Here, we generated a non-gapped specialized CGH microarray representing 18 human chromosome imbalance 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 invaluable 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 further 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 chromosomal imbalance to be our candidate gene database (Table 1). To improve the validity for identifying potential 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 candidate genes involved in these processes may play important roles in lung tumorigenesis. For example, irregular choline transport and metabolism have been involved in growth arrest and apoptosis in human lung ADC . Dysfunction of histone methylation was also shown to contribute to human carcinogenesis . Interestingly, genes involved in Wnt receptor signaling pathway were unique in Asian lung cancer, whereas Caucasian lung cancer was addictive to cell surface receptor linked signal transduction and G-protein coupled receptor protein signaling pathways (Figure1A-B). Genes involved in these pathways warrant further studies.
This study found several candidate genes which are located in the common chromosome alteration regions in both Asian and Caucasian, such as PAFAH1B1 gene on 17p13.3. In array-CGH data, chromosome region 17p13.3 harboring PAFAH1B1 showed gain of copy number in 60% of Asian and 70% of Caucasian patients (Table 1). In addition, PAFAH1B1 DNA copy number and mRNA expression level in tumor tissues was significantly higher than that of the matched-normal tissues in both Asian and Caucasian lung cancer patients (Figure3). The frequency of PAFAH1B1 protein overexpression was 68% in Asian and 70% in Caucasian (Figure4). Interestingly, chromosome region 17p13.3 harboring PAFAH1B1 gene also demonstrated association with advanced tumor stage in array-CGH data (Table 3A). Overexpression of PAFAH1B1 mRNA was found in 59.1% of early-stage and 78.3% of late-stage lung cancer patients of Asian descent ( Additional file 6: Table S5), suggesting that the alteration of PAFAH1B1 gene may be involved in both tumor initiation and progression. More patients will be analyzed to clarify its tumorigenic role.
We also found several candidate genes showing different alteration frequency between Asian and Caucasian patients, such as ARHGAP19 and FRAT2. These two closely located (within a 39.8 kb region at 10q24.1) candidate genes, showed alteration frequency of 57.5% with gain of gene copy number in Asian (Table 1A), whereas the alteration frequency was only 20% in Caucasian patients. In addition, both qPCR and RT-qPCR confirmed significant gene amplification and mRNA overexpression in tumors from Asian but not from Caucasian lung cancer patients (Figure3A-B). Interestingly, in relation to clinical pathological parameters of Asian lung cancer patients, mRNA overexpression of ARHGAP19 and FRAT2 was significantly associated with lung SCC ( Additional file 6: Table S5). ARHGAP19 encodes a Rho GTPase-activating protein that stimulates the intrinsic GTP hydrolysis activity of Rho family proteins . ARHGAP family genes are considered to be cancer-associated genes because their genetic alterations lead to carcinogenesis through the dysregulation of Rho/Rac/Cdc42-like GTPases . The up-regulation of the FRAT2 gene has been reported in human gastric cancer  and has been implicated in carcinogenesis through activation of the Wnt signaling pathway . Our previous study showed that more than 50% of Taiwanese lung cancer patients had alterations in proteins involved in Wnt/β-catenin signaling . It is important to investigate the role of ARHGAP19 and FRAT2 alterations in SCC lung tumorigenesis and their correlations with Rho and Wnt signaling pathways.
Interestingly, ZNF322A on 6p22.1 showed significant gene amplification and mRNA overexpression in Caucasian, and borderline significant in Asian lung cancer patients (Figure3). The DNA copy number validation for ZNF322A gene was lower than the array CGH result because the BAC may harbor some other genes in addition to ZNF322A. ZNF322A has been reported to be a novel human C2H2 Krüppel-like zinc-finger protein, which regulates transcriptional activation in the MAPK signaling pathways. The Krüppel zinc-finger protein is involved in the regulation of normal cell growth, differentiation, embryogenesis, and tumorigenesis . Identification of the role of ZNF322A in lung tumorigenesis is worthy of further study.
Note that no common tumor subtype-specific regions for ADC and SCC were found in Asian and Caucasian lung cancer patients when the genomic alteration profiles were compared (Table 2A). Even in the same racial group, extreme difference of chromosome alterations could occur between ADC and SCC. These data implicated that distinct etiological mechanism in addition to tobacco and environmental exposures are involved in lung cancer of Asian from Caucasian, supporting the epidemiologic observation that lung cancer in Asian shows relatively high percentage of non-smoking female ADC. Notably, Broët et al.  identified molecular differences between NSCLCs from East-Asian and Western European patients by using single nucleotide polymorphism microarray platform, and revealed several copy number aberrations significantly associated with ethnicity (gain on 1p36, 16p13, 16p12, and 16p11 in East-Asian; loss on 19p13 in Western European patients), EGFR mutation (gain on 1p36, 1p35, 7p22-21, 7p15-12, 14q31-32, 16p13, and 16p12 in EGFR mutant tumors; loss on 21q21-22 in EGFR wild-type tumors), but not to K-ras or p53 mutations. Correlation of the mutational spectrum of EGFR
p53, and ALK genes to our array-CGH data warrants further investigations.