Newnham et al. BMC Cancer 2011, 11:93 http://www.biomedcentral.com/1471-2407/11/93 Table 3 Details of genes over-expressed in K-Ras mutant tumours Symbol Gene Name ACOX2 Acyl-Coenzyme A oxidase 2, branched chain ALDH2 Aldehyde dehydrogenase 2 family (mitochondrial) ALDH3B1 Aldehyde dehydrogenase 3 family, member B1 AREG Amphiregulin (schwannoma-derived growth factor) CD55 CD55 molecule, decay accelerating factor for complement (Cromer blood group) CLDN10 Claudin 10 DGKD EGR1 Diacylglycerol kinase, delta 130kDa Early growth response 1 MSLN Mesothelin 16p13.3 MST1R Macrophage stimulating 1 receptor (c-met-related 3p21.3 tyrosine kinase) NPC2 Niemann-Pick disease, type C2 14q24.3 NR4A1 Nuclear receptor subfamily 4, group A, member 1 12q13 PFDN1 Prefoldin subunit 1 5q31 PTGS2 Prostaglandin-endoperoxide synthase 2 (cyclo-oxygenase 1q25.2- 2) q25.3 ST3GAL5 ST3 beta-galactoside alpha-2,3-sialyltransferase 5 2p11.2 TFPI2 UPP1 Tissue factor pathway inhibitor 2 Uridine phosphorylase 1 7q22 7p12.3 Cytoband P value 3p14.3 4.9 E-3 12q24.2 9.8 E-4 11q13 3.9 E-3 4q13-q21 1.4 E-4 1q32 6.9 E-4 13q31- q34 2q37.1 5q31.1 2.9 E-3 1.0 E-3 2.5 E-3 4.61 E-5 4.5 E-3 4.6 E-4 6.2 E-4 1.5 E-3 3.1 E-3 1.4 E-3 4.5 E-4 1.0 E-4 Page 6 of 11 Gene Function Lipid metabolism Alcohol metabolism Alcohol metabolism Autocrine growth factor family member Immune response, protection of cells from complement mediated damage Intercellular tight junctions Intercellular signalling, cell growth. Transcriptional regulation of genes involved in mitogenesis Possible role in cell adhesion Cell motility, positive regulation of cell cycle Regulation of cholesterol transport and storage Transcription factor Cell cycle, transcription factor activity, protein folding Prostaglandin biosynthesis Cell differentiation, cell proliferation, signal transduction. Matrix remodelling, coagulation Nucleotide catabolism common theme of RAS activation represented in ontological and single gene analyses. Included in the differentiating gene list were MAPK1, DUSP11 and DUSP13, PTPN11, and PIK3CB, all having roles in signal transduction and the MAPK pathway. The phos- phatidylinositol signalling pathway was also significantly over-represented in ontology analysis. Expression levels of only 38 genes differed significantly between deceased and surviving patients. 18 of these genes were shared with gene lists of recurrent vs. non- recurrent tumours. Few biological processes were repre- sented by more than one gene, and clear patterns of gene ontology were not apparent. Correlation with External Data Sets Comparison of our differential gene list for recurrence with the discriminating gene list for survival in GSE11117 (tran- scriptional and survival data for 41 NSCLC samples, using Novachip Human 34.5k microarray interrogating ~34,500 transcripts for each sample; http://www.ncbi.nlm.nih.gov/ geo/query/acc.cgi?acc=GSE11117) identified 40 matched transcripts (additional file 5). Log-transformed expression values of the 40 transcripts were used to classify the sam- ples from GSE11117 into two subgroups using a correla- tion, average-linkage hierarchical clustering R package. Kaplan-Meier curves for these external samples using the 40 transcripts matched to our recurrence gene list demon- strated statistically significant survival prediction (figure 3, p < 0.0153)), with 21 and 20 samples in each group. Integration of Genomic and Transcriptional Profiles To determine whether integration of genomic and expres- sion data added to the predictive value of these datasets for histologic classification, we identified 34 genes whose copy number and expression varied between AC and SCC, 24 of which demonstrated concordant differences in copy number and expression (table 4). Notably, 17 of 24 differentiating genes were located on chromosome 3q. Associations with mutation status Examination of transcriptional data from 1p, 1q, 6q, 11p, 11q and 12p (regions of genomic difference) identi- fied 25 genes with concordant differences in copy num- ber and expression between K-Ras mutant and wt tumours (table 5). A number of genes demonstrated reduced copy number and expression in KRAS mutant tumours, including putative tumour suppressor genes (FOXO3, EXTL2, PPP2R1B), negative regulators of the receptor tyrosine kinase oncogenic pathways (PTPRK, DGKZ, NCAM1), and negative regulators of Ras (EPHB2). Several over-expressed genes located in regions of amplification play roles in constitutive KRAS activation (PTGS2/COX2), enhanced transactivation of the EGFR (RGS2), enhanced invasive potential (ECM1), and MAPK/ERK activation (PTGS2/COX2). Associations with metastasis, tumour recurrence and NSCLC-specific survival Investigation of genomic and transcriptional data identi- fied only 2 genes (ARFGEF1 and PENK) whose copy number and expression differentiated ‘metastatic’ from