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 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 expression profiling has allowed the consistently altered by copy number change. Of these, 84 relationship between copy number and 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 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., (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 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. However, the correlation between copy num- importance, and distal 4p which, although deleted in ber and expression is weak for MYCN (r2 ¼ 0.1668), and B20% of neuroblastomas, has not been associated for both DDX1 and NAG, the genes most commonly previously with survival. co-amplified with MYCN (r2 ¼ 0.0791 and 0.01095).

Results Identification of genes differentially expressed in regions of genomic gain and loss Generation of gene expression and DNA copy To identify genes differentially expressed as a result of number data low copy number gains and losses, the dataset was We have used the Affymetrix U133 Plus 2 chip to assess systematically dichotomized with respect to each of the gene expression in neuroblastomas from 30 patients, 10 eight genomic alteration present in at least 25% of the with stages 1, 2, 3 or 4S disease, and 20 with stage 4 tumours analysed, and genes were identified where

Oncogene Genomic and expression microarray analysis in neuroblastoma M Łastowska et al 7434 Table 1 Genomic regions altered in five or more samples 1pÀ 2p+ MYCN 3pÀ 4pÀ 10qÀ 11qÀ 17q+

NB2 pter-56.54 pter-55.49 pter-27.61 30.69-qter 71.43-qter 37.93-qter NB3 pter-51.67 AMP 15.83-qter 32.20-qter NB5 pter-59.65 pter-68.26 64.95-qter 73.56-qter 36.4-qter NB6 pter-43.37 AMP 35.1-qter NB7 pter-28.99 pter-45.02 pter-27.89 43.73-qter NB9 80.46-qter 38.17-qter NB10 pter-115.44 AMP 76.57-qter 40.52-qter NB13 80.08-qter NB18 pter-23.39 AMP pter-40.40 34.2 -qter NB19 pter-87.04 AMP 39.2-qter NB20 pter-52.53 pter-49.77 AMP pter-qter 46.85-qter NB24 pter-68.28 pter-41.1 62.97-qter 43.81-qter NB25 pter-66.9 AMP 55.27-qter NB26 pter-26.23 pter-49.19 AMP 43.83-qter 27.98-qter NB27 pter-116.66 AMP 39.96-qter NB28 pter-79.14 pter-60.47 69.83-qter 32.63-qter NB29 pter-103.28 AMP pter-38.70 64.4-qter NB31 pter-10.22 pter-49.54 pter-5.65 73.74-qter 41.72-qter NB34 pter-188.28 33.66-qter

The extent of unbalanced copy number changes (in megabases), as defined by the closest unaffected SNP, is given to the nearest 10 kb.

expression levels differed according to the presence or absence of each alteration (Wilcoxon test, FDR ¼ 0.05). A list of all genes identified in each of these analyses is provided as Supplementary information (Supplemen- tary Tables S2 and S3). Two of the eight genomic alterations involved gain of chromosomal material; distal 17q and proximal 2p. Because gain of an entire is associated with good prognosis, but unbalanced partial gain of the long arm is associated with poor prognosis (Lastowska et al., 2001), gene expression across this entire chromo- some was analysed. Out of 1656 gene probes, 239 (14%) showed statistically significant differential expression (Figure 2a). Tumours with 17q gain have marked underexpression of differentially expressed genes on 17p and proximal 17q, which is not gained, but marked overexpression of genes within distal 17q, which is gained (Figure 2a). The commonly gained region contains 40 differentially expressed genes (49 probes), all but two of which are upregulated. The difference in mean expression levels between 17q gained and not gained tumours is plotted in Figure 2b, and illustrates that the vast majority of genes on distal 17q show an increase in log2 ratio of between 0.2 and 0.9 (mean ¼ 0.471), consistent with a dosage effect (a log2 ratio of 0.59 represents a fold increase of B1.5). Interestingly, genes on 17p and proximal 17q are underexpressed in tumours with 17q gain relative to Figure 1 Copy number and gene expression within the MYCN 17q normal tumours (mean ¼À0.434). Gain of 2p was amplicon. Genomic copy number estimates derived from flanking observed in seven tumours, with the commonly gained SNPs for each gene are shown in black, fold change in gene region encompassing the distal 45.02 Mb (Table 1). Of expression levels relative to the median value for each gene are shown in red. The most distal 280 genes on 2p are shown in linear the 886 genes on 2p, only 17 were found to be order, left to right (lower scale). The physical scale, in megabases, is significantly altered by the Wilcoxon test. All map therefore only an approximation (upper scale). One probe per gene between 3.0 and 39.1 Mb on 2p and were upregulated in is shown, except for MYCN which is represented by three probes. 2p gained tumours (Supplementary Table S2). Only one gene shows significant variation in expression which is independent of copy number (SLC30A3, visible at B27 Mb in Expression data from the analyses of all five NB25 and NB27). For details see Materials and methods and commonly observed regions of loss are presented in Table 2. SNP, single nucleotide polymorphism. Figure 3. Owing to the huge variation in breakpoint

Oncogene Genomic and expression microarray analysis in neuroblastoma M Łastowska et al 7435 Table 2 Genes co-amplified within MYCN Affymetrix ID Gene name/ID n CN>8 n EXP>4X Mean CN Mean EXP XP Correlation P-value

241535_at LOC388920 1 1 50 64 0.9942 o0.0001 220487_at SNTG2 1 1 99 86 0.9995 o0.0001 210342_s_at TPO 1 1 99 21.7 0.9965 o0.0001 216941_s_at TAF1B 2 2 11.3 13.8 0.7173 0.0195 239046_at Anon 2 2 11.3 7.7 0.5421 0.1055 235878_at FLJ42198 2 2 11.3 10.8 0.7689 0.0093 230948_at yj20d11.s1mRNA 2 2 11.3 8.8 0.6960 0.0254 222830_at LBP-32 3 2 13.5 7 0.8563 0.0016 1553137_s_at TIEG2 3 1 13.5 4.16 0.7963 0.0058 209773_s_at RRM2 3 3 18 7.8 0.8278 0.0031 242556_at Anon 3 0 19 — 0.7540 0.0118 212552_at HPCAL1 3 3 19 7.75 0.9765 o0.0001 200790_at ODC1 3 3 19 6.2 0.8856 0.0007 244728_at Anon 3 0 18 — 0.8472 0.0020 1565577_s_at FLJ34486 2 2 19 10.6 0.9910 o0.0001 222684_s_at FLJ14075 2 2 19 11.6 0.9740 o0.0001 208638_at ATP6V1C2 2 2 19 5.2 0.8986 0.0004 208639_x_at P5 2 2 19 6.8 0.8720 0.0010 216640_s_at FLJ23273 2 2 19 9.1 0.9068 0.0003 210263_at KCNF1 2 0 19 — 0.2528 0.4811 225579_at MGC33602 2 0 34 — À0.1702 0.6382 211504_x_at ROCK2 2 2 34 10.3 0.9187 0.0002 205862_at GREB1 3 3 31 12.5 0.7809 0.0077 206899_at NTSR2 3 1 31 374* 0.9515 o0.0001 234250_at FLJ20410 3 1 44 8.5 0.8528 0.0017 212274_at LPIN1 2 2 19 19.2 0.8888 0.0006 202479_s_at TRB2 3 2 15 15 0.5062 0.1355 1566716_at DKFZp566F0224 3 0 26.5 — 0.5915 0.0717 234331_s_at NSE1 3 2 26.5 21.7 À0.1630 0.6528 231439_at ZD76G03 cDNA 3 2 26.5 6.45 0.7525 0.0120 229546_at LOC400944 9 3 33.1 97* 0.7604 0.0107 240579_at NAG 9 4 33.1 45.8 0.1095 0.7633 207478_at Anon 9 0 38 — 0.1966 0.5861 207477_at Anon 9 1 38 6.1 0.4958 0.1450 201241_at DDX1 9 5 41 6.6 0.0791 0.8280 207028_at NCYM 10 9 35 38.9 0.3067 0.3886 211377_x_at MYCN 10 9 35 33.7 0.1668 0.6452 230276_at DKFZP566A1524 7 0 17.4 — 0.5437 0.1043 226324_s_at SLB 1 0 13 — 0.1363 0.7074 218843_at FRCP1 1 0 17 — 0.2611 0.4662 225050_at ZNF512 1 1 17 8.5 0.0229 0.9533 220321_s_at FLJ13646 1 1 18 20.8 0.0206 0.9581 209313_at XAB1 1 1 18 20 0.0353 0.9280 201838_s_at STAF65 1 1 18 20.6 0.0010 0.9800 218682_s_at SLC4A1AP 1 1 18 22.9 0.0261 0.9470 203781_at MRPL33 1 1 18 10.1 À0.1578 0.6633 57540_at RBSK 1 1 17 14.8 À0.0896 0.8056 1562111_at MAGE:5285600 1 1 13 6.9 À0.0332 0.9274 225262_at FOSL2 1 0 43 — 0.0370 0.9192 235703_at PLB 1 1 43 7.3 0.6513 0.0574 1553310_at Anon 1 1 46 34.8 0.6647 0.0508 231036_at Anon 1 1 46 83.8 0.6569 0.0546 228222_at PPP1CB 1 0 16 — 0.2694 0.4834 221229_s_at FLJ20628 1 0 16 — 0.1656 0.6475 214662_at KIAA0007 1 0 16 — 0.7171 0.0196 238186_at Anon 1 0 16 — 0.4237 0.2224 242710_at Anon 1 1 42 4.8 0.9214 0.0002 226425_at FLJ21069 1 0 42 — 0.8820 0.0007 242964_at Anon 1 1 42 20.9 0.9732 o0.0001 208211_s_at ALK 1 1 42 24.9 0.9891 o0.0001 222408_s_at CGI-127 1 0 19 — 0.4318 0.2128

Columns 3–6 show the number of tumours in which DNA amplification is observed (n CN>8), the number of tumours in which gene expression is increased more than fourfold (n EXP>4X), the mean copy number estimate within amplified tumours (mean CN), and mean fold change in expression within amplified tumours (mean EXP). Correlations between expression and copy number across all samples, together with associated P-values are given. Fold change estimates marked with an asterisk indicate that gene expression levels were too low to be accurately measured in some samples. In column 2, known genes are shown in bold. In columns 7 and 8 correlations which are significant (Po0.05) are shown in bold. A copy number of >8 was chosen to define MYCN amplified tumours as this is comparable to existing measures of MYCN amplification (Ambros and Ambros, 2001) and provided total discrimination between tumours with 2p gain and MYCN amplification for SNPs within the MYCN gene.

Oncogene Genomic and expression microarray analysis in neuroblastoma M Łastowska et al 7436 a 17q not gained 17q gained b Change in mean Log2R -2 -1 0 +1 +2 0 Mb

10 Mb p 10 Mb

20 Mb cen

30 Mb

40 Mb q

50 Mb

60 Mb 64.4 Mb

70 Mb

Figure 2 Differential expression of genes on chromosome 17 with respect to 17q status. Gene expression was analysed in 28 tumours, dichotomized with respect to 17q gain (two tumours with mixed 17 and 17q gain were excluded). (a) Heat map of genes differentially expressed between 17q gained and not gained groups, shown in linear order with respect to chromosomal position (17pter to 17qter). Red indicates overexpression relative to median value and blue indicates underexpression. (b) Difference in mean expression level of each gene observed in the 17q gained group relative to the not gained group, shown with respect to physical position on the chromosome. For ease of presentation, only genes with differences in log2 ratio between À2 and þ 2 are shown. Four genes with more extreme variation are excluded as a result: ATP2A3 (À3.31464), 232887 at (À2.88767) and MGC87631 (4.732143) on 17p and ANKFN1 (À2.56757) on 17q.

position on 1p, the comparison of expression level of 10q deleted tumours (Figure 3d); and on 11q, 291 probes genes in tumours with and without 1p del was (27.7%) were altered, with 274 probes (94%) being performed in a stepwise manner, partitioning the data downregulated in 11q deleted tumours (Figure 3e). To in genomic intervals according to breakpoint position. illustrate the uniform nature of the expression changes Using this approach, 520 probes from chromosome arm associated with chromosomal loss, the difference in 1p (31%) showed significant changes in expression, with mean expression levels between tumours with and 516 of these (99%) showing downregulation in 1p without 11q loss is plotted in Figure 3f. deleted tumours. In some tumours, the expression Because the changes in gene expression are often differences were consistent enough to allow breakpoint relatively small, particularly in regions of gain, it was positions to be inferred directly (for example, NB6, 7 desirable to confirm the gene expression results in a and 26, Figure 3a). The data from chromosomes 3, 4, 10 number of genes of interest using an independent and 11 all show similar patterns, with chromosomal loss method. We therefore reanalysed the expression of four consistently associated with reduced gene expression. genes which showed significant copy number dependent On 3p, 176 probes (19%) were significantly altered, with expression (PMP22, WSB1, BRCA1 and BIRC5) using 100% being downregulated in 3p deleted tumours real-time PCR. These genes were chosen as they map to (Figure 3b). On this chromosome, the small terminal distinct regions of chromosome 17 (17p12, 17q11, 17q21 deletion in tumour 31 defines a 5.6 Mb region of and 17q25, respectively), and because they have been common loss which contains six downregulated genes: implicated previously in neuroblastoma or other cancers CHL1, CNTN4, CRBN, LRRN1, SETMAR and (Chen et al., 2006; Deng, 2006; Miller et al., 2006). ARL8B. On 4p, 81 probes (22%) were significantly Significant correlations were observed between Affyme- altered, all but two being downregulated in 4p deleted trix and real-time PCRdata for all four genes, r2 varying tumours (Figure 3c); on 10q, 215 probes (25%) showed from 0.683 for BRCA1 to 0.961 for PMP22 (Supple- significant expression differences, all downregulated in mentary Figure 2S).

Oncogene hnei log in change ifrnilyepesdgnso 1 nte1qdltdgoprltv otentdltdgop hw ihrsett hsclposition physical log to in respect with change to shown mean group, respect deleted the with not gene, the order to linear each relative in group For deleted shown chromosome. 11q are the the genes in ( on 11q the underexpression.: on indicates case, genes blue each expressed and differentially In value indicated. median to loss relative of ( overexpression region indicates the Red position. to chromosomal respect with dichotomized tumours 3 Figure b h ;pe-e,( pter-cen, 3; Chr ) ifrnilepeso fgnsi ein fcrmsmlls.Ha aso ee ifrnilyepesdbetween expressed differentially genes of maps Heat loss. chromosomal of regions in genes of expression Differential 2 d b a ai cosalgnsi eee einis region deleted in genes all across ratio f -2 -1 2 0 1 10.2 bM 55

c 58.9Mb 10q 3p 5.6Mb h ;pe-e ( pter-cen 4; Chr ) 1p 57 59 0 1 125Mb 115 105 95 85 75 65 d h 0 e-tr ( cen-qter, 10; Chr ) À .12 a 0.0364. var 0.5112, 2 ai ewe 1 eee n 1 omltmusi hw.Mean shown. is tumours normal 11q and deleted 11q between ratio e h 1 e-trad( and cen-qter 11; Chr ) c e eoi n xrsinmcora nlssi neuroblastoma in analysis M microarray expression and Genomic Ł 54.4Mb 11q 4p 27.6Mb astowska tal et f ifrnei enepeso ee of level expression mean in difference ) a h ;pter-cen, 1; Chr ) Oncogene 7437 Genomic and expression microarray analysis in neuroblastoma M Łastowska et al 7438 Correlation with survival identifies candidate genes on 4p and 17q Based on the hypothesis that the expression of genes in regions of loss or gain which are involved in tumour development or progression should correlate more strongly with disease outcome than others, we have analysed the relationship between copy number/expres- sion and patient survival to define further candidate genes. We first analysed the impact of 17q gain, MYCN amplification and 1p del (as detected by FISH) upon overall survival within our dataset using the log-rank test. All three cytogenetic alterations were significantly associated with poor survival, in line with prior expectation, P-values ranging from 0.013 to 0.001 (data not shown). We then ranked all SNPs with respect to their impact upon overall survival in stage 4 tumours (see Materials and methods). The fold over-representa- tion of high-ranking SNPs on each autosomal arm, compared to expectation, is shown in Figure 4a. Chromosome arms 1p and 2p show a greater than 10- fold excess of SNPs at one or more of the percentile ranges analysed, consistent with the survival analyses of FISH data. However, chromosome arm 4p, and to a lesser extent 22q, also shows significant over-representa- tion in this analysis. The physical position of SNPs on chromosomes 1, 2, 4, and 22, which rank within the top 5% are shown in Figure 4b. On , these are distributed throughout the proximal short arm, whereas on , they map precisely to MYCN (B16 Mb) or to ALK (B30 Mb, co-amplified with MYCN in NB10). On , all of the SNPs cluster within the distal 40 Mb of the short arm. The SNPs on chromosome 22 are distributed throughout the long arm of this chromosome. Because some of these copy number changes have not been associated previously with survival, we then used the log-rank test to analyse the impact of each alteration under study upon survival within stage 4 tumours. Only 4p gave a significant result (P ¼ 0.003). To extend this Figure 4 SNP copy number and survival in stage 4 tumours. (a) observation into a larger dataset, we combined results Fold over-representation of SNPs on each autosome arm, relative from the current series with data from all stage 4 to expectation within the top 5, 1 and 0.1% of SNPs ranked tumours of Lastowska et al. (2001) and analysed the according to impact upon overall survival. This analysis is uninformative for 17q gain as all but two stage 4 tumours possess impact of 4p loss on overall survival using the log-rank this alteration. (b) Physical distribution of SNPs ranked within top test. Although copy number in Lastowska et al. (2001) 5% from chromosomes 1, 2, 4 and 22. The position of the was analysed using cytogenetic methods as opposed to centromeres is shown by vertical grey bars. X axis, position in SNPs, the combined data identifies a significant impact megabase; Y axis, inverse of score test P-value for each SNP. (c) Kapan–Meier survival curves for 4p deleted and 4p not deleted of 4p loss on survival, with deletion being associated groups within stage 4 tumours (including data from Lastowska with better response to treatment (Figure 4c, P ¼ 0.013, et al., 2001). SNP, single nucleotide polymorphism 10 tumours with 4p del compared to 38 tumours without 4p del). As chromosome 22 was not altered at high frequency within our dataset, it was not studied further. genome using a Wilcoxon test. Only the minimal regions To directly assess the association of gene expression of chromosomes 2p gain, 4p loss and 17q gain showed within the genomic regions under study upon disease significant enrichment for genes whose expression outcome, we then ranked the expression of all genes correlates with overall survival (P ¼ 0.01, P ¼ 1 Â 10À7 with respect to impact upon overall survival, and and P ¼ 0.032, respectively). Over 45% of all differen- established if each minimal region of gain or loss tially expressed genes from the commonly altered contained more genes whose expression is associated regions on 4p and 17q had expression levels associated with survival than expected by chance. This was with overall survival (Po0.005). However, none of the achieved by comparing the rank of all genes present nine genes on 2p which was differentially expressed with within each region to the rank of all genes in the whole respect to 2p gain correlated with overall survival,

Oncogene Genomic and expression microarray analysis in neuroblastoma M Łastowska et al 7439 indicating that the enrichment on 2p is not due to the 17q. Although genes associated with survival are present genomic alteration under study. The frequency of on both arms of this chromosome (Supplementary Table differentially expressed genes, which correlated with S2), 18 out of 40 differentially expressed probes within overall survival in the other regions under study (1p, 3p, the 14.2 Mb minimal region of gain are associated with 10p and 11q) was also analysed and found to be low, survival (Po0.005, Figure 5c). Furthermore, since varying between 0 and 4%. The results for three mouse and rat neuroblastoma cell lines are known to genomic regions (1p, 4p and 17q) are shown in Figure 5. possess gains of chromosomal regions which are The results for chromosome arm 1p (Figure 5a) are syntenic to human 17q (mouse chromosome 11 and rat presented as an example of a genomic region, where few chromosome 10), it was also possible to assess gene of the differentially expressed genes correlate with expression in these regions relative to rodent neuronal overall survival. Only two of the 19 differentially controls (see Materials and methods). Strikingly, all but expressed genes on the chromosome arm which correlate one of the genes on distal 17q associated with survival with survival (Po0.005) lie within the minimal region of in humans was also expressed at high levels in rodent loss (PRKCZ and CAMTA1). In sharp contrast, 25 out cell lines (SLC25A19, Figure 5c), further supporting of 48 differentially expressed genes within the shortest their candidature for involvement in neuroblastoma region of 4p loss (27.6 Mb) are associated with survival progression. (Po0.005, Figure 5b), including six genes represented This analysis of expression with respect to survival is by more than one probe. A similar result is obtained for complicated by the fact that expression levels are not

Figure 5 Expression and survival in critical regions on 1p, 4p and 17q. The left panel in each case shows the position of breakpoints observed in tumours (black bars), the position of genes which correlate with survival (Po0.005, pink bars), and the minimally altered region on each arm (grey shading). The right panel in each case lists probes which correlate with survival (Po0.005) and their ranking in the analysis. For chromosomes 1 and 4 only genes associated with survival are shown due to space limitations. Genes ranked within the top 500 are highlighted further with darker shading. In addition, the expression of genes marked in violet correlates with survival when the data is stratified for each genomic alteration (see results). For genes on 17q with known orthologues, the fold change of expression in rodent neuroblastoma cell lines relative to appropriate rodent brain controls is also shown. Increase in expression (over twofold) is highlighted in green, an asterisk indicating an absent call in brain tissue (ND: not determined due to absence of orthologous probe on rodent array).

Oncogene Genomic and expression microarray analysis in neuroblastoma M Łastowska et al 7440 independent of the genomic alteration on each chromo- 2004; Wang et al., 2006), as B70% of downregulated some. To address this, we recalculated the correlation genes found in these investigations are also down- with overall survival of all significant genes stratified by regulated in the present study, and extend them due to genomic alteration (for example, for 17q–17q gained the higher resolution chip used in the current analysis and not gained, for 1p–1p loss and no loss). A total of 34 (Affymetrix U133 Plus 2). More importantly, our probes show a significant correlation in this analysis survival analysis has shortlisted 19 genes on this (Po0.05, highlighted in purple in Figure 5) including six chromosome arm whose expression level is associated represented by two or more probes (MACF1, WSHC1, with patient survival. Two, PRKCZ and CAMTA1, are HCAP-G, TSEN54, PTDSR and SFRS2). located within a commonly deleted region, and expres- sion of the latter has been linked recently to survival of patients with neuroblastoma in two independent studies Discussion (Henrich et al., 2006; Asgharzadeh et al., 2006). However, 10 of these genes are located proximal to We have used the combination of FISH, SNP copy 39 Mb, indicating that genes from the proximal region number analysis and microarray expression analysis to of 1p may have additional impact on survival. Among identify genes which map within eight recurrent genomic these, MACF1 is of particular interest because its open copy number alterations in neuroblastoma, and whose reading frame has been found to be disrupted by a expression is significantly changed by these alterations. translocation breakpoint in a neuroblastoma cell line The total number of genes identified represents over (Schleiermacher et al., 2005). 3.5% of all genes analysed, with >1000 genes repre- The combination of expression and SNP microarray sented by >1300 probes being significantly changed. data allows gene expression within MYCN amplicons to This highlights both the significant impact of copy be analysed at the resolution of a single gene for the first number alterations upon the transcriptional programme time. It confirms the coamplification and overexpression of this tumour, and the need for additional criteria to of previously defined amplified genes (Chen et al., 2004; further define candidate genes within such regions. As a Seltzer et al., 2005) and the poor correlation between first step to address this problem, we have used a MYCN expression and copy number (Nisen et al., 1988; combination of clinical and biological features (survival Slavc et al., 1990). While the extensive variation in and expression of orthologues in relevant rodent cell amplicon size and complexity we observe is consistent lines), which have enabled us to define a list of 84 genes with previous reports (Figure 1; Seltzer et al., 2005; in four genomic regions (1p, 2p, 4p and 17q) as being of Stallings et al., 2006), the present analysis defines for the particular interest. Analysis of gene ontology indicates first time over 30 genes as being co-amplified at both the that this list is significantly enriched (Po0.05) for genes DNA and expression level, several of which have been with biological functions of potential relevance to implicated in other neoplasias (Table 3). This complex- cancer, including RNA processing (CROP, DDX1, ity suggests that the relationship between gene expres- DHX15, DUS1L, EIF4A3, NOL14, SFRS1, SFRS2, sion/copy number within the MYCN amplicon and SLBP, TRSPAP1 and TSEN54), DNA metabolism disease outcome, which has produced conflicting results (BRCA1, BRIP1, EME1, KPNA2, RBPSUH, RRM2, using lower resolution methodologies (Weber et al., TK1 and WHSC1) and apoptosis (BIRC5, BRCA1, 2004; De Preter et al., 2005), may warrant renewed CROP, MAEA, NME1, PRKCZ, PTDSR and investigation. This is particularly relevant since in our SH3GLB1). Of these, more than half have been dataset the expression level of MYCN correlated poorly associated previously with other cancers, and eight have with survival, whereas expression of genes co-amplified already been defined as candidate genes for involvement with MYCN (GREB1, LPIN1, ODC1, RRM2 and in neuroblastoma (Table 3). Approximately 20% of TAF1B) showed stronger correlation. these genes have more than one Affymetrix probe The distal long arm of chromosome 17 is of particular associated with survival at po0.005, confirming the interest as gain of this region is strongly associated with consistency of the expression results. Furthermore, the poor prognosis (Bown et al., 1999). Our investigation analysis of expression pinpoints the commonly altered revealed 96 upregulated genes from this region, 22 being regions on 4p and 17q as being significantly enriched for represented by two or more probes. More importantly, genes whose expression correlates with survival, while we identified 17 genes as prime candidates as these the analysis of SNP data within stage 4 tumours are located within the common region of gain, are highlights 1p, 2p and 4p as regions where copy number associated with survival, and have orthologues which correlates with survival (17q is gained in all but two are also overexpressed in mouse and rat neuroblastoma stage 4 tumours, so this analysis is uninformative for cell lines relative to control neuronal tissue (Figure 5). this region). Several of these genes have been implicated previously in Proximal 1p is frequently deleted in progressing neuroblastoma or other cancers including BIRC5, neuroblastomas, but to date no tumour suppressor gene PTDSR, PYCR1 and TK1 (Table 3). in this region has been clearly implicated in disease Our analyses have also identified a high concentration progression. In our study, approximately 330 genes of both genes and SNPs on distal 4p whose expression/ from 1p were downregulated in 1p deleted tumours. copy number correlates with survival. Although dele- These results both confirm the results of two earlier tions of 4p, which occur in B20% of all neuroblastomas analyses of the 1p35–36 region (Janoueix-Lerosey et al., (Caron et al., 1996b), have not been associated

Oncogene Genomic and expression microarray analysis in neuroblastoma M Łastowska et al 7441 Table 3 Genes identified in this study previously implicated in cancer Affymertix ID Gene Symbol Involvement in cancer Referencesa

1p arm 202178_at PRKCZ Glioblastoma; critical for proliferation 1 1555370_a_at CAMTA1 Neuroblastoma; downregulation linked to poor survival 2 and 3 205277_at PRDM2 Pheochromocytoma, AML; downregulated 4 and 5 231110_at FABP3 Breast cancer; candidate tumour suppressor gene 6 208634_s_at MACF1 Neuroblastoma; disrupted by translocation 7 208924_at RNF11 Breast cancer; highly expressed 8 227123_at RAB3B Megakaryoblastic leukaemia, pancreatic carcinoma; upregulated 9 and 10 During differentiation 202741_at PRKACB Ovarian cancer; downregulated 11 209090_s_at SH3GLB1 HeLa cells; proapoptotic, loss may contribute to tumorigenesis 12 4p arm 209492_x_at ATP5I Hepatocellular carcinoma, ovarian cancer; upregulated 10 and 13 40225_at GAK Prostate cancer hormone refractory; upregulated 14 213980_s_at CTBP1 May act as an oncogene 15 206052_s_at SLBP Required for progression through the cell cycle 16 218308_at TACC3 Multiple myeloma; co-ordinately upregulated with WHSC1 17 and 18 Ovarian cancer; upregulated in chemoresistant tumours 222778_s_at WHSC1 Multiple myeloma; upregulation linked to poor prognosis 17 224416_s_at MED28 Various cancers; stimulates cellular proliferation 19 218662_s_at HCAP-G Pancreatic cancer; upregulation contributes to cells motility 20 235554_x_at MGC29898 Glioblastoma; amplified 21 223414_s_at LYARLeukaemia; upregulated, may regulate cell growth 22 201385_at DHX15 MPNSTs and Barrett’s carcinoma; copy number gains 23 and 24 225014_at LOC389203 Glioblastoma; amplified and overexpressed 21 207785_s_at RBPSUH Gastrointestinal carcinoid cells; activation of Notch signalling 25 203343_at UGDH Ovarian carcinoma; associated with CDDP resistance 26 230896_at CCDC4 Neuroblastoma; upregulation linked to poor survival 27 17q arm 212281_s_at MAC30 Meningioma and other cancers; upregulated 28, 29 204531_s_at BRCA1 Breast and ovarian cancers; mutated 30 211603_s_at ETV4 Ewing sarcoma, prostate cancer; fusion gene 31 and 32 208835_s_at CROP Cloned from cisplatin-resistant cell lines 33 201577_at NME1 Neuroblastoma; upregulation in advanced stages; poor prognosis 34 and 35 221703_at BRIP1 Interacts with BRCA1 36 211762_s_at KPNA2 Breast cancer, melanoma; poor prognostic marker 37 and 38 64.4 Mb to qtel 204868_at ICT1 Colon carcinoma; downregulated during the in vitro differentiation 39 217932_at MRPS7 Osteosarcoma; enriched mRNAs compared to normal osteoblasts 40 212723_at PTDSRGlioblastoma; poor prognostic marker 41 200753_x_at SFRS2 Increased expression during mouse fibroblasts transformation 42 1554408_a_at TK1 Proliferation marker in a number of tumours 43 and 44 202095_s_at BIRC5 Neuroblastoma; overexpression linked to poor prognosis 45 201303_at DDX48 Variety of cancers; autoantibodies to DDX48 46 203931_s_at MRPL12 Ovarian cancer; upregulated 11 200656_s_at P4HB HER-2/neu-positive breast cancer, melanoma; upregulated 47 and 48 226414_s_at ANAPC11 Osteosarcoma; enriched mRNAs compared to normal osteoblasts 40 202148_s_at PYCR1 Ovarian cancer; upregulated; antiapoptotic 10 and 49 MYCN coamplified 201241_at DDX1 Neuroblastoma, coamplified with MYCN 50 205862_at GREB1 Breast cancer; suppression blocks estrogen-induced growth 51 212274_at LPIN1 Neuroblastoma, coamplified with MYCN 52 200790_at ODC1 Neuroblastoma, coamplified with MYCN 53 209773_s_at RRM2 Pancreatic cancer; therapeutic target 54 216941_s_at TAF1B MSH-I colorectal carcinoma; mutated in 82% of tumours 55

Genes that are underlined were identified by two or more probes. aReferences are listed in Supplementary Material. previously with survival, the possible impact of dosage with a logarithm of the odds score which peaked in this of genes on 4p upon progression in neuroblastoma is region (Perri et al., 2002). The distal 2 Mb region of 4p supported by gain of the distal 2 Mb of 4p in a complex includes 11 genes whose overexpression is associated unbalanced translocation [t(1;4;17)] in the CLB-Bar with poor survival in this study, including four (ATP5I, neuroblastoma cell line. This line was obtained from a SLBP, TACC3 and WHSC1) where the correlation is relapsed tumour, and the translocation resulted in gain independent of 4p del status. of 4p and 17q and deletion of 1p arm (Schleiermacher We have defined a shortlist of candidate genes within et al., 2005). It is also noteworthy that an analysis of regions of known prognostic importance using gene rare inherited neuroblastomas identified linkage to 4p16 expression profiling, and employed both overall survival

Oncogene Genomic and expression microarray analysis in neuroblastoma M Łastowska et al 7442 and comparative gene expression as additional criteria was established using the NanoDrop ND-1000 spectrophoto- to define genes of particular interest. Given the com- meter (NanoDrop, Rockland, DE, USA). plexities which underpin tumorigenesis, patient survival and species-specific expression differences, it is clear Expression microarrays that no single criterion will be ideal and that additional Expression within human tumours was assessed using the discriminators could be used to further enhance the U133 Plus 2.0 Array (Affymetrix UK Ltd., power of such analyses. Recently, for example, Walker High Wycombe, UK). Gene expression analysis of mouse and et al. (2006) used a combination of SNP and expression rat neuroblastoma cell lines, which harbour gain of regions microarrays to identify 3041 genes whose expression was syntenic to human 17q, was performed using GeneChip Mouse altered in loss and gained regions in multiple myeloma, Expression Set 430 and GeneChip Rat Genome 230 2.0 Array. Cell lines were purchased from ATCC (USA), DSMZ and used expression change during the transition from (Germany) and the Interlab Cell Line Collection (Italy), and normal to myeloma plasma cells as a further criterion cells were cultured in RPMI medium with 10% foetal calf for candidate gene selection, reducing the number of serum. RNA from whole brain preparations of mouse and rat candidate genes to just 47. were used as reference samples for tissues of neuronal origin. The combination of methodologies employed here has Preparation of in vitro transcription products, hybridization identified regions of known prognostic importance in and scanning using the GeneChip scanner 3000 were neuroblastoma (1p, 2p and 17q) and identified specific performed according to Affymetrix protocols using a mini- candidate genes within them for further analysis. Since mum of 1 mg of total RNA to prepare antisense biotinylated these candidate genes were identified in metastatic RNA without a second round of amplification. The quality of tumours from patients with a poor prognosis, it is hybridization was assessed in all samples following the manufacturer’s recommendations. likely that they play a role in tumour progression or Data were analysed with Affymetrix GCOS 1.1.1 software response to treatment. Our analysis has also uncovered using global scaling to a target signal of 500. Data were then a novel region, distal 4p, with a high density of genes imported into GeneSpring GX (Agilent Technologies) for whose expression correlates with both DNA copy subsequent analysis. Expression data for each probe was number and survival. This is particularly striking, as normalized with a ‘per chip normalization’ to the 50th these correlations are observed within metastatic percentile of all values on the chip, and a ‘per gene normal- tumours (stage 4). Furthermore, over 50% of candidate ization’ to the median expression level of the gene across all genes identified by this combined approach within the samples. Probes which gave a present call signal in less than minimal regions of recurrent alteration on 17q and 4p 10% of samples were excluded from analyses. Rodent gene have been implicated previously in cancer. These results expression data were processed in the same way as the human data with the exception that per gene normalization to the strongly suggest that, despite the complexities, the median was replaced by normalization to the reference sample integration of clinical or biological data with gene (brain) and gene expression levels were presented as a fold expression and high resolution estimates of DNA copy change. number may be a powerful tool with which to pinpoint To identify genes that were differentially expressed between candidate genes involved in tumorigenesis in a wide two groups of tumours, with and without specific chromoso- variety of cancers. mal alterations, a Wilcoxon test was applied with a Benjamini and Hochberg false discovery rate (FDR) of 0.05. All genes were also ranked for their association between gene expression and overall survival using a Cox proportional hazard model Materials and methods with a score test (Rao, 1973). To establish if minimal regions of gain or loss contained more genes whose expression is Patient samples and tissue processing associated with survival than expected by chance, the rank of n n Patients attended the Leeds ( ¼ 22) and Newcastle ( ¼ 8) all genes present within each minimal regions of gain or loss NHS Trusts between January 1995 and September 2003, with was compared to the rank of all genes in the whole genome patient age ranging from 1 month to 10 years. Diagnosis and using a Wilcoxon test. Gene ontologies were analysed using the staging were according to the International Neuroblastoma GO Ontology Browser within the Genespring GX analysis et al Staging System (Brodeur ., 1993). Therapy was adminis- platform (Agilent Technologies). tered according to protocols of the United Kingdom Chil- dren’s Cancer Study Group (UKCCSG, now named Children’s Cancer and Leukaemia Group, CCLG), European SNP Microarrays Neuroblastoma Study Group and Localised Neuroblastoma SNP analyses were performed on 19 stage 4 tumours where European Study Group, with similar treatment being adminis- DNA was available, and five constitutional DNA patient tered in both contributing centres. Informed consent was samples obtained from peripheral blood, using Affymetrix obtained to use tumour material for research at both centres. GeneChip Mapping 50 K arrays (Affymetrix UK Ltd.) Primary tumours were snap frozen and stored at À801C. according to the manufacturer’s recommendations. All sam- Cryosections (10 m) were stained with haemotoxylin and eosin ples were analysed using the HindIII 50K array with one and examined by a pathologist to identify tumours with exception, NB20, which was analysed using the XbaI 50K >90% of neuroblastoma cells for subsequent RNA and DNA array. Where DNA quantity was limited, a whole genome extraction. Total RNA was extracted using the RNeasy Micro amplification step (multiple displacement amplification) was kit (Qiagen, Hilden, Germany) and quality was checked using performed before digestion using F29 DNA polymerase an Agilent 2100 Bioanalyser (Agilent Technologies, Palo Alto, according to the manufacturer’s instructions (Quiagen Inc., CA, USA). DNA was extracted from the same tumour Valencia, CA, USA). SNP genotypes were established with material using a phenol/chloroform/isoamyl alcohol extraction GDAS3.0 software using default settings, resulting in an after proteinase K treatment. The quantity of RNA and DNA average of 95% positive SNP calls per sample. Copy number

Oncogene Genomic and expression microarray analysis in neuroblastoma M Łastowska et al 7443 analysis was performed using both the Affymetrix gene chip DNA. Primers, TaqMan TAMRAt probes and specific Copy Number Analysis Tool (CNAT) version 2.0 (Huang conditions are listed in Supplementary Table S1. The mean et al., 2004) with a 500 kb Genome Smoothed Average (GSA) Ct value was normalized against that of the endogenous and using CNAG software (Nannya et al., 2005) with a 10 control gene PPIA (Fischer et al., 2005). Relative gene SNP sliding window. Only copy number changes identified by expression was calculated with the 2ÀDDCt method (Livak and both methods were recorded, except in three polyploid Schmittgen, 2001), using as a reference for each gene the tumours where the data from CNAG was preferred, as it tumour RNA which showed the median expression level for was found to be more robust (as copy number can be defined that gene as measured by microarray analysis (tumour 24 for relative to one or more regions of known ploidy, defined by PMP22, tumour 10 for WSB1, tumour 3 for BRCA1 and cytogenetic analysis). No discrepancies involving gains or BIRC5). losses of >2 Mb were observed between the two methods. The consistency of copy number estimation was assessed in four Molecular cytogenetic analysis cases through additional hybridization to the XbaI 50K of MYCN mapping array (Affymetrix), and in five cases through direct Presence amplification and status of chromosomes 1 comparison of tumour and constitution samples. All copy and 17 were analysed in all 30 tumours using FISH. Short- number changes >2 Mb in size were confirmed by these term culture of primary tumour cells, harvesting and slide preparation were performed according to published protocols analyses. Genome smoothed copy number estimates from all (Bown et al., 1994). Conditions for FISH hybridization, HindIII arrays (n ¼ 18) were also ranked to investigate the association between copy number and overall survival using a washing, detection and the probes used for 17q, 1p and MYCN Cox proportional hazard model with a score test (Rao, 1973). analyses have been described elsewhere (Lastowska et al., 1997, 2002). Real-time reverse transcriptase–PCR Real-time PCR for BRCA1, PPIA, WSB1, PMP22 and PPIA Acknowledgements was performed using the 50 nuclease assay on the ABI PRISMt 7700 Sequence detector (Perkin-Elmer, Applied The financial support of the Neuroblastoma Society UK, Biosystems, Foster City, CA, USA). Oligonucleotides were Newcastle Healthcare Charity, the Candlelighters Trust Leeds designed using Primer Express software (v.3.0, PE Biosystems, and Cancer Research UK is gratefully acknowledged. The Foster City, CA, USA); forward and reverse primers were authors also thank the CCLG for access to constitutional selected in adjacent exons to avoid amplification of genomic DNA samples.

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Supplementary Information accompanies the paper on the Oncogene website (http://www.nature.com/onc).

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