Microarray Analyses Reveal Strong Influence of DNA Copy Number

Microarray analyses reveal strong inﬂuence of DNA copy number alterations on the transcriptional patterns in pancreatic cancer: implications for the interpretation of genomic ampliﬁcations

Markus Heidenblad*,1, David Lindgren1, Joris A Veltman2, Tord Jonson1, Eija H Mahlama¨ ki3, Ludmila Gorunova1, Ad Geurts van Kessel2, Eric FPM Schoenmakers2 and Mattias Ho¨ glund1

1Department of Clinical Genetics, Lund University Hospital, SE-221 85 Lund, Sweden; 2Department of Human Genetics, University Medical Center Nijmegen, PO Box 9101, 6500 HB Nijmegen, The Netherlands; 3Laboratory of Cancer Genetics, University of Tampere and Tampere University Hospital, Tampere, Finland

DNA copy number alterations are believed to play a major 1997). The consistent presence of these genomic altera- role in the development and progression of human tions indicates that an apparently tumor-type indepen- neoplasms. Although most of these genomic imbalances dent selective advantage is added to the transformed have been associated with dysregulation of individual cell, most probably through modified expression levels genes, their large-scale transcriptional consequences of genes in the affected segments. This is supported by remain unclear. Pancreatic carcinomas frequently display studies of breast cancer, where parallel microarray gene copy number variation of entire chromosomes as well analyses of mRNA (expression profiling) and DNA as of chromosomal subregions. These changes range from (genomic profiling) have shown a consistent association homozygous deletions to high-level amplifications and are between gene copy numbers and expression levels believed to constitute key genetic alterations in the (Hyman et al., 2002; Pollack et al., 2002). These studies cellular transformation of this tumor type. To investigate indicate that DNA copy number has a major impact on the transcriptional consequences of the most drastic transcription, especially in high-level amplifications genomic changes, that is, genomic amplifications, and to where as many as 44–62% of the affected genes also analyse the genome-wide transcriptional effects of DNA show overexpression. However, similar data from colon copy number changes, we performed expression profiling cancer indicate that only 4% of the amplified genes of 29 pancreatic carcinoma cell lines and compared the respond in terms of increased expression (Platzer et al., results with matching genomic profiling data. We show 2002). In the present study, we set out to determine that a strong association between DNA copy numbers and whether the strong gene dosage/expression correspon- mRNA expression levels is present in pancreatic cancer, dence is restricted to breast cancer, or reflects a more and demonstrate that as much as 60% of the genes within common phenomenon that also applies to other tumor highly amplified genomic regions display associated over- types. Pancreatic carcinomas frequently show imbal- expression. Consequently, we identified 67 recurrently ances of entire chromosomes as well as of smaller overexpressed genes located in seven precisely mapped genomic segments. The DNA copy number variations, commonly amplified regions. The presented findings which range from homozygous deletions to high-level indicate that more than one putative target gene may be amplifications, are believed to constitute key genetic of importance in most pancreatic cancer amplicons. alterations in the development and progression of this Oncogene (2005) 24, 1794–1801 doi:10.1038/sj.onc.1208383 tumor type. To investigate the genome-wide transcrip- Published online 31 January 2005 tional effects of gene copy number alterations, in particular of genomic amplifications, we performed Keywords: expression profiling; genomic profiling; mi- expression profiling analyses of 29 pancreatic carcinoma croarray; pancreatic cancer cell lines using cDNA microarrays, and compared the results with matching genomic profiling data (Heiden- blad et al., 2004). The analysis of the combined genomic and expression data sets allowed us to (i) determine the A common feature in human malignancies is the genome-wide association between gene copy number presence of genomic imbalances. Even though some of and expression levels in pancreatic cancer, (ii) identify 67 these are tumor specific, the majority can be found in a recurrently overexpressed genes in seven precisely large number of different tumor types (Mertens et al., mapped commonly amplified regions, and (iii) analyse the genome-wide mRNA effects two of the most frequently amplified regions in pancreatic cancer, *Correspondence: M Heidenblad; E-mail: [email protected] 8q23-24 and 12p11-12. Received 26 July 2004; revised 15 November 2004; accepted 18 To evaluate the global transcriptional consequences November 2004; published online 31 January 2005 of gene copy number alterations, paired gene expression Transcriptional patterns in pancreatic cancer M Heidenblad et al 1795 and DNA copy number ratios from 10 of the cell lines respectively, did not result in any major changes of were analysed. A total of 79 927 paired observations, the expression levels. However, when the fold-changes representing 13 720 different cDNA clones, were in- exceeded 2.0, a large effect was seen, both on the cluded. Based on gene copy number ratios, these were number of genes affected and on the maximum subdivided into seven categories with fold-changes expression levels. This pattern was even more o0.7, 0.7–0.9, 0.9–1.1, 1.1–1.5, 1.5–2.0, 2.0–2.5, pronounced in the category with >2.5-fold DNA and >2.5. In Figure 1a, all expression levels amplification. within the respective categories are plotted. Gene copy To investigate specifically the relationship between fold-changes below 0.9, and between 1.1 and 2.0, gene copy numbers and proportion of over-/under- corresponding to gains or losses of 1–2 gene copies, expressed genes, these were defined as the upper and lower 7th percentiles (Hyman et al., 2002) of the B80 000 expression measurements. The fraction of over- and underexpressed genes within each gene copy a 8 number category revealed that the former showed a 7 strong positive association with DNA copy numbers, 6 and that the latter showed a less pronounced negative 5 association with the number of genomic copies present (Figure 1b). The category with gene copy fold-changes 4 o0.7 showed over- and underexpression of 5.1 and 3 12%, respectively, of the genes in this group, whereas 2 the equivalent fractions in the highest copy number Expression ratio 1 category were 53 and 3.4%. In the normal copy number group (ratios 0.9–1.1), 6.2% of the genes were over- 0 expressed and 6.5% underexpressed. To evaluate the -1 effects of the most highly amplified genomic sequences < 0.7 0.7-0.9 0.9-1.1 1.1-1.5 1.5-2.0 2.0-2.5 > 2.5 Copy number ratio in more detail, we raised the threshold ratio of the highest DNA copy number category to 3.0 and 3.5. This b 60 analysis showed that 62% of the genes in the former and Overexpressed 60% of the genes in the latter were overexpressed, Underexpressed 50 suggesting that a maximum fraction of susceptible genes seems to be affected when fold-changes exceed 3.0, 40 equivalent to approximately 10 gene copies in the present microarray platform. A consequence of these 30 findings is that a change from a low- to a high-level amplification not only results in further upregulation of 20 genes already overexpressed at low gene copy numbers Proportion ESTs (%) but also in additional genes within a given segment 10 becoming upregulated. Hence, the biological outcome of 0 high-level amplifications may be very different from that < 0.7 0.7-0.9 0.9-1.1 1.1-1.5 1.5-2.0 2.0-2.5 > 2.5 of gains and low-level amplifications of the same Copy number ratio segment. The maximum of 60% affected genes in high- Figure 1 Association between gene copy number and expression level amplifications is in agreement with observations in levels. To obtain a reliable and representative assembly of paired breast cancer using array-based comparative genomic genomic and transcriptional observations for the association hybridization (CGH) for amplicon mapping (Hyman analysis, we used the following selection criteria. First, based on et al., 2002; Pollack et al., 2002), but differs substantially the genomic aberration spectra and hybridization qualities of both hybridizations, 10 tumor cell lines were selected; AsPC-1, DANG, from findings in colon cancer based on traditional CGH HupT3, LPC3p, LPC4p, LPC5 m, LPC10 m, LPC11p, LPC13p, for amplicon delineation (Platzer et al., 2002). A partial and LPC14p. Second, to enrich high-quality observations within explanation for this discrepancy could be that CGH this subset of tumor cell lines, we used a signal-to-noise filter of 3.0 analyses on metaphase chromosomes generally over- for both dyes, and for both experiments. Only cDNA clones with at least one paired observation remaining after this filtration were estimate the size of amplified segments, potentially included in the association analysis (a total of 13 720 of 25 468). In leading to analysis of a large number of unamplified total, 79 927 paired ratios from the 10 cases were analysed. For genes, which in turn would reduce the frequency of detailed information on cell lines and hybridizations, see legends to affected genes. Figure 2. (a) Box-Whisker plots illustrating the transcriptional We previously determined the exact genomic bound- distributions of 79 927 observations divided into seven different copy number categories: o0.7 (N ¼ 2943), 0.7–0.9 (N ¼ 21 325), aries of 60 amplicons in the investigated cell lines 0.9–1.1 (N ¼ 31 885), 1.1–1.5 (N ¼ 21 427), 1.5–2.0 (N ¼ 1971), 2.0– (Heidenblad et al., 2004). In total, 491 (36%) of the 2.5 (N ¼ 230), and >2.5 (N ¼ 146). Boxes show the central quartiles UniGene clusters harboring clones in these segments in each group (25–75%), with medians denoted as black points in were found to show more than twofold increased bold. Extremes and outliers are excluded. (b) Percentage of over- and underexpressed genes in each copy number category. Thresh- expression (Supplementary Table 1). By using a criterion old values for overexpression were 2.004 (global upper 7% of of more than twofold overexpression in at least two expression ratios) and 0.418 (global lower 7% of expression ratios) cases with genomic amplification, a final list of 67

Oncogene Transcriptional patterns in pancreatic cancer M Heidenblad et al 1796 putative target genes from seven different genomic ulator of TGFB signaling, was overexpressed in two regions was identiﬁed (Figure 2). All of the regions cases with 7q22–31 ampliﬁcations (Figure 2), whereas contained genes that could be of importance to cellular MET (HGFR) represented a borderline case showing transformation (Figure 2). SMURF1, a negative reg- more than twofold increased expression in one case and

Log2Ratio

-3.0 0.0 3.0

Gene/EST Band Mb LPC1p LPC2p LPC3p LPC4p LPC5m LPC6p LPC7m LPC8p LPC10m LPC11p LPC12m LPC13p LPC14p LPC15p AsPC-1 BxPC-3 Capan-2 CFPAC DANG Hs700T Hs766T HupT3 HupT4 PANC-1 PaTu8902 PaTu8988S PaTu8988T SU.86.86 SW1990 HLA-A (IMAGE: 450358) 6p22.1 30.02 IMAGE: 2114351 (Hs.122390) 6p22.1 30.08 TRIM26 (IMAGE: 823982) 6p22.1 30.26 c6orf134 (IMAGE: 378372) 6p22.1 30.72 CREBL1 (IMAGE: 385017) 6p21.3 32.15 HBS1L (IMAGE: 1685029) 6q23.3 135.35

AKAP9 (IMAGE: 2306682) 7q21.2 91.35 BET1 (IMAGE: 203351) 7q21.3 93.24 PON2 (IMAGE: 781019) 7q21.3 94.65 SMURF1 (IMAGE: 725407) 7q22.1 98.24 CBLL1 (IMAGE: 281442) 7q31.1 106.96 DLD (IMAGE: 813648) 7q31.1 107.12 HIC (IMAGE: 2403355, 148810) 7q31.2 114.21 TM4SF12 (IMAGE: 2283651) 7q31.3 119.98

MAL2 (IMAGE: 565319) 8q24.1 120.21 NOV (IMAGE: 1505534, 2502789) 8q24.1 120.39 MGC5528 (IMAGE: 1394099) 8q24.1 120.80 MGC21654 (IMAGE: 1641737) 8q24.1 124.12 ZHX1 (IMAGE: 609283) 8q24.1 124.22 PRO2000 (IMAGE: 2800375) 8q24.1 124.29 LOC286052 (IMAGE: 1534493) 8q24.1 125.28 LOC157378 (IMAGE: 811020)8q24.1 125.31 IMAGE: 206986 (Hs.440163) 8q24.1 125.60 NSE2 (IMAGE: 278572, 1650748) 8q24.2 127.52 IMAGE: 175533 (Hs.32501) 8q24.2 130.81 PTK2 (IMAGE: 724892, 2107322) 8q24.3 141.64 CYC1 (IMAGE: 813830) 8q24.3 145.26 MAF1 (IMAGE: 897727)8q24.3 145.27 HT002 (IMAGE: 470099)8q24.3 146.08

CMAS (IMAGE: 897301) 12p12.1 22.11 SIAT8A (IMAGE: 610012)12p12.1 22.25 KIAA0528 (IMAGE: 2296063) 12p12.1 22.49 EKI1 (IMAGE: 1635221) 12p12.1 22.73 IMAGE: 383185, 281191 (Hs.29464) 12p12.1 22.73 BCAT1 (IMAGE: 1470169, 2009885) 12p12.1 24.89 c12orf2 (IMAGE: 289794) 12p12.1 26.12 BHLHB3 (IMAGE: 595078, 712544)12p12.1 26.17 SSPN (IMAGE: 265868, 814259, 281190) 12p12.1 26.28 IMAGE: 505243 (Hs.406751)12p11.2 26.38 ITPR2 (IMAGE: 128020) 12p11.2 26.45 DKFZP564O1863 (IMAGE: 323623, 1628142)12p11.2 27.01 TM7SF3 (IMAGE: 66978, 813631) 12p11.2 27.02 SURB7 (IMAGE: 567265)12p11.2 27.07 IMAGE: 1639294 (Hs.184156) 12p11.2 27.08 PPFIBP1 (IMAGE: 2010014) 12p11.2 27.69 IMAGE: 256513 (Hs.125229) 12p11.2 27.73 IMAGE: 1637759 (Hs.269836)12p11.2 27.74 KIAA1340 (IMAGE: 435776, 249755)12p11.2 27.84 FLJ11088 (IMAGE: 786169, 1048795) 12p11.2 28.55 FLJ10462 (IMAGE: 754334) 12p11.2 29.36 PTX1 (IMAGE: 825603) 12p11.2 29.41 ARG99 (IMAGE: 81316) 12p11.2 29.55

FLJ12760 (IMAGE: 266218) 17q23.3 61.00 TLK2 (Hs.897751) 17q23.3 61.16

CAPN12 (IMAGE: 194161) 19q13.2 43.91 ECH1 (IMAGE: 2580311, 769537, 1556151)19q13.2 44.00 MRPS12 (IMAGE: 131653)19q13.2 44.12 PAK4 (IMAGE: 756450)19q13.2 44.36 FLJ10211 (IMAGE: 731258)19q13.2 44.53 PD2 (IMAGE: 1625869) 19q13.2 44.57 SUPT5H (IMAGE: 506270) 19q13.2 44.66 DLL3 (IMAGE: 1469966) 19q13.2 44.69 MGC20452 (IMAGE: 448820) 19q13.2 44.72 FBL (IMAGE: 855755) 19q13.2 45.02 PSMC4 (IMAGE: 810558) 19q13.2 45.17 LOC284323 (IMAGE: 2108364) 19q13.2 45.28 AKT2 (IMAGE: 2072862) 19q13.2 45.43

Oncogene Transcriptional patterns in pancreatic cancer M Heidenblad et al 1797 1.9-fold upregulation in another (data not shown). The systematically searching for overexpressed genes within increase in SMURF1 expression is noteworthy in precisely mapped amplicons in these cell lines, it became light of the frequent inactivation of the TGFB-signaling evident that several genes in each amplified segment pathway in pancreatic carcinomas (Bardeesy and showed significant transcriptional response to the DePinho, 2002). Among the 14 identified candidate corresponding copy number increase. Accordingly, in genes in 8q24, the most frequently overexpressed were many of the amplified segments where established LOC286052 and LOC157378, upregulated more than oncogenes have been identified, additional genes with twofold in three and four of the cases with this possible tumorigenic effects are colocalized and over- amplification, respectively (Figure 2). No molecular expressed. This indicates that more than one gene may functions have been assigned to these genes. The MYC be of selective advantage in each commonly amplified oncogene, amplified in seven cases, unexpectedly region. showed decreased expression in four of these and more Apart from the amplified regions listed in Figure 2, we than twofold overexpression in only one case. The identified 22 unique amplicons and three genomic microarray results for MYC were verified by RT–PCR segments that were recurrently amplified but did not (data not shown). show any commonly overexpressed genes as determined We previously identified BHLBH3 (DEC2), KRAS2 by the array analyses. Three of these regions were and PPFIBP1 as possible target genes for 12p amplifica- selected for more detailed analysis (Figure 3a–c). The tions in pancreatic carcinomas (Heidenblad et al., 2002). first, located to 6p21.1, was amplified in two cases, In the present study, as many as 20 additional putative LPC3p and SU.86.86. As the commonly amplified target genes/ESTs were identified in this region segment contained CCND3, highly overexpressed in (Figure 2), four of which (EKI1, BCAT, CMAS, and LPC3p (Figure 3a) but not in SU.86.86 as determined by SURB7) have also been shown to be overexpressed in the array study, RT–PCR analysis was performed. This testicular germ cell tumors with similar amplifications confirmed the microarray results for LPC3p (Figure 3d) (Rodriguez et al., 2003). Both cases with 17q23 and indicated a 2.2-fold increased expression in amplifications showed more than twofold upregulated SU.86.86 (data not shown). The second region involved expression of FLJ12760 and TLK2. No function has two unique 8p amplifications in DANG (Figure 3b). As been assigned to FLJ12760, whereas TLK2 is a serine/ 8p is lost in approximately 60% of pancreatic carcino- threonine kinase activated by cell cycle-dependent mas (Hahn et al., 1995), and adverse genomic instability phosphorylation. Interestingly, both genes showed is frequent in this tumor type (Gorunova et al., 1998), increased expression exclusively in cases with amplifica- we hypothesized that these alterations could be biolo- tion, indicating a strong link between DNA copy gically less significant side effects of the genomic number and gene expression (Figure 2). Amplification instability. However, the expression profile of this and overexpression of AKT2 in 19q13.2 is well case revealed five genes with highly increased established in pancreatic cancer (Cheng et al., expression (>4-fold) in the distal amplicon, and six 1996). In the present investigation, we identified 12 genes with more than twofold overexpression in the additional amplified and overexpressed genes in this proximal amplicon (Figure 3b and Supplementary region, including PAK4 (Mahlama¨ ki et al., 2004), Table 1). Among these, at least BAG4, a member of involved in the reorganization of the cytoskeleton the BAG1-related protein family that inhibits apoptosis, and in inducing antiapoptotic effects. Thus, by may contribute to cellular transformation. The

Figure 2 List of putative amplification target genes. The figure shows 67 genes/ESTs identified as amplified and overexpressed more than twofold in at least two cases. Expression levels are shown for all 29 cases and are depicted on a log2-based pseudocolor scale (color code on top). Red and green squares indicate over- and underexpression, respectively, and gray areas denote missing values. Surrounding boxes in yellow show cases in which the respective genes are amplified. To the right, gene symbols, clone identities, and chromosomal localizations are given. In cases where genes are represented by more than one cDNA clone on the microarray, the mean expression of the clones listed is shown. For cDNA clones lacking gene symbol annotation, clone identities are shown with the corresponding UniGene cluster number. The 29 pancreatic carcinoma cell lines analysed comprised LPC1p, LPC2p, LPC3p, LPC4p, LPC5m, LPC6p, LPC7m, LPC8p, LPC10m, LPC11p, LPC12m, LPC13p, LPC14p, and LPC15p, which have been previously described (Jonson et al., 1999; Heidenblad et al., 2004), and the established cell lines AsPC-1, BxPC-3, Capan-2, CFPAC-1, DANG, Hs700T, Hs766T, HupT3, HupT4, PANC-1, PaTu8902, PaTu8988S, PaTu8988T, SU.86.86, and SW1990 obtained from cell repositories. Expression profiling was performed using cDNA microarrays, containing 25 648 different cDNA clones (17,494 UniGene clusters) obtained from the Swegene DNA microarray resource center at Lund University (http://swegene.onk.lu.se). Amplified tumor cell line and reference RNA (RiboAmp RNA amplification kit, Arcturus, Mountain View, CA, USA) was differentially labeled (CyScribe Post Labeling kit, Amersham Biosciences, Uppsala, Sweden) with Cy3- and Cy5-dUTP, respectively. As reference, a pool containing equal amounts of all amplified RNA samples was used. Labeled cDNAs were hybridized to the microarrays for 18 h. Pre- and post-treatments of microarray slides were performed according to Universal Hybridization Kit manual (Corning, Acton, MA, USA). Fluorescence intensities were quantified on an Agilent G2565AA microarray scanner (Agilent technologies, Palo Alto, CA, USA) and the raw images were analysed using the GenePix Pro 4.0 software (Axon Instruments, Inc., Foster City, CA, USA). Background corrected data were Lowess normalized (smoothing factor 0.33) and filtered (signal-to-noise ratio >3.0) using the web- based database BASE (Saal et al., 2002). For mapping and annotations of cDNA clones, data available through the UCSC genome browser (http://genome.ucsc.edu, July 2003 freeze) and NCBI (http://www.ncbi.nlm.nih.gov/UniGene, UniGene Build 164) were used. Genomic profiling information from the 29 cell lines was extracted from previously published data (Heidenblad et al., 2004). The data sets are available at http://www.klingen.lu.se/E/research

Oncogene Transcriptional patterns in pancreatic cancer M Heidenblad et al 1798 overexpression of this gene was validated by RT–PCR proposed as target genes (Albertson et al., 2000). Even (Figure 3d). This suggests that small and infrequent though both of these genes were located in the amplifications may also harbor genes of potential amplification maximum of LPC3p, ZNF217 showed importance for tumor development. no transcriptional response, and CYP24A1 showed The third region, located at 20q13.2, was amplified in considerable under- rather than overexpression LPC3p (Figure 3c) and PaTu8988T. Amplification of (Figure 3c). In contrast, PFDN4, also localized within this segment is frequent in various malignancies, and in the amplification, displayed a pronounced (>4-fold) breast cancer, ZNF217 and CYP24A1 have been overexpression (Figure 3c). RT–PCR analysis of

a 6.0 b 6.0 LPC3p (Exp.) DANG (Exp.) LPC3p (aCGH) CCND3 DANG (aCGH) 4.0 4.0 BAG4 2.0 2.0

0.0 0.0

Log2Ratio -2.0 Log2Ratio -2.0

-4.0 -4.0

-6.0 -6.0 0 102030405060 0105201525 30 35 40 45 Mb position Mb position

c 6.0 d 6.0 LPC3p (Exp.) RT-PCR LPC3p (aCGH) Microarray 4.0 4.0 PFDN4 2.0 2.0

0.0 0.0 CCND3 BAG4 PFDN4 ZNF217 CYP24A1 ZNF217 Log2Ratio -2.0 Log2Ratio -2.0

CYP24A1 -4.0 -4.0

-6.0 -6.0 30 35 40 45 50 55 60 65 Mb position Gene Figure 3 Composite gene copy number/expression profiles, and RT–PCR validation results. The log2 test/reference profiles demonstrate the transcriptional impact of genomic amplifications on (a) 6p in LPC3p, (b) 8p in DANG, and (c) 20q in LPC3p. For visualization purposes, the genomic profiles (connected black circles) are displayed as a moving average (symmetric four-nearest neighbors), whereas the unconnected gray circles illustrate the individual expression level of each cDNA clone. Each gene may be represented by several cDNA clones, which are shown according to their Mb positions in the UCSC genome browser (July 2003 freeze). Genes within the amplicons selected for semiquantitative RT–PCR validations are marked in the profiles. (d) Expression levels from both analyses (RT–PCR and microarray) are shown for the genes CCND3, BAG4, PFDN4, ZNF217, and CYP24A1. For RT– PCR validations, including MET and MYC, two multiplex PCR reactions were performed for each gene, one including ACTB, and the other GAPD as internal standard. PCR conditions conformed to standard procedures, and quantification was performed by phosphorimaging as described previously (Heidenblad et al., 2002). The expression levels were calculated as the mean of the ratios between the assayed genes and internal standards, normalized against the reference. As reference, a combination of cDNA samples from all 29 cases was used, equivalent to the reference applied in the expression profiling

Figure 4 Gene expression signatures associated with amplifications of (a) 8q24 and (b) 12p11–12. The figure shows 98/71 genes/ESTs differentially expressed, as determined by a Bonferroni adjusted t-test (Po0.05) using the TIGR MeV suite of software (Saeed et al., 2003), in cases with 8q24/12p11–12 amplifications as compared with their unaffected counterparts. The cases were selected based on previous conventional and array-based CGH data (Mahlama¨ ki et al., 1997; Heidenblad et al., 2004). Expression levels are presented on a log2-based pseudocolor scale (color code on top), where red and green indicate over- and underexpression, respectively, and gray denotes missing values. Genes included in the respective amplicons are enclosed in yellow boxes. To the right, gene symbols, clone identities, and chromosomal localizations according to the UCSC genome browser (July 2003 freeze) are given. In cases where genes are represented by more than one cDNA clone on the microarray, the mean expression of the clones listed is shown. For cDNA clones lacking gene symbol annotation, clone identities are shown with the corresponding UniGene cluster number

Oncogene a 303.0 -3.0 LPC6p LPC12m LPC15p Log2Ratio Hs700T 0.0 HupT3 LPC4p LPC8p LPC14p BxPC-3 MG:825 H.34)41.57 IMAGE: 812053(Hs.43047) RAE1 SLC9A8 IMAGE: 2017423(Hs.158604) KCNN4 XRCC1 YF13H12 SLC14A1 NAPG VAPA MRCL3 XRCC3 LRP10 AK000009 SAP18 IMAGE: 2273362 (Hs.407903) PRICKLE1 FZD4 MDK CLN2 ADAM8 NT5C2 HPS1 MGC13047 FER1L3 BTAF1 IMAGE: 294136(Hs.125029) HFL1 IMAGE: 22500(Hs.403972) ABL1 FLJ10493 UNC13 FLJ10842 TULP4 FLJ31349 NT5C2L1 IMAGE: 268818(Hs.26904) IMAGE: 1424961(Hs.159388) C6orf35 LOC339929 IMAGE: 2028599(Hs.14074) GPC1 NAB1 COL5A2 LAPTM5 LOC339400 SFRS11 IMAGE: 1758937(Hs.13944) PRSS15 NPL4 HLF BCL6B KIF22 TRAP1 DNASE1 FLJ12484 BAG5 DNCH1 ALKBH AHSA1 MED6 IMAGE: 242007(Hs.191334) TROAP SLC36A4 IMAGE: 234320(Hs.42050) IMAGE: 884653(Hs.433616) UBE2R2 ALAD FLJ14825 IMAGE: 134918(Hs.390240) MYO6 HIST1H2AM TINAG MGAT1 KIAA0676 CDC25C GTF2H2 FLJ13611 SMN2 ZSIG11 EMILIN3 RAB9P40 MAPKAPK3 IFRD2 MAP4 SEC22L3 SH3BP5 IMAGE: 209118,(Hs.19977) PDK1 IMAGE: 1031548(Hs.515975) MTA3 LOC126731 SLC16A1 LOC51064 ATF4 NEK6 DNAJB6 LOC15543 eeETCr Mb Chr. Gene/EST (IMAGE: 41684) IAE 549)46.37 (IMAGE: 1574594) IAE 525)38.16 (IMAGE: 2562955) (IMAGE: 450060) (IMAGE: 489968) (IMAGE: 454698) (IMAGE: 2131058) (IMAGE: 855385) IAE 050 56.61 (IMAGE: 609530) (IMAGE: 415525, 767828) (IMAGE: 306798) (IMAGE: 435208) (IMAGE: 796876) (IMAGE: 1645668) (IMAGE: 49475) IAE 316)102.02 (IMAGE: 769942) (IMAGE: 2321066) (IMAGE: 594556) (IMAGE: 812033) (IMAGE: 586839) 2569196, (IMAGE: 782797) (IMAGE: 1573157) (IMAGE: 810992) (IMAGE: 470216) (IMAGE: 809946) (IMAGE: 1869112) (IMAGE: 378475) (IMAGE: 2497644) IAE 076 19.52 (IMAGE: 209756) (IMAGE: 784085) (IMAGE: 725076) (IMAGE: 308115) (IMAGE: 2514552) (IMAGE: 1031747) (IMAGE: 754649) (IMAGE: 2541460) IAE 624 48.75 (IMAGE: 756708) (IMAGE: 760224) (IMAGE: 2551144) (IMAGE: 451757) (IMAGE: 704254) (IMAGE: 276871) (IMAGE: 796986) (IMAGE: 242578) (IMAGE: 415406) (IMAGE: 1636061) (IMAGE: 810441) (IMAGE: 2401917) (IMAGE: 131791) (IMAGE: 2028266) (IMAGE: 796613) (IMAGE: 855890) (IMAGE: 2566815) (IMAGE: 626343) IAE 524)88.36 (IMAGE: 1582148) (IMAGE: 753313) (IMAGE: 770289) (IMAGE: 1568318) (IMAGE: 415102) (IMAGE: 2466211) (IMAGE: 240062) (IMAGE: 376217) (IMAGE: 34294) (IMAGE: 281127) (IMAGE: 486175) IAE 466 123.34 (IMAGE: 249606) (IMAGE: 284120) (IMAGE: 195557) (IMAGE: 289016) 86.91 (IMAGE: 431214) (IMAGE: 243088) (IMAGE: 788494) (IMAGE: 2100930) IAE 692 73.91 (IMAGE: 469952) (IMAGE: 758309) (IMAGE: IAE 662)142.44 (IMAGE: 1636523) (IMAGE: 786053) (IMAGE: 758314) (IMAGE: 2406134) (IMAGE: 261473) (IMAGE: 46287) (IMAGE: 52031) (IMAGE: 1687138) 22 19 17 17 17 16 16 16 15 14 14 14 14 14 12 12 11 10 10 22 21 20 20 20 19 19 19 18 18 18 18 14 14 13 13 12 12 11 11 11 10 10 10 10 10 10 7 7 7 7 6 6 6 6 6 6 3 3 2 2 2 1 1 1 1 1 9 9 9 9 9 9 8 6 6 6 6 5 5 5 5 5 5 3 3 3 3 3 3 3 2 2 2 1 1 9 9 2 241.73 191.76 190.10 100.44 129.04 103.85 124.19 140.76 158.84 154.76 116.61 108.23 189.19 172.77 194.09 136.29 180.35 179.42 137.70 173.65 226.42 112.75 102.15 134.55 104.51 122.48 156.60 154.99 140.39 108.01 35.40 33.91 38.19 41.39 30.71 80.28 53.88 29.85 37.14 48.00 92.59 34.91 82.22 76.60 27.97 70.56 64.94 51.70 50.65 50.29 47.85 42.55 15.28 47.31 42.91 49.19 48.96 48.70 41.57 10.54 21.34 64.93 41.14 86.38 65.98 99.84 99.81 94.74 93.46 37.08 76.13 75.92 69.04 1.69 5.64 7.13 9.95 3.24 NA NA NA NA NA NA NA NA b 303.0 -3.0

LPC4p Heidenblad cancer M pancreatic in patterns Transcriptional LPC5m DANG

SU.86.86 Log2Ratio tal et LPC8p 0.0 LPC10m BxPC-3 HupT3

HupT4 SW1990 NOLA1 MGC3232 (IMAGE:LOC285458 1572723) IMAGE: 195836(Hs.151251) HUMAGCGB VILL IMAGE: 435055(Hs.179864) IMAGE: 447700(Hs.444948) MGC4309 IMAGE: 1591154(Hs.172844) TNFRSF8 RPGR AP1S2 RNU22 PANK1 (IMAGE:LOC285958 470232) CDC2L5 E11s1 GABRP DKFZP56401863 c12orf2 IMAGE: 383185(Hs.29464) NGP SPA17 CTSC FLJ11336 1558108) (IMAGE: ABCC8 KIA1010 SARA1 GEM FLJ11767 KCNK5 C6orf35 ANXA6 FLJ35779 IL7R DKFZP434D146 PPP1R7 ACTR3 XCL1 ARHGEF2 ZBED4 ZNF42 IMAGE: 377279(Hs.355739) FLJ31100 LGALS4 JUNB MGC45408 ZFP161 SYNGR2 FLJ20920 IMAGE: 1896335(Hs.444858) LOC90110 IMAGE: 1636837(Hs.409362) LOC161291 IMAGE: 452483(Hs.122063) RPA1 IMAGE: 1639640(Hs.447456) DREV1 LOC123803 EIF2B2 IMAGE: 418307(Hs.22100) PTX1 FLJ11088 TM7SF3 ADSL PKNOX1 CBR3 LOC284296 DRIL1 KIF1C eeETCr Mb Chr. Gene/EST (IMAGE: 2435159) (IMAGE: 80344, 841238) (IMAGE: 590264) (IMAGE: 767765) (IMAGE: 825603) (IMAGE: 1896981) IAE 084 127.65 (IMAGE: 309864) (IMAGE: 2222825) (IMAGE: 813280) (IMAGE: 1948085) (IMAGE: 845355) (IMAGE: (IMAGE: 2013094) (IMAGE: 753080) (IMAGE: (IMAGE: 462113) (IMAGE: IAE 023)37.16 (IMAGE: 2012234) IAE 988)63.77 (IMAGE: 1908389) IAE 548 15.21 (IMAGE: 757428) (IMAGE: 503866) IAE 538 48.50 (IMAGE: 250328) (IMAGE: 593251) (IMAGE: 1635032) (IMAGE: 1055460) (IMAGE: 1591154) (IMAGE: 1475633) (IMAGE: 713031) (IMAGE: 187266) (IMAGE: 289794) (IMAGE: (IMAGE: 1901562) (IMAGE: 842839) (IMAGE: (IMAGE: 563598) (IMAGE: 824511) (IMAGE: 1636061) (IMAGE: 586685) (IMAGE: 66978) (IMAGE: 795379) (IMAGE: 814508) (IMAGE: (IMAGE: 824937) (IMAGE: 1947972) (IMAGE: 2509911) (IMAGE: 897529) (IMAGE: 347742) (IMAGE: 360065) (IMAGE: 814515) (IMAGE: 269788) (IMAGE: 786169) (IMAGE: 505538) (IMAGE: 814443) (IMAGE: 1570427) (IMAGE: (IMAGE: 769975) (IMAGE: 328342) (IMAGE: (IMAGE: 1520448) (IMAGE: (IMAGE: 322511) (IMAGE: 455179) (IMAGE: (IMAGE: 50299) (IMAGE: (IMAGE: 275653) (IMAGE: 361659) (IMAGE: 1628142) 22 19 19 19 19 19 19 18 17 17 17 15 14 12 11 10 17 17 17 16 16 14 13 12 12 12 12 12 12 12 11 11 11 11 10 10 22 21 21 19 19 17 8 8 6 6 5 5 5 3 2 2 1 1 7 7 7 5 4 4 4 3 3 3 3 2 1 1 1 X X 198.67 204.28 170.19 111.20 169.38 150.51 150.98 112.75 124.10 173.79 101.30 51.39 38.01 33.10 87.68 15.28 39.00 43.34 36.44 60.63 39.88 29.94 57.75 52.81 76.77 49.02 38.10 37.78 38.99 59.73 62.40 91.01 44.76 63.50 62.57 43.98 52.96 11.91 49.69 39.24 75.05 35.92 15.53 24.24 11.48 16.57 15.31 21.59 73.46 29.40 28.35 27.02 27.01 26.12 22.73 14.93 87.71 33.05 71.25 95.22 0.92 5.13 2.01 N N N A A A Oncogene 1799 Transcriptional patterns in pancreatic cancer M Heidenblad et al 1800 ZNF217, CYP24A1 and PFDN4 corroborated the strategy to identify putative target genes but microarray findings (Figure 3d). The expression also provided an opportunity to analyse potential patterns of all three genes in PaTu8988T were similar indirect consequences of the 8q23–24 and 12p11–12 to the patterns seen in LPC3p, showing that even amplifications. though the same genomic segments are amplified in The presented data have shown that the standard different tumor types, their transcriptional outcome may approach to analyse genomic amplifications has vary. several limitations. First, high-level amplifications are A limitation in the above used selection of amplifica- likely to affect a large number of genes. This, and the tion targets is that it does not take the dynamic currently limited amount of functional data available expression range of the individual genes into con- for many genes, complicate the selection of primary sideration; genes showing less than twofold upregulation putative target sets. Second, cases with normal copy may also be of biological importance. Moreover, numbers may also overexpress genes within commonly identified target genes may also be upregulated in amplified regions, making the link between increased cases with normal gene copy numbers. As an alternative expression and amplification less significant. This strategy to identify genes more strongly associated means that elevated expression of an amplified gene with genomic amplifications, we used a t-test between cannot alone be considered as strong and sufficient cases with and without amplification. As the whole evidence for being a candidate oncogene. Furthermore, data set was used, genes at genomic locations other the presented data point to an alternative interpretation than the amplified ones, possibly affected by the of genomic amplifications, namely that the target may amplifications, were also identified. The inclusion consist of several genes, and that the spectrum of criterion of at least four cases per evaluated affected genes may vary both among tumor cases group limited the analyses to 8q23–24 and 12p11–12 in and between different tumor types. Taken together, the present data set. The t-test identified one gene the dynamics of altered gene expression due to increased in the commonly amplified 8q region, FLJ14825 gene copies as well as the complex structure of amplicon (Figure 4a), a gene with unknown function that profiles makes the identification of possible target showed increased expression when amplified. Notably, genes based on amplicon mapping, patterns of altered this gene was not identified using the previous strategy. gene expression, and gene annotations a delicate In addition, 97 genes in locations other than 8q matter. showed significantly altered expression; 46 upregulated and 51 downregulated (Figure 4a). The analysis of 12p identified six genes located within the commonly Acknowledgements amplified region (Figure 4b), all of which were also This work was supported by the Swedish Cancer Society, the identified in the previous analysis. Moreover, 33 American Cancer Society, the EC COST Action B19, the upregulated and 32 downregulated genes in other Crafoord Foundation, the John and Augusta Persson Foun- genomic locations were identified (Figure 4b). Hence, dation, the Eric Philip So¨ rensen Foundation, and the Royal these analyses not only complemented the previous Physiographic Society.

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Oncogene