for assistance with the pathology specimens, and Dr. Kenneth Buetow for support. Author Contributions Conceived and designed the experiments: SW JJ ML JS PB NC. Performed the experiments: TD HL FM JF MH SM. Analyzed the data: SW JJ ML JF JS DC AP PB TD MR AD AB NC. Contributed reagents/materials/ analysis tools: JJ PY ML PB NC. Wrote the paper: ML. Other: Participated in data interpretation: DC PB PY AP AD JF TD HL AB SM JJ NC JS SW MR. Read and approved the manuscript: JJ NC DC PB PY AP JF AB SM HL TD AD JS SW MR. Interpreted the data: ML. 1. Bryant A, Cerfolio RJ (2007) Differences in epidemiology, histology, and survival between cigarette smokers and never-smokers who develop non-small cell lung cancer. Chest 132: 185–192. 2. Benjamini Y, Hochberg Y (1995) Controlling the False Discovery Rate: a practical and powerful approach to multiple testing. J Royal Stat Soc Ser B 57: 289–300. 3. Ashburner M, Ball CA, Blake JA, Botstein D, Butler H, et al. (2000) Gene ontology: tool for the unification of biology. The Gene Ontology Consortium. Nat Genet 25: 25–29. 4. Subramanian A, Tamayo P, Mootha VK, Mukherjee S, Ebert BL, et al. (2005) Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles. Proc Natl Acad Sci U S A 102: 15545–15550. PLoS ONE | www.plosone.org 7 February 2008 | Volume 3 | Issue 2 | e1651