The database of chromosome imbalance regions and genes resided in lung cancer from Asian and Caucasian identified by array-comparative genomic hybridization

Abstract

Background: Cancer-related genes show racial differences. Therefore, identification and characterization of DNA copy number alteration regions in different racial groups helps to dissect the mechanism of tumorigenesis.

Methods: Array-comparative genomic hybridization (array-CGH) was analyzed for DNA copy number profile in 40 Asian and 20 Caucasian lung cancer patients. Three methods including MetaCore analysis for disease and pathway correlations, concordance analysis between array-CGH database and the expression array database, and literature search for copy number variation genes were performed to select novel lung cancer candidate genes. Four candidate oncogenes were validated for DNA copy number and mRNA and protein expression by quantitative polymerase chain reaction (qPCR), chromogenic in situ hybridization (CISH), reverse transcriptase-qPCR (RT-qPCR), and immunohistochemistry (IHC) in more patients.

Results: We identified 20 chromosomal imbalance regions harboring 459 genes for Caucasian and 17 regions containing 476 genes for Asian lung cancer patients. Seven common chromosomal imbalance regions harboring 117 genes, included gain on 3p13-14, 6p22.1, 9q21.13, 13q14.1, and 17p13.3; and loss on 3p22.2-22.3 and 13q13.3 were found both in Asian and Caucasian patients. Gene validation for four genes including ARHGAP19 (10q24.1) functioning in Rho activity control, FRAT2 (10q24.1) involved in Wnt signaling, PAFAH1B1 (17p13.3) functioning in motility control, and ZNF322A (6p22.1) involved in MAPK signaling was performed using qPCR and RT-qPCR. Mean gene dosage and mRNA expression level of the four candidate genes in tumor tissues were significantly higher than the corresponding normal tissues (P<0.001~P=0.06). In addition, CISH analysis of patients indicated that copy number amplification indeed occurred for ARHGAP19 and ZNF322A genes in lung cancer patients. IHC analysis of paraffin blocks from Asian Caucasian patients demonstrated that the frequency of PAFAH1B1 protein overexpression was 68% in Asian and 70% in Caucasian.

Conclusions: Our study provides an invaluable database revealing common and differential imbalance regions at specific chromosomes among Asian and Caucasian lung cancer patients. Four validation methods confirmed our database, which would help in further studies on the mechanism of lung tumorigenesis.

Figure 1

Pie chart of significantly altered biological processes in Asian (A) and Caucasian (B) lung cancer patients. The data were analyzed by DAVID bioinformatics resources. The common biological processes in both Asian and Caucasian marked with asterisk (*).

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.

Figure 3

DNA copy number validation by qPCR (A) and mRNA expression validation by RT-qPCR (B) of four candidate genes in clinical samples. The Y-axis is the mean DNA dosage ratio (A) or mean mRNA expression ratio (B) between candidate gene and the internal control gene in all samples analyzed. The P values of comparison between normal (N) and tumor (T) samples for each gene is as indicated.

Figure 4

DNA copy number and protein expression level validation in specimens from lung cancer patients. (A) Representative CISH images of ARHGAP19 and ZNF322A genes in paraffin-embedded lung tumor tissue. Images taken from tumor without gene copy number amplification (left) and with gene copy number amplification (right). The enlarged image of one nucleus is shown on the lower right corner. CISH images: 400X magnification. (B) Representative IHC images of PAFAH1B1 protein in tissue array. Patients with protein overexpression are indicated by + symbol. Patients with normal expression are indicated by – symbol. IHC images: 200X magnification.