Cheng et al. BMC Medical Genomics 2012, 5:14 http://www.biomedcentral.com/1755-8794/5/14 RESEARCH ARTICLE Open Access Identification of genes with a correlation between copy number and expression in gastric cancer Lei Cheng1,4†, Ping Wang2†, Sheng Yang1, Yanqing Yang1, Qing Zhang3, Wen Zhang4, Huasheng Xiao4, Hengjun Gao4* and Qinghua Zhang1,4* Abstract Background: To elucidate gene expression associated with copy number changes, we performed a genome-wide copy number and expression microarray analysis of 25 pairs of gastric tissues. Methods: We applied laser capture microdissection (LCM) to obtain samples for microarray experiments and profiled DNA copy number and gene expression using 244K CGH Microarray and Human Exon 1.0 ST Microarray. Results: Obviously, gain at 8q was detected at the highest frequency (70%) and 20q at the second (63%). We also identified molecular genetic divergences for different TNM-stages or histological subtypes of gastric cancers. Interestingly, the C20orf11 amplification and gain at 20q13.33 almost separated moderately differentiated (MD) gastric cancers from poorly differentiated (PD) type. A set of 163 genes showing the correlations between gene copy number and expression was selected and the identified genes were able to discriminate matched adjacent noncancerous samples from gastric cancer samples in an unsupervised two-way hierarchical clustering. Quantitative RT-PCR analysis for 4 genes (C20orf11, XPO5, PUF60, and PLOD3) of the 163 genes validated the microarray results. Notably, some candidate genes (MCM4 and YWHAZ) and its adjacent genes such as PRKDC, UBE2V2, ANKRD46, ZNF706, and GRHL2, were concordantly deregulated by genomic aberrations. Conclusions: Taken together, our results reveal diverse chromosomal region alterations for different TNM-stages or histological subtypes of gastric cancers, which is helpful in researching clinicopathological classification, and highlight several interesting genes as potential biomarkers for gastric cancer. Keywords: Copy number variations, Gene expression profile, Correlation, Biomarkers, Gastric cancer Background gastric cancer patients barely reach below 30% in most Despite its steady declining trend worldwide, gastric cancer countries [3]. It is of great clinical importance to identify is still the second most common cause of cancer related new biomarkers for early diagnosis, targeted treatment and deaths with 700,000 cases annually [1]. Due to no symp- prognosis evaluation in gastric cancer. toms at the early stage of gastric cancer, it is often detected Gastric cancers can be divided into two main histo- at the advanced stage and the prognosis for treatment at logical subtypes, differentiated and poorly differentiated that time is poor [2]. Therapeutic interventions to treat (PD) adenocarcinomas. Differentiated adenocarcinoma is such late stage carcinomas are usually restricted to non- defined by tubular or glandular formation with cancer curative gastrectomy, lymphadenectomy and postoperative cells similar to the intestinal metaplasia, whereas the PD chemoradiotherapy. Thus, five-year relative survival rates of type is characterized by disruption of tubular formation due to reduction or loss of cell-cell interaction [4]. The PD adenocarcinomas occur in relatively young indivi- * Correspondence: [email protected]; qinghua_zhang@shbiochip. com duals and often metastasize to the peritoneum or lymph †Equal contributors nodes, resulting in a poor prognosis [5]. Despite the 1 State Key Laboratory of Medical Genomics and Shanghai Institute of aggressive nature of PD gastric cancer, little is known Hematology, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, China about the precise mechanisms of carcinogenesis or pro- 4National Engineering Center for Biochip at Shanghai, Shanghai, China gression and specific therapeutic targets. Full list of author information is available at the end of the article © 2012 Cheng et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Cheng et al. BMC Medical Genomics 2012, 5:14 Page 2 of 13 http://www.biomedcentral.com/1755-8794/5/14 It is currently realized that multiple genetic aberra- microdissection (LCM) to reduce the contamination of tions accumulating during the long process of carcino- cancer cells by non-cancer cells. We also analyzed aber- genesis are responsible for the initiation and progression ration patterns of different gastric cancer histopathology of cancers [6]. DNA copy number variations (CNVs) are subtypes to highlight molecular markers with potential important influential factors for altered gene expression clinical significance. levels in cancer. Recently, integration of genome-wide array-based comparative genomic hybridization (aCGH) Results and gene expression microarray data has provided a new DNA copy number variations in gastric cancer insight about the molecular mechanisms underlying The 27 pairs of gastric samples were analyzed by aCGH gene expression alterations [7-10]. In previous studies, as shown in Additional file 1: Table S3. The CNVs of all various microarrays (cDNA, BAC or PAC clone, oligo) the chromosomes were displayed in Figure 1A. The fre- were applied to investigate CNVs of gastric cancer. Due quency of CNVs was detected across the entire genome to the limit of resolution, sample size and preparation (Figure 1B). Noticeably, chromosomes 8, 20, and 7 con- method, the impact of CNVs on gene expression tained more genes undergoing frequent copy number remains poorly understood. amplifications, whereas the high frequent copy number In this study, we performed genome-wide DNA copy deletions were observed on chromosomes 6, 3, 4, and number and gene expression profiling of 25 pairs of gas- 18. CNVs frequently detected in gastric cancer were tric tissues to identify genes that show correlated pat- summarized in Table 1. The gained regions detected in terns of variations. Our study applied laser capture at least 25% of the samples were located at 8p11-q24, Figure 1 DNA copy number change profiles in the 27 pairs of gastric samples. (A) Summary of chromosomal aberrations was shown. (B) The CNVs frequency of the whole genome was analyzed by aCGH. Gains were marked in red and losses in green. Cheng et al. BMC Medical Genomics 2012, 5:14 Page 3 of 13 http://www.biomedcentral.com/1755-8794/5/14 Table 1 Chromosomal copy number gains and losses different regions in copy number variations between PD detected in at least 25% of gastric cancer samples and MD type. Moreover, we found that the MD type could Chromosomal aberration Samples, n = 27 (%) be classified by amplification of C20orf11 at 20q13.33 Gains (two-sample t-test, P < 0.05) (Figure 3). 8p11-q24 19 (70%) Besides, we also performed the comparisons in copy 20q11-q13 17 (63%) number changes for T1-2 (n = 11) versus T3-4 (n = 13) 7q21-q22 12 (44%) as well as N0 (n = 14) versus N1-3 (n = 11). We found that the frequencies of loss at 4p16.1, 4p14, 4q13.2, 7p12-p11 11 (41%) 5q21.1, 9q21.13, 9q22.31, 10q22.1, 12q15, 14q24.2, 20p12-p11 11 (41%) 22q11.21, and 22q12.2 were significantly higher in T3-4 7p21 10 (37%) stages than in T1-2 stages, while one region (10q22.1) 7q11 10 (37%) showed more gains in T1-2 stages. One region, 6q15 13q13-q14 9 (33%) had both significant gains and losses in T3-4 stages com- 6p21 8 (30%) pared to T1-2 stages (Figure 2B). DNA copy number 6p12 8 (30%) variation profiling of N0 and N1-3 stages also revealed 7p15 8 (30%) 18 significantly altered genomic regions (1q32.2, 13q12 8 (30%) 1q42.12, 2q14.1, 2q14.2, 2q35, 3p25.3, 3q26.2, 5q33.3, 20p13 8 (30%) 6q16.3, 6q22.33, 7q11.22, 9p23, 9q33.2, 11q23.3, 11q25, 14q11.2, 14q32.11, and 15q24.1) which showed more 1q42 7 (26%) aberrations in N1-3 stages compared with N0 stage 7p22 7 (26%) (Figure 2 C). Losses 4q34 9 (33%) Copy number associated gene expression changes 6p25 9 (33%) We found that 163 individual genes showed at least a 18q12 8 (30%) 1.3-fold copy number associated alteration in their ex- 18q22 8 (30%) pression (range 1.3 – 9.8, median 1.4) (Additional file 2: 3p14 7 (26%) Table S4). Of these, there was no gene located in the re- current regions of copy number loss. The gene showing 20q11-q13, 7q21-q22, 7p12-p11, 20p12-p11, 7p21, 7q11, the highest correlation was PI3 (FC = 9.8). PI3 (peptidase 13q13-q14, 6p21, 6p12, 7p15, 13q12, 20p13, 1q42, and inhibitor 3, skin-derived (SKALP)) gene, amplified in the 7p22 in decreasing order of frequency (Table 1). Regions 20q12-q13.2 region, displayed the strongest copy num- of loss detected in at least 25% of the samples were ber amplification correlated overexpression in gastric located at 4q34, 6p25, 18q12, 18q22, and 3p14 in de- cancer. Generally, the highest gene expression fold creasing order of frequency (Table 1). Minimal common changes between tumor samples with and without copy regions of these copy number aberrations were shown in number amplifications were detected at the 6p region Table 2, including the size, frequency, possible target since out of the 20 genes showing >1.7-fold copy genes, and chromosomal position of the alteration in number associated changes in their expression, 11 base pairs. Possible target genes were selected with at (55.0%) were located in the 6p region (Additional file 2: least two-fold copy number associated