Lo et al. BMC Cancer 2012, 12:235 http://www.biomedcentral.com/1471-2407/12/235 Page 8 of 13 Figure 2 Flowchart summarizing the strategy of candidate genes selection. First, a total of 818 genes with more than 50% frequency of chromosome alteration were analyzed by MetaCore software. Second, the candidate genes analyzed with MetaCore were further integrated with the gene expression database. Integrating our array-CGH database with the expression array databases, 134 genes showing correlation between DNA copy number and mRNA expression patterns were found. Finally, literature search was performed to select 54 novel lung cancer candidate genes for following validation assays at the DNA, and RNA levels. for following validation assays at the DNA, and RNA levels. A good correlation is found between array-CGH data and various validation methods including CISH, qPCR, RT-qPCR, and IHC Four methods were employed for gene validation in 147 lung cancer patients. The clinical characteristics and the validation method conducted for each patient are shown in the Additional file 1: Table S1. qPCR for DNA copy number was performed for four candidate genes in tumors from 30 Asian and 30 Caucasian lung cancer patients, whose samples were analyzed in array-CGH. The mean gene dosage of lung cancer candidate genes ARHGAP19, FRAT2, PAFAH1B1, and ZNF322A in tumor tissues was significantly higher than the corre- sponding normal tissues in Asian lung cancer. Gene dos- age of PAFAH1B1 and ZNF322A was significantly higher in tumor tissues than autopsy tissues from Caucasian (Figure 3A). RT-qPCR of the four genes was conducted with cDNA from tumor and corresponding normal tis- sues of 45 Asian lung cancer patients. Only two candi- date genes, PAFAH1B1 and ZNF322A, were analyzed in tumor and corresponding normal lung tissues from 24 Caucasian lung cancer patients due to the mRNA ina- vailability. The mean mRNA expression level of all genes analyzed in the tumor tissues was significantly higher than in the corresponding normal tissues in Asian and Caucasian patients (Figure 3B). In addition, the validation data showed concordant results with array-CGH. CISH analysis of patients indicated that copy number amplification indeed occurred for ARHGAP19 and ZNF322A genes in lung cancer patients (Figure 4A). To determine protein expression level of the selected candi- date genes in lung cancer, IHC was performed for PAFAH1B1 protein on paraffin blocks from 28 Asian patients and tissue arrays of Caucasian patients includ- ing 37 lung cancer patients (Figure 4B). The frequency of PAFAH1B1 protein overexpression was 68% in Asian and 70% in Caucasian. Discussion Here, we generated a non-gapped specialized CGH microarray representing 18 human chromosome imbal- ance hotspot regions in lung cancer. This specialized CGH array identified the chromosomal regions whose structural alteration may play critical roles in lung tumorigenesis. Our array-CGH study provided an in- valuable database of chromosomal imbalance regions and candidate genes in Asian and Caucasian lung cancer patients. CISH, qPCR, RT-qPCR, and IHC were employed for gene validation of four novel genes with amplification. The putative lung cancer-related genes identified in our array-CGH database would help in fur- ther studies on the mechanism of lung tumorigenesis. Using the UCSC Genome browser, we have annotated a total of 476 genes for Asian patients and 459 genes for Caucasian patients showing high frequency of chromo- somal imbalance to be our candidate gene database (Table 1). To improve the validity for identifying poten- tial cancer-related gene, the genes residing within the chromosomal imbalance regions were integrated with gene expression databases. Among the genes selected, the ion transport and chromatin remodeling are the two main common biological processes altered in both Asia and Caucasian populations, suggesting that the candi- date genes involved in these processes may play import- ant roles in lung tumorigenesis. For example, irregular choline transport and metabolism have been involved in growth arrest and apoptosis in human lung ADC [21]. Dysfunction of histone methylation was also shown to contribute to human carcinogenesis [22]. Interestingly, genes involved in Wnt receptor signaling pathway were