Identification of novel candidate target genes in amplicons of Glioblastoma multiforme tumors detected by expression and CGH microarray profiling

1476-4598-5-39 1476-4598 Research Identification of novel candidate target genes in amplicons of Glioblastoma multiforme tumors detected by expression and CGH microarray profiling Ruano Yolanda yruano@sescam.jccm.es Mollejo Manuela mmollejov@sescam.jccm.es Ribalta Teresa TRIBALTA@clinic.ub.es Fiaño Concepción concepcion.fiano.valverde@sergas.es Camacho I Francisca fcamachoc@sescam.jccm.es Gómez Elena megomez@cnio.es de Lope Rodríguez Angel angelr@sescam.jccm.es Hernández-Moneo Jose-Luis jlhernandez@sescam.org Martínez Pedro pmartinez@sescam.jccm.es Meléndez Bárbara bmelendez@sescam.jccm.es

Genetics Department, Hospital Virgen de la Salud, Avda. Barber 30, 45004-Toledo, Spain

Department of Pathology, Hospital Virgen de la Salud, Avda. Barber 30, 45004-Toledo, Spain

Department of Pathology, Hospital Clinic, Barcelona, C/Villarroel, 170, 08036-Barcelona, Spain

Department of Pathology, Complejo Hospitalario Xeral-Cies, C/Pizarro, 22, 36204-Vigo, Spain

Banco de Tumores, Spanish National Cancer Centre (CNIO), c/Melchor Fernéndez Almagro 6, 28029-Madrid, Spain

Neurosurgery, Hospital Virgen de la Salud, Avda. Barber 30, 45004-Toledo, Spain

Molecular Cancer 1476-4598 2006 5 1 39 http://www.molecular-cancer.com/content/5/1/39 17002787 10.1186/1476-4598-5-39 08 9 2006 26 9 2006 26 9 2006 2006 Ruano et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Background

Conventional cytogenetic and comparative genomic hybridization (CGH) studies in brain malignancies have shown that glioblastoma multiforme (GBM) is characterized by complex structural and numerical alterations. However, the limited resolution of these techniques has precluded the precise identification of detailed specific gene copy number alterations.

Results

We performed a genome-wide survey of gene copy number changes in 20 primary GBMs by CGH on cDNA microarrays. A novel amplicon at 4p15, and previously uncharacterized amplicons at 13q32-34 and 1q32 were detected and are analyzed here. These amplicons contained amplified genes not previously reported. Other amplified regions containg well-known oncogenes in GBMs were also detected at 7p12 (EGFR), 7q21 (CDK6), 4q12 (PDGFRA), and 12q13-15 (MDM2 and CDK4). In order to identify the putative target genes of the amplifications, and to determine the changes in gene expression levels associated with copy number change events, we carried out parallel gene expression profiling analyses using the same cDNA microarrays. We detected overexpression of the novel amplified genes SLA/LP and STIM2 (4p15), and TNFSF13B and COL4A2 (13q32-34). Some of the candidate target genes of amplification (EGFR, CDK6, MDM2, CDK4, and TNFSF13B) were tested in an independent set of 111 primary GBMs by using FISH and immunohistological assays. The novel candidate 13q-amplification target TNFSF13B was amplified in 8% of the tumors, and showed protein expression in 20% of the GBMs.

Conclusion

This high-resolution analysis allowed us to propose novel candidate target genes such as STIM2 at 4p15, and TNFSF13B or COL4A2 at 13q32-34 that could potentially contribute to the pathogenesis of these tumors and which would require futher investigations. We showed that overexpression of the amplified genes could be attributable to gene dosage and speculate that deregulation of those genes could be important in the development and progression of GBM. Our findings highlight the important influence in GBM of signaling pathways such as the PI3K/AKT, consistent with the invasive features of this tumor.

Background

Glioblastoma multiforme (GBM) is the commonest and most malignant of the primary central nervous system tumors in the human adult. Mean survival of GBM patients treated with the current standard therapy is approximately one year 1.

Glioblastomas, like other solid tumors, are characterized by changes in the expression of oncogenes and tumor suppressor genes, often as a consequence of numerical chromosomal abnormalities (genomic amplifications, gains, and losses) that occur during the tumoral process. Conventional and molecular cytogenetic techniques, such as comparative genomic hybridization (CGH), have led to the identification of recurrent genomic copy number changes that play an important role in the malignancy of GBM. Aberrations that occur with high frequency include gains of chromosomes 7, 19, and 20, and losses of chromosomes 6q, 9p, 10, 13q, and 14q 23. Nevertheless, the low resolution of these techniques, which is restricted to the chromosome level, together with the large number of genes located within these regions, makes difficult the identification of candidate genes.

High-level DNA copy number changes in tumors are restricted to chromosome regions that show more than 5- to 10-fold copy number increases (regions of amplification, or amplicons). Some of these amplicons contain well-known oncogenes that are also overexpressed. While this is the case for oncogenes associated with the development of GBM, such as Epidermal Growth Factor Receptor (EGFR) (7p12), Cyclin-Dependent Kinase 4 (CDK4) (12q14), and the human homolog of the Mouse Double Minute 2 (MDM2) (12q15) 234, other regions of amplification and/or other relevant genes located within these or other regions remain unknown or incompletely described.

New high-throughput genomic technologies, such as cDNA microarray CGH 5, use conventional cDNA microarrays that are normally used in expression profiling, to examine genomic copy number imbalances. In this way, thousands of genes can be reviewed in a high-resolution analysis to define amplicons and identify candidate genes showing recurrent genomic copy number changes. Parallel expression profiling experiments then allows the identification of relevant target genes whose aberrant expression could suggest its involvement in the pathogenesis of the tumors 678910.

The objective of our study was to define at high resolution regions of amplification in GBMs, and through integration of copy number and gene expression data, to identify possible candidate target genes that could give insights into the pathology of GBM. In addition, we also aimed to analyze in detail the gene copy number changes associated with these tumors, since this is not feasible using classic chromosomal CGH.

For that purpose, we surveyed for changes in DNA copy number and expression levels throughout the genomes of 20 primary GBMs by using cDNA microarray CGH and expression profiling experiments. The most significant alterations found were validated in additional series of primary GBMs using locus-specific fluorescence in situ hybridization (FISH), and immunohistochemical analyses.

Methods

Patients and tissue samples

The genomic-profiling study involved 20 cases of primary GBMs. Expression profiling experiments were carried out in 17 of the GBMs for which RNA material was available. Thirteen of the patients were males and 7 were females with a mean age of 61 years (range, 39 to 81 years). The clinical information of the patients is summarized in Table 1.

Table 1

Clinical and molecular data of the patients.

Case

Sex

Age

Site

Status

7p EGFR

4p/4q

12q CDK4

12q MDM2

13q

-10q

C29

C34

P-O

C35

C35b

C36

T-P

C38b

T-P

C42

T-P-TA

C47

C33

C26

F-T-P

C36b

C48

C28

C39b

C32

T-P

C30

T-P

C31

C37b

C43

P-O

C46

Clinical data: sex (M male, F female), tumor site (T temporal, P parietal, O occipital, F frontal and T thalamus), overall survival (OS) in months, and status (D death, A Alive).

Molecular data: (A) amplification, (L) loss.

To validate our results, 111 primary GBMs were arrayed in tissue microarrays (TMAs) and subjected to immunohistochemical and FISH analyses. Samples were collected from Virgen de la Salud Hospital (Toledo, Spain), Clinic Hospital (Barcelona, Spain) and Xeral-Cies Hospital (Vigo, Spain). All samples were reviewed by means of tissue sections stained with hematoxylin and eosin (H&E) to verify tumor viability and confirm the diagnosis according to the WHO guidelines by M.M., T.R., C.F., and F.-I.C.

cDNA microarray CGH and expression profiling

The cDNA microarrays used in this study (Oncochip v2.0) were purchased from Centro Nacional de Investigaciones Oncológicas 11. These microarrays contained 27,454 cDNA clones, including 9,900 known genes and uncharacterized ESTs related to tumorigenesis.

High molecular weight genomic DNA from tumors and normal human lymphocytes (used as reference DNA) were extracted following standard phenol/chloroform purification protocols. CGH experiments on cDNA microarrays were performed as described 51012. Briefly, 20 μg of genomic tumoral and reference DNAs were digested for 14–18 hours with AluI and RsaI (Life Technologies, Inc., Rockville, MD) and purified by phenol-chloroform extraction. Six μg of purified digested tumor DNA and reference DNA were labeled with Cy5-dUTP and Cy3-dUTP (Amersham Biosciences, Piscataway, NJ), respectively, using the Bioprime Labeling Kit (Life Technologies, Inc.). Labeled tumor and sex-matched reference DNA were co-hybridized at 58°C for 14–16 hours. Post-hybridization washes were carried out in 2×S SC/0.03% SDS at hybridization temperature, 1× SSC and 0.2× SSC at room temperature for 5 minutes each.

For expression profiling experiments total RNAs were extracted with Tri Reagent (Molecular Research Center, Cincinnati, OH) and amplified using a T7-based method, as previously described (11). Five μgr of total RNA were used to produce double-stranded cDNA (Superscript Choice System, Life technologies, Inc.) and amplification of mRNAs was performed using the Megascript T7 in vitro transcription kit (Ambion, Austin, TX). A pool of aRNAs obtained from the Universal Human RNA (Stratagene, La Jolla, CA) was used as a standard reference in all hybridizations. Test or reference amplified RNAs (aRNAs) were labeled with fluorescent Cy5 and Cy3, respectively, and hybridized at 42°C for 15 hours. Two control RNAs obtained from non-tumoral brain (one of them from Stratagene) were used for normalization purposes.

After hybridizations, slides were scanned using an Axon GenePix 4100A confocal scanner. Image analysis was performed using GenePix Pro 6.0 software (Axon Instruments Inc., Union City, CA). Cy5/Cy3 fluorescence ratios were normalized for each microarray using the print-tip loess method and background subtraction with the Diagnosis and Normalization Array Data (DNMAD) tool 13.

Microarray data analysis

Data were preprocessed using Gene Expression Preprocessing Analysis Suite (GEPAS) 14 [see Additional file 1]. Cut-off points for defining gains and losses of genetic material in the test hybridizations were established as reported before 710. The mean log₂-transformed ratios derived from the self versus self experiments of normal genomic DNA in control hybridizations allowed us to establish the cut-off points for defining gains and losses. A value of the mean ratio +/- two standard deviations showed a normal range of variation corresponding to log₂-transformed values of -0.42 to 0.42. In order to ensure a fluorescence ratio of gain or loss, we considered gene gain to be when log₂ratios were ≥ 0.5, and gene loss to be when log₂ratios were ≤-0.5. Log₂fluorescent ratios ≥ 2 were considered to represent gene amplification.

Additional file 1

Genomic and expression row data. Genomic and expression row data of the GBM tumors. Gene symbol, gene description and localization are provided.

Click here for file

Fluorescence in situ hybridization and immunohistochemistry on TMAs

TMAs were constructed using formalin-fixed paraffin-embedded archival tissue blocks as reported 15. Five non-tumoral controls (4 normal brain and one tonsil tissue samples) were included. H&E-stained full sections from each donor block were used for morphological selections of the representative areas of each case.

FISH assays were performed as described previously 15 using gene-specific and control BAC clones selected from the EnsEMBL 16 and UCSC 17 databases (Table 2). Gene probes were labeled with SpectrumOrange-dUTP (red) and control probes with SpectrumGreen-dUTP (green) using the CGH Nick Translation Kit (Vysis). Hybridizations were done overnight at 37°C after deparaffinization of 4-μm-thick sections of the TMAs, target retrieval by pressure-cooking with 1 mM EDTA for 10 minutes, and pepsin digestion (4 mg/ml at 37°C for 30 minutes). After post-hybridization washes, tissue was counterstained with DAPI in antifade solution (Oncor, Gaithersburg, MD).

Table 2

Clones used for validation by FISH.

Gene name

Gene-specific-BAC clones

Location

Control clones

Location

EGFR

RP11-339F13

7p12

p275α7^a

7 cen

CDK4

RP11-571M6

12q13-q14

RP11-467M14

12p13

MDM2

RP11-611O2, RP11-450G15

12q15

RP11-467M14

12p13

ESTs on 13q

RP11-94P17, RP11-406G20

13q32.2

RP11-364F8, RP11-151A6, RP11-113J24

13q33.3

^aplasmid probe located at the centromere (cen) of chromosome 7. The specificity and location of the probes were confirmed by FISH on normal metaphases before hybridization on the TMAs. When clones were available, and in order to increase the signal, two or three BAC clones around the test gene or EST were hybridized together. Control FISH experiments on normal interphase nuclei were performed beforehand in order to ensure a unique signal when two BAC clones were hybridized together. BAC clones on 13q were purchased from the BACPAC Resources Center (Children's Hospital Oakland Research Institute, CHORI). All other clones were generously provided by Dr. Rocchi.

Fluorescence signals were scored (Y.R., B.M.) in accordance with previous reports 18. In each sample, only well-defined nuclei were analyzed, and the numbers of single-copy gene and control probe signals were scored. Tumors were considered as amplified when five or more unbalanced gene copies, or more than three times as many gene signals as control signals were found in more than 5% of tumor cells.

Immunophenotypic analysis was performed on deparaffinized TMA sections. For antigen retrieval, a heating step in a solution of 10 mM sodium citrate buffer at pH:6 in a pressure cooker was included before incubation with antibodies. Tissues were immunohistochemically stained by the Labeled Streptavidin Biotin (LSAB) (DAKO, Glostrup, Denmark), or alkaline phosphatase-conjugated EnVision (DAKO) method, using the TechMate 500 (DAKO) automatic immunostaining device. The primary antibodies used were EGFR, MDM2 (Oncogene Research Products, Boston, MA) and TNFSF13B (BAFF or BlySS). Tumors were considered positive when membranous (EGFR), nuclear (MDM2), and membrane-bound or cytoplasmic (TNFSF13B) staining was observed in ≥ 5% of the tumor cells.

Results

Impact of copy number alteration on gene expression

The gene frequencies of gain and loss of genetic material found in the 20 primary GBMs were calculated, and plotted relative to the position along the chromosome (Figure 1). Chromosomes 7, 19, and 20 most frequently showed gains in copy number, while chromosomes 10 and 13 most frequently had losses (Table 3).

Figure 1

Frequency of copy number change

Frequency of copy number change. (A) Frequency of gene amplification (black), gain (red), and loss (green) in the 20 GBMs relative to their map position in the EnsEMBL database (calculated as the number of GBMs showing amplified, gained, or lost genes vs. the number of tumors analyzed for each gene). The white vertical bars represent the separation between chromosomes. Only the most representative chromosomes are shown. Map positions for each cDNA clone were obtained from the EnsEMBL database using the IDconverter tool [45].

Table 3

Frequent copy number and expression altered genes¹.

GAINED AND OVEREXPRESSED GENES

SYMBOL

GENE DESCRIPTION

MEDIAN

LOCATION

ATP2B4

ATPase, Ca++ transporting, plasma membrane 4

20.00

1.17

1q25-q32

LOC153222

Adult retina protein

25.00

1.32

GBAS

Glioblastoma amplified sequence

20.00

1.61

7p12

GUSB

Glucuronidase, beta

20.00

1.42

7q21.11

RFC2

Replication factor C (activator 1) 2, 40kDa

26.32

1.06

7q11.23

MCM7

MCM7 minichromosome maintenance deficient 7 (S. cerevisiae)

35.29

1.02

7q21.3-q22.1

GIMAP6

GTPase, IMAP family member 6

20.00

1.29

LR8

LR8 protein

21.05

2.34

7q36.1

APOE

Apolipoprotein E

22.22

1.48

19q13.2

EMP3

Epithelial membrane protein 3

33.33

1.36

19q13.3

FPR1

Formyl peptide receptor 1

25.00

1.94

19q13.4

LOST AND UNDEREXPRESSED GENES

SYMBOL

GENE DESCRIPTION

MEDIAN

LOCATION

CYP1B1

Cytochrome P450, family 1, subfamily B, polypeptide 1

21.05

-2.87

2p21

TDE2

Tumor differentially expressed 2

23.53

-1.86

6q22.32

OPTN

Optineurin

41.18

-1.86

10p14

DNAJC12

DnaJ (Hsp40) homolog, subfamily C, member 12

25.00

-1.16

SIRT1

Sirtuin (silent mating type information regulation 2 homolog) 1

27.27

-1.27

10q22.1

PPP3CB

Protein phosphatase 3 (formerly 2B), catalytic subunit, beta isoform

35.00

-2.08

10q21-q22

GHITM

Growth hormone inducible transmembrane protein

37.50

-1.55

10q23.2

CYP26A1

Cytochrome P450, family 26, subfamily A, polypeptide 1

21.05

-1.08

10q23-q24

LGI1

Leucine-rich, glioma inactivated 1

45.00

-1.32

10q24

SLK

STE20-like kinase (yeast)

26.32

-1.11

10q25.1

ADD3

Adducin 3 (gamma)

68.42

-1.04

10q24.2-q24.3

MXI1

MAX interactor 1

40.00

-1.11

10q24-q25

ATRNL1

Attractin-like 1

30.77

-1.65

KIAA1598

21.05

-1.83

10q26.12

NDFIP2

Nedd4 family interacting protein 2

33.33

-1.22

13q22.2

MAP2K4

Mitogen-activated protein kinase kinase 4

23.53

-1.30

17p11.2

DSG2

Desmoglein 2

31.25

-1.94

18q12.1

TPTE

Transmembrane phosphatase with tensin homology

22.22

-1.67

21p11

¹Percentage of tumors showing copy number and expression alterations (%). Median of expression log₂ratios of copy number altered genes (median).

The global effect of copy number alterations on gene expression was evaluated in 17 of the primary tumors. Up to 33% of gained genes (>2.5-fold change in copy number), or up to 56% with >4-fold, were overexpressed. Nevertheless, 8% of the genes with normal copy numbers were overexpressed (Figure 2A). Conversely, approximately 8% of the transcripts with high-level expression (>10-fold) showed amplification (Figure 2B).

Figure 2

Impact of gene copy number on global gene expression levels

Impact of gene copy number on global gene expression levels. (A) Percentage of over- or underexpressed genes (Y axis) according to copy number ratios (X axis). (B) Percentage of amplified and deleted genes according to expression ratios.

Gene amplifications in primary GBMs

Amplicons were identified on chromosomes 1, 4, 7, 12, and 13 due to the presence of more than four contiguous amplified genes. EGFR (7p12) was the most commonly amplified gene (7p12), showing amplification by microarray CGH in 50% (10/20) of the GBMs (Table 1).

The amplicon detected on chromosome 13 in tumor 39 (Figure 3A) contained contiguous amplified clones covering a region of about 2.5 Mb at 13q32-34 that included TNFSF13B (a ligand of the tumor necrosis factor superfamily), and collagen type IV genes (COL4A1 and COL4A2). Among the amplified genes or ESTs, the most important fold-changes in expression levels were those of TNFSF13B, COL4A2 and FLJ10769. In addition, two other tumors had centromeric-amplified clones including ESTs AA706834, AA994053, and AI093016. However, these ESTs were not overexpressed.

Figure 3

Amplification at 13q

Amplification at 13q. (A) DNA copy number and expression log₂ratios (black and red dots, respectively) of tumor 39b plotted according to the map position. Moving average of log₂-genomic ratios over five neighboring genes are plotted and shown with a grey line. In the table below, amplified and overexpressed genes (in bold), with their corresponding log₂-ratios, are detailed. Lost and amplified regions are indicated by green and red bars, respectively, under the graph. The amplified region at 13q32-34 is indicated by a grey square. (B) Amplification at 13q observed in one of the tumors of the TMA by using BAC probes located at 13q32.3 (labeled in red) and 13q33.2 (labeled in green). (C) Amplification on 13q32.3 in another GBM (BAC probes labeled in red). Amplified cells are indicated by arrows. (D) Photomicrograph of tumour tissue with positive expression for TNFSF13B showing cytoplasmic pattern (original magnification ×1000).

To determine the frequency of 13q amplification in a larger series of tumors, we carried out FISH assays in an independent set of 111 GBM samples. We found 8,5% of tumors (6/70) showing amplification (Figure 3B, 3C). In this same series, we examined the protein expression of TNFSF13B as one of the putative target genes of the 13q amplification. We detected TNFSF13B immunostaining positivity in 20,6% of the samples (20/97). Half of the 13q-amplified tumors (3/6) showed TNFSF13B positive expression, and 7 out of 59 (11,8%) non 13q-amplified tumors showed TNFSF13B positivity (Figure 3D).

Amplicons were also detected on 1q, 4p, 4q, 7q, and 12q. Chromosome 4 had two separated regions of amplification (Figure 4A), one of about 6 Mb at 4p15, and the other of approximately 2.2 Mb at 4q12. The 4p15 amplicon is described here for the first time and contained several genes and ESTs not previously reported as being amplified in GBMs. The amplicon at 4q12 contained PDGFRA, whose amplification in GBMs is already well known. Among the amplified genes, SLA/LP, LOC389203, STIM2, SGCB, RASL11B, and PDGFRA seem to respond to gene dosage, presenting high fold-changes in expression levels (Figure 4A). Likewise, amplification of several contiguous clones on chromosome 1q32 included ATP2B4, KIAA0663, KISS1, PPP1R15B, PIK3C2B, and MDM4. Among them, ATP2B4, KIAA0663, PPP1R15B, PIK3C2B were overexpressed in the expression analyses (Figure 4B).

Figure 4

Amplification at chromosomes 4 and 1

Amplification at chromosomes 4 and 1. (A) DNA copy number ratios and expression log₂ratios (black and red dots, respectively) plotted with respect to their map position obtained for chromosome 4 of tumor 39b, and (B) for chromosome 1 of tumor 35b. Average log₂genomic values over five neighboring genes are shown with a grey line as a function of the location of the clones. Described in the corresponding tables below each graph are the amplified and overexpressed (in bold) genes together with their corresponding copy number and expression log2 values.

Chromosome 12 showed two regions of amplification at 12q13-15 (Table 4), one of about 180 Kb at 12q13 containing CDK4 (10%, 2/19) and another one of about 700 Kb containing MDM2 (15%, 3/20) at 12q14.3-q15. These results were validated by FISH analyses with specific probes containing CDK4 or MDM2 and immunohistochemical analyses for MDM2 onto paraffin sections of the samples in a set of 111 primary GBMs, allowing confirmation of the microarray results (Table 1). We found alterations (either amplification or overexpression) of MDM2 and CDK4 in 11% of the tumors for which data were available (11/98 and 7/66, respectively). Except for one tumor, the two regions were not amplified simultaneously. These results suggest that MDM2 and CDK4 may be independently amplified in most GBM tumors and confirm those of other authors 19. Finally, amplification at 7q included PEX1, and CDK6 (7q21) as overexpressed putative amplification targets (Table 5).

Table 4

Amplicon at 12q.

SYMBOL

GENE NAME

POSITION

MEDIAN* GENOMIC

MEDIAN* EXPRESSION

AMPLICON 1

SLC26A10

Solute carrier family 26, member 10

56290368

0.49

0.44

GALGT

UDP-N-acetyl-alpha-D-galactosamine

56305945

1.73

0.33

SAS

Sarcoma amplified sequence

56425051

0.49

2.92

CDK4

Cyclin-dependent kinase 4

56428272

2.44

4.12

CYP27B1

Cytochrome P450, family 27, subfamily B, polypeptide 1

56442389

2.35

1.93

TSFM

Ts translation elongation factor, mitochondrial

56462849

1.02

-0.26

AVIL

Advillin

56477704

1.84

0.53

AMPLICON 2

GNS

Glucosamine (N-acetyl)-6-sulfatase

63393491

0.48

0.47

CGI-119

CGI-119 protein

64817459

0.94

5.38

IFNG

Interferon, gamma

66834816

0.76

-0.02

MDM2

Mdm2, transformed 3T3 cell double minute 2

67488247

2.72

1.64

* Median of genomic or expression log₂ratios of gained genes.

Table 5

Amplicon at 7q.

SYMBOL

GENE DESCRIPTION

POSITION

GENOMIC

EXPRESSION

PFTK1

PFTAIRE protein kinase 1

89870462

1.68

-0.58

FZD1

Frizzled homolog 1 (Drosophila)

90538434

3.05

2.91

PEX1

Peroxisome biogenesis factor 1

91760991

3.13

4.04

CDK6

Cyclin-dependent kinase 6

91878888

1.53

0.43

Genomic and expression log₂ratios of gained genes.

Discussion

The overall impact of copy number on gene expression analyzed in GBMs reflects the importance of recurrent gene copy number changes in the development and progression of these brain tumors. Our results in GBMs extend previous studies in breast and prostate cancers 102021, and confirm that the effects of gene copy number on expression levels were more relevant for high-level amplifications on a gene-by-gene analysis (56% of highly-gained genes were overexpressed).

Gene amplification is regarded to reflect genetic instabilities in solid tumor cells 22. It has been proposed that activation of proto-oncogenes by amplification plays an important role in the development of many human solid tumors. Therefore, detection of specific gene amplifications in tumor cells can lead to the identification of genes putatively involved in growth control and tumorigenesis.

While only a few candidate genes could be investigated at a time in previous studies 42324 we have used cDNA microarray technology to search the whole genome for gene copy number alterations in GBMs. Our results from the approximately 10,000 genes and ESTs related to the tumoral process that could be analyzed in this study, confirm the previous conclusion that EGFR is the principal oncogene amplified in GBMs. However, in this study several other chromosomal regions showing gene amplification have also been detected in GBMs at 1q, 4p, 4q, 7q, 12q, and 13q. Amplification at 13q32-34 has been reported before from chromosomal CGH studies in malignant gliomas, cell lung carcinoma, head and neck squamous carcinoma, and systemic lymphomas 202526272829. Here we have used microarray CGH to study this region at higher resolution (gene level) and have narrowed down the region to 2.5 Mb, showing that this amplification could affect a small fraction of GBMs (8%). Several known genes and uncharacterized ESTs are contained in the 13q amplicon. Among these, COL4A2 has also been found to be overexpressed in our study and in other microarray expression-profiling studies of GBM biopsies and GBM cell lines 303132. We speculate that this overexpression may have been caused by amplification of COL4A2, which may be a putative target of the amplicon. In agreement with this hypothesis, very recently, Tso and coworkers 33 have shown that COL4A2 is one of 15 highly expressed genes that is shared between primary and secondary GBMs. In addition, this gene was found to be involved in glioma progression and associated with vascular proliferation. However, immunohistochemical analyses revealed that collagen IV is mainly expressed in association with the tumor blood vessels and not from the tumor cells 32, suggesting that the COL4A2 overexpression detected in GBMs may not be as a result of gene amplification in tumoral cells. This may point out to TNFSF13B as a likely candidate for the target of amplification. Immunohistochemical analyses of 111 primary GBMs revealed that TNFSF13B could be affected in about 20% of the tumors. The TNFSF13B ligand (or BAFF) is a member of the TNF cytokine family that activates nuclear factor (NF)-κB, phosphatidylinositol-3 kinase (PI3K)/AKT, and mitogen-activated protein kinase (MAPK) pathways in myeloma multiple cells, and induce strong up-regulation of Mcl-1 and Bcl-2 antiapoptotic proteins 34.

On another hand, the gene IRS2 (which codes a cytoplasmic adaptor protein that facilitates intracellular signal transduction) is also located within the amplified region close to COL4A2 and TNFSF13B. Although we have no data available for this gene, one study suggests that IRS2 is a novel but rare amplification target at 13q34 in GBMs 35. Thus, we propose here that genes such as COL4A2 or TNFSF13B could be additional putative targets for the 13q amplicon, and therefore would warrant further detailed analyses in GBMs.

It is of particular note that two of the tumors showing this amplification in the telomeric region of chromosome 13 had loss of the rest of the chromosome (Figure 3A), as was also described by Weber and coworkers in the cases in which amplification was revealed by chromosomal CGH 36. This finding is consistent with the finding that gene amplification may be accompanied by loss of genetic material in the proximity of the amplification site 37.

Chromosome 4 has two amplicons, located close together. One contains PDGFRA as the putative target of the amplicon, and the other contains SLA/LP, STIM2, and two ESTs. To our knowledge, this is the first report of amplification of SLA/LP and STIM2 genes, which are both overexpressed. However, further analyses are required to identify the amplification gene target(s) and to determine the relevance of this novel amplification in GBM. STIM2 codes for a transmembrane phosphoprotein whose structure is unrelated to that of any other known protein 38 and whose biological function has not been thoroughly studied. STIM1 is the other member of this family of proteins and it is thought to regulate cell growth control and function within a signaling cascade, although the precise pathway is not known 38.

There is controversy concerning the amplification on chromosome 1, as to whether 1q32 has two independent amplification targets or a single one affecting both MDM4 and CNTN2 genes 394041. We observed amplification covering a small region of about 800 Kb that excluded CNTN2. Thus, our results support the proposal of Riemenschneider and coworkers that MDM4 is the main amplification target gene at 1q32 40. However, other genes among those contained in the amplicon, such as PIK3C2B, which encodes a catalytic subunit of the PI3K, could be of importance in GBMs. This gene was also found amplified and overexpressed in GBMs by others 3241 and has a crucial role in the PI3K/AKT signaling pathway, which regulates a number of cellular processes such as cell growth and proliferation, apoptosis, migration and invasion, and angiogenesis 39. Thus, our study excludes CNTN2 and shows that other interesting candidate genes together with MDM4 may be important gene targets for this 1q32 amplification.

Chromosome 7 was the most frequently gained chromosome in GBMs, as already known. Our results showed a large number of overexpressed gained-chromosome 7 genes which may suggest the importance of the complete gain of this chromosome in primary GBMs. Further studies, however, should be carried out to assess the importance of chromosome 7-candidate genes, other than EGFR, in the pathogenesis of GBM. Likewise, chromosome 10 was the most frequently lost chromosome in GBMs. The most frequently lost and underexpressed genes mapped at 10q and involved candidate genes in gliomas such as ADD3, between others. Downregulation of ADD3 expression was associated with increased migratory activity of human glioma cells in vitro 42, and decreased expression of ADD3 has been described in astrocytomas 43. Chromosome 19 also showed frequent gene gains, mainly located on 19q. One of the possible candidates located in this chromosome could be FPR1 due to this gene is expressed in malignant glioma and appears to mediate motility, growth, and angiogenesis of GBM 44.

Conclusion

In summary, our results show that the cDNA microarray CGH technique in parallel with expression profiling allows the comprehensive, rapid and reliable analysis of the whole genome in GBM tumors and enables the refined and detailed study of amplicons and regions of recurrent copy number change. This approach makes it possible to identify putative glioma oncogenes/tumor-suppressor genes that may deserve further investigation. Our findings highlight the important influence in GBM of signaling pathways such as the PI3K/AKT, consistent with the invasive features of this tumor. In this context, we identify candidate target genes of amplification that may help to direct therapeutics for the treatment of GBM.

Authors' contributions

YR carried out selection of the GBM samples, performed nucleic acid extraction, DNA and RNA hybridizations onto the microarrays, FISH experiments, and participated in the discussion of the results. MM, TR, CF, and FIC carried out histopathological analyses and pathological diagnosis. MM and FIC participated in the design of the study, and in the analysis and discussion of the results. EG participated in selection of the samples, DNA and RNA extraction, and carried out construction of the tissue arrays. AR-L and J-LH-M recovered clinical data of the patients and carried out clinical diagnostics. PM participated in the design and discussion of the results BM carried out statistical analysis of the microarray results, conceived of the study, drafted the manuscript, participated in its design, and carried out coordination. All authors read and approved the final manuscript.

Acknowledgements

We would like to acknowledge the assistance of Lydia Sanchez (CNIO) with the immunohistochemical studies. Some of the BAC clones were generously provided by Dr. Mariano Rocchi (University of Bari, Italy).

This work was partially supported by grants from the Fondo de Investigaciones Sanitarias FIS 02/3006 and FIS 03/0727, and from the Consejería de Sanidad Junta de Comunidades de Castilla-La Mancha SESCAM GC3011 and SESCAM 04032.

Impact of morphology and biology on the prognosis of patients with gliomas Gudinaviciene I Pranys D Juozaityte E Medicina (Kaunas) 2004 40 112 120 15007269 Genetic aberrations defined by comparative genomic hybridization distinguish long-term from typical survivors of glioblastoma Burton EC Lamborn KR Feuerstein BG Prados M Scott J Forsyth P Passe S Jenkins RB Aldape KD Cancer Res 2002 62 6205 6210 12414648 Chromosomal abnormalities in human glioblastomas gain in chromosome 7p correlating with loss in chromosome 10q Inda MM Fan X Munoz J Perot C Fauvet D Danglot G Palacio A Madero P Zazpe I Portillo E Tunon T Martinez-Penuela JM Alfaro J Eiras J Bernheim A Castresana JS Mol Carcinog 2003 36 6 14 10.1002/mc.10085 12503074 Detection of multiple gene amplifications in glioblastoma multiforme using array-based comparative genomic hybridization Hui AB Lo KW Yin XL Poon WS Ng HK Lab Invest 2001 81 717 723 11351043 Genome-wide analysis of DNA copy-number changes using cDNA microarrays Pollack JR Perou CM Alizadeh AA Eisen MB Pergamenschikov A Williams CF Jeffrey SS Botstein D Brown PO Nat Genet 1999 23 41 46 10.1038/14385 10471496 Chromosomal localization of DNA amplifications in neuroblastoma tumors using cDNA microarray comparative genomic hybridization Beheshti B Braude I Marrano P Thorner P Zielenska M Squire JA Neoplasia 2003 5 53 62 12659670 Genomic profiles in stage I primary non small cell lung cancer using comparative genomic hybridization analysis of cDNA microarrays Jiang F Yin Z Caraway NP Li R Katz RL Neoplasia 2004 6 623 635 10.1593/neo.04142 15548372 High-resolution genomic and expression profiling reveals 105 putative amplification target genes in pancreatic cancer Mahlamaki EH Kauraniemi P Monni O Wolf M Hautaniemi S Kallioniemi A Neoplasia 2004 6 432 439 10.1593/neo.04130 15548351 Microarray analysis reveals a major direct role of DNA copy number alteration in the transcriptional program of human breast tumors Pollack JR Sorlie T Perou CM Rees CA Jeffrey SS Lonning PE Tibshirani R Botstein D Borresen-Dale AL Brown PO Proc Natl Acad Sci USA 2002 99 12963 12968 130569 12297621 10.1073/pnas.162471999 High-resolution analysis of gene copy number alterations in human prostate cancer using CGH on cDNA micro arrays, impact of copy number on gene expression Wolf M Mousses S Hautaniemi S Karhu R Huusko P Allinen M Elkahloun A Monni O Chen Y Kallioniemi A Kallioniemi OP Neoplasia 2004 6 240 7 10.1593/neo.03439 15153336 Identification of genes involved in resistance to interferon-alpha in cutaneous T-cell lymphoma Tracey L Villuendas R Ortiz P Dopazo A Spiteri I Lombardia L Rodriguez-Peralto JL Fernandez-Herrera J Hernandez A Fraga J Dominguez O Herrero J Alonso MA Dopazo J Piris MA Am J Pathol 2002 161 1825 1837 12414529 Comprehensive copy number and gene expression profiling of the 17q23 amplicon in human breast cancer Monni O Barlund M Mousses S Kononen J Sauter G Heiskanen M Paavola P Avela K Chen Y Bittner ML Kallioniemi A Proc Natl Acad Sci USA 2001 98 5711 5716 33278 11331760 10.1073/pnas.091582298 DNMAD, web-based diagnosis and normalization for microarray data Vaquerizas JM Dopazo J Diaz-Uriarte R Bioinformatics 2004 20 3656 3658 10.1093/bioinformatics/bth401 15247094 Gene expression data preprocessing Herrero J Diaz-Uriarte R Dopazo J Bioinformatics 2003 19 655 656 10.1093/bioinformatics/btg040 12651726 Immunohistochemical characteristics defined by tissue microarray of hereditary breast cancer not attributable to BRCA1 or BRCA2 mutations, differences from breast carcinomas arising in BRCA1 and BRCA2 mutation carriers Palacios J Honrado E Osorio A Cazorla A Sarrio D Barroso A Rodríguez S Cigudosa JC Diez O Alonso C Lerma E Sanchez L Rivas C Benitez J Clin Cancer Res 2003 9 3606 3614 14506147 The Ensembl database http://www.ensembl.org The UCSC Genome Bioinformatics Site http://genome.ucsc.edu Tissue microarrays for gene amplification surveys in many different tumor types Schraml P Kononen J Bubendorf L Moch H Bissig H Nocito A Mihatsch MJ Kallioniemi OP Sauter G Clin Cancer Res 1999 5 1966 1975 10473073 Refined mapping of 12q13-q15 amplicons in human malignant gliomas suggests CDK4/SAS and MDM2 as independent amplification targets Reifenberger G Ichimura K Reifenberger J Elkahloun AG Meltzer PS Collins VP Cancer Res 1996 56 5141 5145 8912848 Microarray analysis reveals a major direct role of DNA copy number alteration in the transcriptional program of human breast tumors Pollack JR Sorlie T Perou CM Rees CA Jeffrey SS Lonning PE Tibshirani R Botstein D Borresen-Dale AL Brown PO Proc Natl Acad Sci U S A 2002 99 12963 12968 130569 12297621 10.1073/pnas.162471999 Impact of DNA amplification on gene expressionpatterns in breast cancer Hyman E Kauraniemi P Hautaniemi S Wolf M Mousses S Rozenblum E Ringner M Sauter G Monni O Elkahloun A Kallioniemi OP Kallioniemi A Cancer Res 2002 62 6240 6245 12414653 Oncogene amplification in solid tumors Schwab M Semin Cancer Biol 1999 9 319 325 10.1006/scbi.1999.0126 10448118 Gene amplification as a prognostic factor in primary and secondary high-grade malignant gliomas Galanis E Buckner J Kimmel D Jenkins R Alderete B O'Fallon J Wang CH Scheithauer BW James CD Int J Oncol 1998 13 4 717 724 9735401 Gene amplification as a prognostic factor in primary brain tumors Olson JJ Barnett D Yang J Assietti R Cotsonis G James CD Clin Cancer Res 1998 4 215 222 9516974 Molecular cytogenetic characterization of non-Hodgkin lymphoma cell lines Mehra S Messner H Minden M Chaganti RS Genes Chromosomes Cancer 2002 33 225 234 10.1002/gcc.10025 11807979 Localisation of a novel region of recurrent amplification in follicular lymphoma to an approximately 6.8 Mb region of 13q32-33 Neat MJ Foot N Jenner M Goff L Ashcroft K Burford D Dunham A Norton A Lister TA Fitzgibbon J Genes Chromosomes Cancer 2001 32 236 243 10.1002/gcc.1187 11579463 Chromosomal and gene amplification in diffuse large B-cell lymphoma Rao PH Houldsworth J Dyomina K Parsa NZ Cigudosa JC Louie DC Popplewell L Offit K Jhanwar SC Chaganti RS Blood 1998 92 234 240 9639522 Mapping of multiple DNA gains and losses in primary small cell lung carcinomas by comparative genomic hybridization Ried T Petersen I Holtgreve-Grez H Speicher MR Schrock E du Manoir S Cremer T Cancer Res 1994 54 1801 1806 8137295 Comparative genomic hybridization detects novel deletions and amplifications in head and neck squamous cell carcinomas Speicher MR Howe C Crotty P du Manoir S Costa J Ward DC Cancer Res 1995 55 1010 1013 7866983 Integrated array-comparative genomic hybridization and expression array profiles identify clinically relevant molecular subtypes of glioblastoma Nigro JM Misra A Zhang L Smirnov I Colman H Griffin C Ozburn N Chen M Pan E Koul D Yung WK Feuerstein BG Aldape KD Cancer Res 2005 65 1678 1686 10.1158/0008-5472.CAN-04-2921 15753362 Systematic variation in gene expression patterns in human cancer cell lines Ross DT Scherf U Eisen MB Perou CM Rees C Spellman P Iyer V Jeffrey SS Van de Rijn M Waltham M Pergamenschikov A Lee JC Lashkari D Shalon D Myers TG Weinstein JN Botstein D Brown PO Nat Genet 2000 24 3 227 235 10.1038/73432 10700174 Characterization of gene expression profiles associated with glioma progression using oligonucleotide-based microarray analysis and real-time reverse transcription-polymerase chain reaction van den Boom J Wolter M Kuick R Misek DE Youkilis AS Wechsler DS Sommer C Reifenberger G Hanash SM Am J Pathol 2003 163 1033 1043 12937144 Distinct transcription profiles of primary and secondary glioblastoma subgroups Tso CL Freije WA Day A Chen Z Merriman B Perlina A Lee Y Dia EQ Yoshimoto K Mischel PS Liau LM Cloughesy TF Nelson SF Cancer Res 2006 66 159 167 10.1158/0008-5472.CAN-05-0077 16397228 BAFF and APRIL protect myeloma cells from apoptosis induced by interleukin 6 deprivation and dexamethasone Moreaux J Legouffe E Jourdan E Quittet P Reme T Lugagne C Moine P Rossi JF Klein B Tarte K Blood 2004 103 3148 3157 10.1182/blood-2003-06-1984 15070697 Genetic alterations and aberrant expression of genes related to the phosphatidyl-inositol-3'-kinase/protein kinase B (Akt) signal transduction pathway in glioblastomas Knobbe CB Reifenberger G Brain Pathol 2003 13 507 518 14655756 Characterization of genomic alterations associated with glioma progression by comparative genomic hybridization Weber RG Sabel M Reifenberger J Sommer C Oberstrass J Reifenberger G Kiessling M Cremer T Oncogene 1996 13 5 983 94 8806688 Amplification at 12q13-14 in human malignant gliomas is frequently accompanied by loss of heterozygosity at loci proximal and distal to the amplification site Reifenberger G Reifenberger J Ichimura K Collins VP Cancer Res 1995 55 731 734 7850781 Identification and characterization of the STIM (stromal interaction molecule) gene family, coding for a novel class of transmembrane proteins Williams RT Manji SS Parker NJ Hancock MS Van Stekelenburg L Eid JP Senior PV Kazenwadel JS Shandala T Saint R Smith PJ Dziadek MA Biochem J 2001 357 673 685 1221997 11463338 10.1042/0264-6021:3570673 Pten signaling in gliomas Knobbe CB Merlo A Reifenberger G Neuro-oncol 2002 4 3 196 211 10.1215/15228517-4-3-196 12084351 Amplification and overexpression of the MDM4 (MDMX) gene from 1q32 in a subset of malignant gliomas without TP53 mutation or MDM2 amplification Riemenschneider MJ Buschges R Wolter M Reifenberger J Bostrom J Kraus JA Schlegel U Reifenberger G Cancer Res 1999 59 6091 6096 10626796 Refined mapping of 1q32 amplicons in malignant gliomas confirms MDM4 as the main amplification target Riemenschneider MJ Knobbe CB Reifenberger G Int J Cancer 2003 104 752 757 10.1002/ijc.11023 12640683 Glioma cell motility is associated with reduced transcription of proapoptotic and proliferation genes, a cDNA microarray analysis Mariani L Beaudry C McDonough WS Hoelzinger DB Demuth T Ross KR Berens T Coons SW Watts G Trent JM Wei JS Giese A Berens ME J Neurooncol 2001 53 161 176 10.1023/A:1012253317934 11716068 Gene <it>expression profiling of low-grade diffuse astrocytomas by cDNA arrays</it> Huang H Colella S Kurrer M Yonekawa Y Kleihues P Ohgaki H Cancer Res 2000 60 6868 6874 11156382 Formylpeptide Receptor FPR and the Rapid Growth of Malignant Human Gliomas Zhou Y Bian X Le Y Gong W Hu J Zhang X Wang L Iribarren P Salcedo R Howard OMZ Farrar W Wang JM J Natl Cancer Inst 2005 97 823 835 15928303 Gene Expression Pattern Analysis Suite http://idconverter.bioinfo.cipf.es