Integration of DNA Copy Number Alterations and Transcriptional Expression Analysis in Human Gastric Cancer

Background: Genomic instability with frequent DNA copy number alterations is one of the key hallmarks of carcinogenesis. The chromosomal regions with frequent DNA copy number gain and loss in human gastric cancer are still poorly defined. It remains unknown how the DNA copy number variations contributes to the changes of gene expression profiles, especially on the global level. Principal Findings: We analyzed DNA copy number alterations in 64 human gastric cancer samples and 8 gastric cancer cell lines using bacterial artificial chromosome (BAC) arrays based comparative genomic hybridization (aCGH). Statistical analysis was applied to correlate previously published gene expression data obtained from cDNA microarrays with corresponding DNA copy number variation data to identify candidate oncogenes and tumor suppressor genes. We found that gastric cancer samples showed recurrent DNA copy number variations, including gains at 5p, 8q, 20p, 20q, and losses at 4q, 9p, 18q, 21q. The most frequent regions of amplification were 20q12 (7/72), 20q12-20q13.1 (12/72), 20q13.1-20q13.2 (11/72) and 20q13.2-20q13.3 (6/72). The most frequent deleted region was 9p21 (8/72). Correlating gene expression array data with aCGH identified 321 candidate oncogenes, which were overexpressed and showed frequent DNA copy number gains; and 12 candidate tumor suppressor genes which were down-regulated and showed frequent DNA copy number losses in human gastric cancers. Three networks of significantly expressed genes in gastric cancer samples were identified by ingenuity pathway analysis. Conclusions: This study provides insight into DNA copy number variations and their contribution to altered gene expression profiles during human gastric cancer development. It provides novel candidate driver oncogenes or tumor suppressor genes for human gastric cancer, useful pathway maps for the future understanding of the molecular pathogenesis of this malignancy, and the construction of new therapeutic targets. © 2012 Fan et al.published_or_final_versio

DNA copy number analysis of representative amplicons and homozygous deletions.

<p>Clones were ordered by their position from pter (left) to qter (right). The log2 ratios of every clone in these specific cases were plotted as broken line graphs with different color. Multiple clear copy number changes (gains, losses, amplifications and deletions) can be recognized. The center of amplicon and homozygous deletion cores were indicated together with genes in each core region. (<b>A</b>) Amplification in 17q11.2–17q21. (<b>B</b>) Amplification in 19q12–19q13.1. (<b>C</b>) Amplification in 8q24.1–8q24.2. (<b>D</b>) Homozygous deletion in 18q21.1. (<b>E</b>) Homozygous deletion in 9p21. (<b>F</b>) Homozygous deletion in 16q23. (<b>G</b>) Homozygous deletion in 18q12.</p

Hierarchical clustering of gastric tumors.

<p>(<b>A</b>) Hierarchical clustering the patterns of variation in expression of 333 candidate oncogene and tumor suppressor genes (from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029824#pone.0029824.s018" target="_blank">Table S6</a>) in 62 gastric tumors<b>.</b> Each row represented a separate cDNA clone on the microarray and each column represented the expression pattern in a separate tumor or tissue sample. The ratio of abundance of transcripts of each gene to its mean abundance across all tissue samples was depicted according to the color scale shown at the bottom. Gray indicated missing or excluded data. The dendrogram at the top of the figure represented the hierarchical clustering of the tumors based on similarity in their pattern of expression of these genes. (<b>B</b>) <b>to</b> (<b>H</b>) compared DNA copy number changes with the corresponding gene expression values in selected gene clusters in each individual tumor sample. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029824#pone.0029824.s020" target="_blank">Table S8</a> for full data.</p

Ingenuity networks in gastric cancer samples.

<p>Ingenuity networks generated by mapping the candidate oncogenes and tumor suppressor genes identified by integrated analysis of expression array and aCGH data. Each network was graphically displayed with genes or gene products as nodes (different shapes represented the functional classes of the gene products) and the biological relationships between the nodes as edges (lines). The length of an edge reflected the evidence in the literature supporting that node-to-node relationship. The intensity of the node color indicated the degree of up- (red) or downregulation (green) of the respective gene. Genes in uncolored notes were not identified as differentially expressed in our experiment and were integrated into the computationally generated networks on the basis of the evidence stored in the IPA knowledge memory indicating a relevance to this network. A solid line without arrow indicated protein-protein interaction. Arrows indicated the direction of action (either with or without binding) of one gene to another.</p

DNA copy number alterations by aCGH.

<p>(A) Overall frequency of DNA copy number alterations by aCGH. Frequency analysis measured as a fraction of cases gained or lost over all the BAC clones on the arrays. Data presented was ordered by chromosomal map position of the clones. Lower bars represented losses and upper bars represented gains. The purple vertical bars represented the boundary between each chromosome. (B) DNA copy number alterations in each gastric cancer samples. 72 tumor samples were ordered from top to bottom. Red columns represented copy number gains and green columns represented copy number losses.</p