Newnham et al. BMC Cancer 2011, 11:93 http://www.biomedcentral.com/1471-2407/11/93 Combined Lung Service between February 2004 and July 2006 and planned for curative resection were invited to participate. Exclusion criteria included age <18 years, administration of neoadjuvant chemotherapy, and inability to provide informed consent. Integrated demographic, radiological, pathological and outcome data was collected for all consenting patients. In addition, a small number of samples collected ear- lier and stored in the Peter MacCallum (PeterMac) tis- sue bank were utilised after approval by the PeterMac Tissue Management Committee. Microarray analyses Samples of tumour (≥1 cm 3) were selected from fresh specimens, and then stored whole at -180°C. Only those specimens containing >75% tumour cells and <25% necrosis were used in molecular studies. Both RNA and DNA were isolated from each sample for analysis using established protocols (see additional files 1 and 2). Tran- scriptional profiling was performed using 10,500 ele- ment cDNA microarrays (PeterMac, Melbourne, Australia) [2]. Genomic profiling using 2400 element bacterial artificial chromosome (BAC) arrays was com- pleted at the University of San Francisco, California, USA [3]. Detailed description of transcriptional and genomic profiling is included in additional files 3 and 4. Mutation analyses All samples were tested for TP53 mutations and all ade- nocarcinoma (AC) and large cell carcinoma (LCC) sam- ples were screened for KRAS mutations using high resolution melting analysis [4,5] with or without DNA sequencing. Bioinformatic analyses The effect of histology, presence or absence of KRAS or TP53 mutation, tumour size and metastasis status, recur- rence within 12 months of surgery and survival on gene expression was explored. After removing control genes, analysis was conducted in CRAN, R Bioconductor using the LIMMA [6,7] package to generate detailed lists of gene expression differences with significance p values between each subgroup of interest. To account for multiple testing, p values of <0.005 were considered statistically significant. Gene lists were then interrogated using publicly available programs (Intelligent Systems and Bioinformatics Labora- tory, Wayne State University, Detroit, MI, USA, http://vor- tex.cs.wayne.edu/projects.htm) to identify gene ontology and molecular pathway patterns. Changes in the normalized and smoothed genomic data [8] were assigned stepwise copy change levels from -2 to 3 (-2 = homozygous loss, -1 = heterozygous loss, 1 = single copy gain, 2 = gain of two copies, 3 = high- level gain, 0 = normal copy number). Using these Page 2 of 11 standardised copy number values, it was possible to make comparisons of the frequency of each level of copy change at each BAC location for specific groups of interest. To compare our data with that of other groups of patients with early stage NSCLC, we performed compar- isons with publicly available external data sets obtained via the NCBI Gene Expression Omnibus (GEO) website. Different platforms were reconciled using HUGO approved gene symbols, and extracted gene expression data were log2 transformed, centred and scaled across samples in order to emphasise relative expressions as opposed to absolute values. Integration of transcriptional and genomic profiles Integration of transcriptional and genomic datasets was performed by investigating levels of expression for genes located in regions of copy number variation between groups of interest. Genes whose differences in expression varied in the same direction as differences in copy num- ber between two groups (e.g. relative over-expression of genes in a region of increased copy number) were viewed as genes of interest. Results Molecular and clinical data was available for 69 patients who underwent surgery for NSCLC between May 1999 and July 2006. Demographic and pathologic data are included in table 1. Median follow-up for surviving patients exceeds 35 months. After a median follow-up of 36 months (1 - 80) for all patients, 28/68 patients developed recurrent disease, and 23/68 patients died of NSCLC (one patient with disseminated disease at diag- nosis excluded from analysis). Comparable to other ser- ies of early stage NSCLC [9], five year overall survival rates approximated 55%. Genomic Analysis Aneuploidy All samples demonstrated significant chromosomal instability with an average of 43.6 chromosomal break- points per sample (defined by a change in the stepwise copy number along a chromosome), with over 100 and 150 regions of high-level gain (+3) and loss (-3) respec- tively. There was also a very high rate of low-level geno- mic alteration (both gains and losses). On average, over 10 whole arm losses or duplications were seen per sam- ple, with a rate of isochromosome formation of 1.6 per genome (duplication of one arm with loss of the oppos- ing arm of the same chromosome). These results are consistent with the highly disordered nature of lung cancer genomes. Comparisons between clinical sub- groups of interest revealed remarkably similar degrees of aneuploidy and chromosomal disorder in almost all