Oncogene (2003) 22, 4287–4300 & 2003 Nature Publishing Group All rights reserved 0950-9232/03 $25.00 www.nature.com/onc Profiling, comparison and validation of gene expression in gastric carcinoma and normal stomach Karin A Oien*,1,2, J Keith Vass3, Ian Downie2, Grant Fullarton4 and W Nicol Keith1 1Cancer Research UK Department of Medical Oncology, Cancer Research UK Beatson Laboratories, University of Glasgow, Garscube Estate, Switchback Road, Glasgow G61 1BD, UK; 2University Department of Pathology, Glasgow Royal Infirmary, 84 Castle Street, Glasgow G4 OSF, UK; 3Beatson Institute for Cancer Research, Cancer Research UK Beatson Laboratories, Garscube Estate, Switchback Road, Glasgow G61 1BD, UK; 4Department of Surgery, Gartnavel General Hospital, 1053 Great Western Road, Glasgow G12 0YN, UK Gastric carcinoma is the fourth most common cause of Introduction cancer death worldwide but its molecular biology is poorly understood. We catalogued the genes expressed in two Gastric carcinoma is a common tumour with a high gastric adenocarcinomas and normal stomach, using serial mortality (Fuchs and Mayer, 1995; Blok et al., 1997). analysis of gene expression (SAGE), and compared the Worldwide, it is the fourth most common cause of profiles on-line with other glandular epithelia. Candidates cancer death (Parkin, 2001). It ranks fifth in Scotland, were validated by Northern blotting and immunohisto- where the overall 5-year survival for gastric carcinoma is chemistry. A total of 29 480 transcripts, derived from only around 12%, which compares poorly with 44% for 10 866 genes, were identified. In all, 1% of the genes were colonic carcinoma (Harris et al., 1998). The reason is differentially expressed (Xfivefold difference plus P-value that most patients with gastric carcinoma in the Western p0.01) between cancers and normal stomach. The most world develop symptoms and are diagnosed when abundant transcripts included ribosomal and mitochon- disease is locally advanced, often with metastases drial proteins, of which most were upregulated in the (Roukos, 2000). Curative surgery is then no longer tumours, as were other widely expressed genes including possible, and effective adjuvant therapies are not yet transcription factors, signalling molecules (serine/threo- well-established. Much better survival figures of over nine protein kinases), thymosin beta 10 and collagenase I. 80% can be achieved, however, when the tumour is Transcripts abundant in normal stomach were functionally identified and treated at an earlier clinical stage important, including gastrin, immunoglobulin alpha, (Roukos, 2000). lysozyme, MUC5, pS2 and pepsinogens, which were Over 90% of cancers in the stomach are carcinomas among 55 gastric-specific genes. Many transcripts were (specifically, adenocarcinomas), but these are hetero- minimally characterized or new, some cancer-associated geneous (Fuchs and Mayer, 1995; Blok et al., 1997). genes reflected their intestinal morphology, and some Many classifications exist: the two most common normal gastric genes had previously been considered as subdivide tumours by anatomical location (Owen, pancreatic carcinoma markers. The gastric carcinoma 1986), into distal and proximal, and by histopathology, profiles resembled other tumours’, supporting the ex- into intestinal and diffuse cancers (Laure´ n, 1965). These istence of common cancer-associated targets. These data tumour subtypes differ in their epidemiology and provide a catalogue from which to develop markers for aetiology, as well as in their molecular biology. Most better diagnosis and therapy of gastric carcinoma. of the classic cancer-associated molecular alterations Oncogene (2003) 22, 4287–4300. doi:10.1038/sj.onc.1206615 have been found in gastric carcinomas (Hanahan and Weinberg, 2000), and some, such as loss of functional Keywords: stomach; stomach neoplasms; adenocarcino- p53 and telomerase activation, are common to all ma; gene expression profiling; serial analysis of gene tumour subtypes (Blok et al., 1997; Chan et al., 1999). ONCOGENOMICS expression (SAGE) Downregulation of cell adhesion molecules, such as cadherins, however, is associated mainly with diffuse cancers; E-cadherin mutations were recently the first germline changes to be identified as predisposing to (diffuse) gastric carcinoma (Guilford et al., 1998). Conversely, such abnormalities are uncommon in tumours of intestinal type, which in molecular terms *Correspondence: KA Oien, Cancer Research UK Department of resemble colonic carcinomas so frequently containing Medical Oncology, Cancer Research UK Beatson Laboratories, adenomatous polyposis coli (APC) mutations (Chan University of Glasgow, Garscube Estate, Switchback Road, Glasgow G61 1BD, UK; E-mail: [email protected] et al., 1999). Received 13 August 2002; revised 21 March 2003; accepted 24 March Such classical molecular studies of gastric carcinoma 2003 have been extremely fruitful, but have of necessity Gene expression in gastric carcinoma and normal stomach KA Oien et al 4288 focused on small numbers of genes. Changes in the propriate tags (resulting from duplicate ditags or linker global pattern of gene transcription may be equally sequences), we obtained a total of 29 480 tags, or significant, and technological advances now permit their transcripts, derived from 10 866 different genes (unique investigation (Carulli et al., 1998). Gastric carcinoma tags). The libraries from tumour 1, tumour 2 and has been studied using differential display, Clontech normal stomach contained 10 222, 10 825 and 8433 tags, cDNA membrane arrays and cDNA microarrays (Ebert respectively (Table 1). To permit direct comparison, et al., 2000; Jung et al., 2000; Yoshikawa et al., 2000; El- each library was normalized to a total of 10 000 tags (at Rifai et al., 2001; Wang et al., 2001; Hippo et al., 2002; which level, a tag present 10 times has an abundance of Mori et al., 2002). However, we chose to use serial 0.1%). The normalized, rather than absolute, tag counts analysis of gene expression (SAGE), which produces are used henceforth. comprehensive, quantitative and reproducible gene expression profiles (Velculescu et al., 2000). Global analysis of gastric SAGE libraries SAGE is based on generating clones of concatenated (linked) short sequence tags derived from mRNA from Table 1 presents a global analysis of the three SAGE the target tissue (Velculescu et al., 1995; Zhang et al., libraries. The most common tag was CTCCCCCAA, 1997). Each tag is 9 or 10 bp long and represents a single which was found 781 times (7.8%) in normal stomach mRNA transcript; each clone insert contains up to 40 and represents immunoglobulin alpha (IgA). Only tags joined together. Sequencing of multiple concate- around seven genes were expressed at levels of 1% or nates therefore efficiently describes the pattern and over in each library. In contrast, the vast majority (97– numbers of genes expressed. The mRNA transcript 98%) of transcripts, including the classical house- corresponding to the short SAGE tag is identified via keeping genes, were present at levels in single figures genetic databases. As SAGE is labour-intensive and (below 0.1%). The global figures for each library were hence limited to small numbers of specimens, resulting similar. candidate genes are usually validated in a larger sample set. SAGE has already been applied to the other Global comparison of gastric carcinoma and normal common adenocarcinomas, of colon, pancreas, breast, SAGE libraries ovary and prostate, and to squamous lung carcinoma and other cancers (Velculescu et al., 1995; Zhang et al., Differential expression between libraries was defined as 1997; Hibi et al., 1998; Lal et al., 1999; Nacht et al., a difference in tag ratio of fivefold or more, combined 1999; Velculescu et al., 1999; Hough et al., 2000; with a P-value of 0.01 or less. When tags from the two Waghray et al., 2001) but, to our knowledge, no SAGE tumours were pooled and compared with normal data for gastric carcinoma have yet been published in stomach, 106 tags were differentially expressed (0.97% the literature. of the total genes in the three libraries), of which 47 were Since the most common subtype of gastric carcinoma higher in the tumours and 59 were higher in normal is distal and intestinal (Fuchs and Mayer, 1995), we have stomach. These genes are listed in Table 2. When generated SAGE profiles of two such tumours, along compared individually with normal stomach, 76 tags with one sample of normal gastric antral (distal) (1.09%) and 125 tags (1.53%) were differentially mucosa. From the resulting catalogues of gene expres- expressed in tumours 1 and 2, respectively, of which sion, we have highlighted abundant, differentially 85 tags were upregulated in either tumour. The two expressed and gastric-specific transcripts and have tumours themselves differed by 64 tags (0.75%). These validated selected genes in a wider panel of normal results are in keeping with their histopathology: tumour and tumour gastrointestinal tissues. 1 was better differentiated and of an earlier stage. Global comparison of gastric libraries digitally with Results SAGE libraries from other glandular epithelial tissues and mesothelium Generation of gastric SAGE tag libraries The normal gastric SAGE library was compared SAGE libraries were created from two gastric adeno- digitally with normal breast, colonic, ovarian, pancrea- carcinomas, both of distal, intestinal subtype, and from tic and prostatic tissue and with normal mesothelium. In normal gastric antral mucosa. After excluding