Lo et al. BMC Cancer 2012, 12:235 http://www.biomedcentral.com/1471-2407/12/235 Background Lung cancer is the leading cause of cancer death world- wide [1,2]. There is mounting evidence of difference be- tween Asian and Caucasian lung cancer patients in clinical and epidemiological aspects. For example, Lee et al. [3] reported that lung cancer in Taiwan and South China is characterized with a high percentage of female adenocar- cinoma (ADC) and nonsmokers. In addition, response to certain anticancer treatments such as gefitinib is much greater in Asian patients than Caucasians [4]. How- ever, these are rare large-scale attempts to identify the molecular characteristics of lung cancer between Asian and Caucasian. Therefore, identification and characterization of the chromosome regions with DNA copy number alter- ation in Western and Asian countries is important to unravel the mechanism underlying lung tumorigenesis. Genomic DNA copy number variation is a hallmark of cancer and can lead to alteration in the expression and functions of genes residing within the affected chromo- somal region [5]. Such a segment in the tumor genome may harbor either oncogenes or tumor suppressor genes depending on whether they are present in increased or decreased copy number, respectively. Identification of regions with copy number aberrations and the genes involved thus offers a basis for better understanding of cancer development [6]. Array-comparative genomic hybridization (array-CGH) can measure relative copy number at specific loci, which are represented by arrays of mapped bacterial artificial chromosomes (BAC) clones, cDNA clones, or various oligonucleotide-based formats [7-9]. The resolution of array-CGH is deter- mined by the genomic spacing of the array elements thus is much more improved compared to previous con- ventional CGH [10-12]. To identify the chromosome regions with DNA copy number alteration in patients from Western and Asian countries, we constructed a non-gapped array containing 3604 BAC clones covering 18 lung cancer-related chromosome imbalance hotspot regions identified in our previous study [13]. DNA from 40 Asian and 20 Caucasian non-small cell lung cancer (NSCLC) patients was analyzed using this specialized array to identify lung cancer-related genes either common or differential in these two ethnic groups. The Bayes statistical and comparative genomic approaches were used to identify genes with DNA copy number alterations. Our study provides a comprehensive database for chromosomal regions and genes with copy number imbalance in both Asian and Caucasian lung cancer patients. In addition, gene validation was performed using quantitative polymerase chain reaction (qPCR), reverse transcriptase-qPCR (RT-qPCR), chromogenic in situ hybridization (CISH), and immunohistochemis- try (IHC) on specimens from the Asian and Caucasian lung cancer patients. Page 2 of 13 Methods Clinical samples preparation and DNA/RNA extraction Tissues were collected after obtaining appropriate institu- tional review board permission (VGHIRB No. 201001019IA) and informed consent from the recruited patients. Surgically resected tumor tissue and corresponding normal tissue were collected from 40 patients diagnosed with primary non- small cell lung cancer (NSCLC) admitted to Taipei Veterans General Hospital, Taiwan. Twenty NSCLC tumor tissues from Caucasian patients were obtained from the University of Chicago, USA. The list of these 60 patients along with the patients used for further validations is described in the Additional file 1: Table S1. Histological classification was determined according to the WHO classification system and the tumor-node-metastasis system. Infor- mation on the age, sex, tumor type, tumor stage and smoking history of the patients was obtained from hos- pital records. The genomic DNA was prepared using pro- teinase K digestion and phenol-chloroform extraction. Total RNA was prepared from tumors and normal lung tissues, using TRIzol reagent (Invitrogen, Carlsbad, CA). cDNA was generated using SuperScript reverse transcript- ase (Invitrogen). Array-CGH The genomic microarray used contained 3604 BAC clones representing the human genome at 18 non-gap chromosome regions (Additional file 2: Table S2). Clone mapping was analyzed using the BAC end pairs database of UCSC Genome browser (http://genome. ucsc.edu/, build 36 - hg18). The BAC library used was RP-11. The extracted BAC DNA was amplified to 10 μg using Phi29 DNA polymerase reaction (TempliPhi DNA Sequencing Template Amplification Kit, Amersham, Princeton, NJ), then digested with DNaseI (New England Biolabs, Ipswich, MA), and purified with MultiScreen- PCR Plates (Millipore, Danvers, MA). After digestion, the DNA fragments were resolved by gel electrophoresis and the fragment sizes were optimized to within 0.5–3 kb. Finally, the purified BAC DNA fragments were spotted in triplicate of 72 blocks onto the glass slides (Corning, NY) with spotting solution (50% DMSO, 50% dH2O). Representative array images are shown in the Additional file 3: Figure S1. Approximately 500 ng of tumor genomic DNA and reference DNA [for Asian samples: 28 matched-normal genomic DNA pool; for Caucasian samples: male human genomic DNA (Promega, Leiden, the Netherlands)] were labeled by random priming using BioPrime DNA Label- ing System (Invitrogen) with Cy3-dCTP (reference) or Cy5-dCTP (tumor). Labeled tumor and reference DNA were combined together with 100 μg human Cot-1 DNA (Invitrogen). Hybridization was performed using MAUI hybridization system (BioMcro Systems, Salt Lake City,