Modern Pathology (2006) 19, 854–863 & 2006 USCAP, Inc All rights reserved 0893-3952/06 $30.00 www.modernpathology.org Comprehensive analysis of 19q12 amplicon in human gastric cancers Suet Yi Leung1, Coral Ho2, I-Ping Tu3, Rui Li4, Samuel So4, Kent-Man Chu5, Siu Tsan Yuen1 and Xin Chen2 1Department of Pathology, The University of Hong Kong, Queen Mary Hospital, Pokfulam, Hong Kong; 2Department of Biopharmaceutical Sciences, University of California, San Francisco, CA, USA; 3The Institute of Statistical Science, Acdemia Sinica, Taipei, Taiwan, ROC; 4Department of Surgery, Stanford University, Stanford, CA, USA and 5Department of Surgery, The University of Hong Kong, Queen Mary Hospital, Pokfulam, Hong Kong Amplification at 19q12 has been observed in multiple tumor types, while cyclin E1 (CCNE1) has been considered to be the key oncogene within this amplicon. We have previously applied cDNA microarray analysis to systematically characterize gene expression patterns of gastric tumor and nontumor samples. We identified a cluster of five tightly coregulated genes all located at chromosome 19q12, including CCNE1. We found that the 19q12 gene cluster is highly expressed in gastric tumors compared to nontumor gastric samples. Array based comparative genomic hybridization and real-time PCR was used to define the boundary of the 19q12 amplicon to a region of approximately 200 kb. Interestingly, we found that in some cases amplification at 19q12 was not associated with DNA copy number gain at CCNE1, suggesting that some other genes within the 19q12 amplicon may also have important function during gastric tumorigenesis. We found high expression of the 19q12 gene cluster to be statistically correlated with the cell proliferation gene signature. Using the SAM software, we identified a set of 577 genes whose expression levels positively correlated with the 19q12 gene cluster. GO term analysis revealed that this genelist is enriched with genes involved in cell cycle regulation and cell proliferation. In conclusion, expression array analysis combined with array comparative genomic hybridization and real-time PCR provides a new and powerful tool to identify clusters of genes which may be regulated by genomic DNA aberrations. In addition, our study indicates that amplification at 19q12 is associated with cell proliferation in vivo. Modern Pathology (2006) 19, 854–863. doi:10.1038/modpathol.3800593; published online 31 March 2006 Keywords: microarrays; array CGH; gastric cancer; amplification Carcinogenesis is a multistep process involving observed in tumor samples. Among these amplicons many genetic and epigenetic events. The former many have well-characterized oncogenes, for exam- can take the forms of amplification of oncogenes and ple, Myc at 8q24, ErbB2 at 17q12 and cyclin D at deletion or mutation of tumor suppressor genes. 11q13. Mutation of genes involved in maintaining genomic Amplification at chromsome 19q12 has been integrity results in catastrophic events, leading to observed in multiple tumor types2–5 (also see rapid accumulation of chromosomal aberrations.1 www.helsinki.fi/cmg/cgh_data). Cyclin E1 (CCNE1), The occurrence of genomic instability is expected to an E type cyclin, is located at 19q12, and has affect global gene expression profiles, and provide a traditionally been considered to be the target of ground for selection of genetic traits that confer a 19q12 amplicon. CCNE1 associates with CDK2 growth advantage important for tumor progression. and CDK3 and directs the cell from the late As a result, most human solid tumors are aneuploid, G1 phase into the S phase. It also controls the accumulating numerous gains and losses of chro- initiation of DNA replication and centrosome mosomal regions. In some cases, recurrent high duplication.6,7 As unchecked cell growth and levels of DNA copy number amplification can be proliferation is the hallmark of human cancers, the CCNE1/CDK2 kinase system has been shown to be misregulated in multiple tumor types.2,8 For Correspondence: Dr X Chen, PhD, Department of Biopharmaceu- example, CCNE1 is overexpressed in breast, ovarian, tical Sciences, University of California, San Francisco, 513 pancreatic, bladder, endometrial and colon can- Parnassus Avenue, CA 94143-0446, USA. 3–5,9–11 E-mail: [email protected] cers. In many cases, the overexpression of Received 3 October 2005; revised and accepted 27 February 2006; CCNE1 is associated with amplification at the 19q12 published online 31 March 2006 locus.2–5 19q12 amplicon in gastric cancers SY Leung et al 855 Gastric cancer is the second most common cause mately 5200 unique genes for hierarchical cluster- of cancer death worldwide.12 Environmental and ing, were generated as described.24,25 genetic factors are both important in gastric carcino- genesis. Gastritis, caused most commonly due to Helicobacter pylori (H. pylori) infection, is strongly Genomic DNA Extraction associated with the development of gastric cancer.13 Comparative genomic hybridization (CGH) has been Genomic DNA was extracted from gastric tumor applied to study DNA copy number variation among samples using QiaAmp kit (Qiagen, Valencia, CA, gastric cancers.14–18 Like many other cancer types, USA). In all cases, DNA was isolated from adjacent CCNE1 is found to be amplified at 19q12 and blocks from the same tumors that RNA was extracted overexpressed in a subset of gastric cancer sam- and used for microarray analysis. ples.19–21 The biological and clinical significance of CCNE1 amplification and overexpression remains controversial: while some studies reported the Array CGH and Analysis correlation of CCNE1 expression with poor patient 20,22 23 The arrays used in the study were prepared survival, others found no such correlation. and hybridized as described previously.26 In Recently, we reported the systematic characteriza- brief, human 1.14 arrays were obtained from the tion of gene expression in a large-scale analysis of UCSF Cancer Center Array Core (http://cc.ucsf.edu/ gastric adenocarcinomas vs non-neoplastic gastric 24,25 microarray/). The arrays consisted of 2463 bacterial mucosa using cDNA microarrays. Hierarchical artificial chromosome (BAC) clones that covered the clustering of global expression profiles of human human genome at 1.5 Mb resolution. For hybridiza- gastric cancers revealed a tightly coregulated cluster tion, 1 mg of tumor DNA and 1 mg of gender-matched of five genes, all located at chromosome 19q12, reference DNA (isolated from normal donor lym- including CCNE1. In this paper, we report an in phocytes) was labeled by random priming using depth analysis of this 19q12 gene cluster and Cy3-dCTP and Cy5-dCTP, respectively, and hybri- demonstrate that high expression of the 19q12 gene dized to the arrays. Three single-color intensity cluster is associated with 19q12 locus amplification. images (DAPI, Cy3 and Cy5) were collected from Using array-based CGH (array CGH) and real-time each array using a charge-coupled device camera. PCR we further refine the boundary of 19a12 UCSF SPOT software27 was used to automatically amplicon to approximately 200 kb along chromo- segment the spots based on DAPI images, perform some 19q. We found that high expression of the local background correction and to calculate various 19q12 gene cluster statistically correlates with the measurement parameters, including log2 ratios of cell proliferation gene signature. In addition, we the total integrated Cy3 and Cy5 intensities for each identified a set of 577 genes whose expression levels spot. A second custom program, SPROC (http:// positively correlate with the 19q12 gene cluster. The jainlab.ucsf.edu/Downloads.html) was used to asso- study therefore indicates that amplification at 19q12 ciate clone identities and a mapping information file is associated with cell proliferation in vivo. with each spot, so that the data could be plotted relative to the position of the BACs. Chromosomal aberrations were classified as a gain when the Experimental methods normalized log2 Cy3/Cy5 ratio was higher than 0.225; and as a loss when the ratio was lower than Patient Samples, RNA Preparation, cDNA Microarray À0.225. Steep copy number changes with the graph and Hierarchical Clustering showing a peak rather than a plateau, and a minimal normalized log Cy3/Cy5 ratio of 0.9 or higher, were Samples of tumor and normal gastric mucosa were 2 classified as amplifications. Likewise, log2 Cy3/Cy5 collected from gastrectomy specimens from Depart- ratio of À0.8 or lower were classified as homozygous ment of Surgery, Queen Mary Hospital, The deletions. University of Hong Kong. Tissues were frozen in liquid nitrogen within half an hour after they were resected. Non-neoplastic mucosa from stomach Quantitative Real-Time PCR to Determine DNA Copy was dissected free of muscle, and histologically Number Changes confirmed to be tumor free by frozen section. Total RNA was extracted using Trizol (Invitrogen, Sybergreen-based real-time PCR was performed to Carlsbad, CA, USA). A detailed description of assay the DNA copy number variation among nine DNA microarray procedure has been previously loci at 19q12. House keeping gene GAPDH was used reported.24,25 Raw data are available at both the as the reference control. The detailed method of Stanford Microarray Database (http://genome- quantitative real-time PCR is the same as previously www5.stanford.edu/MicroArray/SMD/) and the described.28 Assays were carried out using the ArrayExpress online data repository (http://www. software supplied with the ABI 7900 (Applied