Oncogene (2007) 26, 7432–7444 & 2007 Nature Publishing Group All rights reserved 0950-9232/07 $30.00 www.nature.com/onc ORIGINAL ARTICLE Identification of candidate genes involved in neuroblastoma progression by combining genomic and expression microarrays with survival data M Łastowska1, V Viprey2, M Santibanez-Koref1, I Wappler1, H Peters1, C Cullinane3, P Roberts4, AG Hall5, DA Tweddle5, ADJ Pearson6, I Lewis7, SA Burchill2 and MS Jackson1 1Institute of Human Genetics, University of Newcastle upon Tyne, Newcastle upon Tyne, UK; 2Children’s Cancer Research Laboratory, Cancer Research UK Clinical Centre, St. James’s University Hospital, Leeds, UK; 3Department of Pathology, St. James’s University Hospital, Leeds, UK; 4Department of Cytogenetics, St. James’s University Hospital, Leeds, UK; 5Northern Institute for Cancer Research, University of Newcastle upon Tyne, Newcastle upon Tyne, UK; 6Institute of Cancer Research, Sutton, Surrey, UK and 7Department of Paediatric Oncology and Haematology, St. James’s University Hospital, Leeds, UK Identifying genes, whose expression is consistently altered may be useful in the identification of critical genes within by chromosomal gains or losses, is an important step in regions of loss or gain in many human cancers. defining genes of biological relevance in a wide variety of Oncogene (2007) 26, 7432–7444; doi:10.1038/sj.onc.1210552; tumour types. However, additional criteria are needed to published online 28 May 2007 discriminate further among the large number of candidate genes identified. This is particularly true for neuroblasto- Keywords: neuroblastoma; expression arrays; SNP ma, where multiple genomic copy number changes of arrays; candidate genes proven prognostic value exist. We have used Affymetrix microarrays and a combination of fluorescent in situ hybridization and single nucleotide polymorphism (SNP) microarrays to establish expression profiles and delineate copy number alterations in 30 primary neuroblastomas. Introduction Correlation of microarray data with patient survival and analysis of expression within rodent neuroblastoma cell The presence of recurrent chromosomal copy number lines were then used to define further genes likely to be alterations in many solid tumours which correlate with involved in the disease process. Using this approach, we disease outcome suggests that changes in the expression identify >1000 genes within eight recurrent genomic of specific genes within these regions are critical to the alterations (loss of 1p, 3p, 4p, 10q and 11q, 2p gain, 17q disease process (Popescu and Zimonjic, 1997). Recently, gain, and the MYCN amplicon) whose expression is microarray gene expression profiling has allowed the consistently altered by copy number change. Of these, 84 relationship between copy number and gene expression correlate with patient survival, with the minimal regions of in such regions to be analysed in detail in a variety of 17q gain and 4p loss being enriched significantly for such tumours including prostate, glioblastoma, multiple genes. These include genes involved in RNA and DNA myeloma and colon carcinoma (Wolf et al., 2004; Nigro metabolism, and apoptosis. Orthologues of all but one of et al., 2005; Tsafrir et al., 2006; Walker et al., 2006). All these genes on 17q are overexpressed in rodent neuro- these analyses indicate that expression of a significant blastoma cell lines. A significant excess of SNPs whose fraction of genes within gained or deleted regions are copy number correlates with survival is also observed on altered in a manner consistent with the underlying proximal 4p in stage 4 tumours, and we find that deletion genomic alteration, with increased expression in regions of 4p is associated with improved outcome in an extended of gain and decreased expression in regions of loss. cohort of tumours. These results define the major impact Although gene ontology and expression profiles within of genomic copy number alterations upon transcription tissues of interest can be used to further define candidate within neuroblastoma, and highlight genes on distal 17q genes, the extensive copy number dependence of gene and proximal 4p for downstream analyses. They also expression suggests that additional information will be suggest that integration of discriminators, such as survival required to identify those genes which are likely to be and comparative gene expression, with microarray data critical for tumour biology (Bussey et al., 2006; Walker et al., 2006). The potential value of additional criteria to assess candidate genes in regions of loss and gain is particu- Correspondence: Dr MS Jackson, Institute of Human Genetics, larly clear in neuroblastoma, the most common extra- International Centre for Life, Central Parkway, Newcastle upon Tyne cranial childhood solid tumour, where multiple genomic NE1 3BZ, UK. E-mail: [email protected] alterations of proven prognostic value have been Received 14 December 2006; revised 22 March 2007; accepted 19 April identified. These alterations, which include MYCN 2007; published online 28 May 2007 amplification (Seeger et al., 1985), 1p deletion (del) Genomic and expression microarray analysis in neuroblastoma M Łastowska et al 7433 (Caron et al., 1996a), 11q del (Attiyeh et al., 2005) and disease. To correlate gene expression and DNA copy 17q gain (Bown et al., 1999), help to define three non- number the status of MYCN, 1p and 17q was overlapping clinicogenetic subtypes of neuroblastoma: established in all tumours using fluorescence in situ The first is characterized by the presence of numerical hybridization (FISH). The breakpoint positions of chromosomal abnormalities and low stage of disease; unbalanced gains and losses were delineated further at the second by the presence of 17q gain, 11q and 3p del; high resolution using Affymetrix 50 K HindIII SNP the third by the presence of 17q gain, 1p del and MYCN arrays and validated through comparison with data amplification (Lastowska et al., 2001; Chen et al., 2004; from constitutional DNA samples and 50K XbaI arrays Vandesompele et al., 2005). Among these alterations, (see Materials and methods). Since low-stage tumours 17q gain is the single most important indicator of poor are characterized by numerical changes in whole prognosis (Lastowska et al., 2001; Vandesompele et al., chromosomes (Lastowska et al., 2001; Chen et al., 2005). The relevance of genes within these regions of 2004; Vandesompele et al., 2005), this analysis was recurring copy number alteration is supported further confined to stage 4 tumours where DNA was available by the fact that many chromosomal gains and losses (n ¼ 19). The SNP results were consistent with the FISH observed in a transgenic mouse model of neuroblastoma data in all cases, except that the SNP analysis identified are syntenic to regions altered in the human disease an additional 17q gain in one tumour (NB29), where the (Hackett et al., 2003). Recently, two analyses have region gained (64.41Mb-qter) lies distal to the probe integrated genomic copy number estimates obtained used in FISH analyses (MPO, 53.7Mb). Results showing from BAC arrays or PCRwith microarray expression breakpoint delineation on chromosome 1 in three analyses to specifically investigate 1p del (Janoueix- tumours, and confirmation of 2p gain using direct Lerosey et al., 2004), and 1p del, 11q del and 17q gain comparison between tumour and constitutional DNA, (Wang et al., 2006). These investigations generated lists are shown in Supplementary Figure S1. In total, the of genes whose expression is significantly altered by SNP analysis delineated 132 gains and losses, 71 of copy number changes, and established that the expres- which (B54%) were accounted for by eight recurring sion of between 15 and 61% of all genes in these regions alterations each of which was present in at least 25% (5) are copy number dependent, highlighting further the of the tumours analysed: MYCN amplification, gains of need to use additional criteria for candidate gene 17q and 2p, and losses of 1p, 3p, 4p, 10q and 11q appraisal. (Table 1). We therefore restricted our analysis of the Here, we present the results of an integrated single relationship between gene expression and copy number nucleotide polymorphism (SNP) and expression micro- to these eight genomic alterations. array analysis of 30 primary neuroblastomas, which define deregulated genes within eight recurrent chromo- Identification of genes coamplified and overexpressed somal copy number alterations. Collectively, the genes with MYCN identified represent over 3.5% of all genes analysed. Expression and DNA copy number data for distal 2p in Since it has been shown previously that gains of the 10 MYCN amplified tumours are shown in Figure 1. chromosomal regions syntenic to human 17q occur in Diploid copy number estimates (black) for the MYCN rodent neuroblastoma (Lastowska et al., 2004), we have gene range from 30 to 200 (position B16 Mb), with the used gene expression data from mouse and rat amplicon extending proximally for 6–8 Mb in three neuroblastoma cell lines to further pinpoint candidates tumours (NB6, 25 and 19), and additional peaks of from this genomic region. Furthermore, since many of amplification clearly visible in NB26 (B2 Mb) and the genomic alterations analysed have been shown to be NB10 (28–31 Mb). Excluding MYCN and its antisense of prognostic importance, we have also used the extent NCYM, a total of 59 genes have a DNA copy number to which gene expression and SNP copy number estimate of >8 in one or more tumour samples (Table 2) estimates correlate with patient survival as an additional and of these, 43 (73%) show a >4-fold increase in gene criterion to prioritize differentially expressed genes in expression in one or more tumours where there is an these regions. This greatly reduced the number of increase in DNA copy number. Of these, 30 genes show candidate genes for downstream analysis from over a significant (P 0.05) and positive (r2>0.7) correlation 1000 to 84. Genes in two regions stand out in these o between gene expression and copy number across all analyses; distal 17q gain, which is of known prognostic samples.