Comparative genomic hybridization in ganglioneuroblastomas

Cancer Genetics and Cytogenetics 132 (2002) 36–40

Comparative genomic hybridization in ganglioneuroblastomas Ahter Dilsad Toramana, I˙brahim Kesera,*, Güven Lülecia, Nurdan Tunalıc, Tekinalp Gelenb a

Department of Medical Genetics, School of Medicine, Akdeniz University, TR-07070 Antalya, Turkey b Department of Pathology, Akdeniz University, Antalya, Turkey c Department of Pathology, Cukurova University, Adana, Turkey Received 5 March 2001; received in revised form 29 May 2001; accepted 30 May 2001

Abstract

The ganglioneuroblastoma are rare lesions with widespread neuronal differentiation that have been classified as intermediate stages between neuroblastoma and ganglioneuroma. To identify overall chromosome aberrations in ganglioneuroblastoma, we performed comparative genomic hybridization. All of the five tumor samples were found to exhibit multiple gains involving different chromosomal regions. Chromosomal gains displayed by chromosomes and chromosome loci were 2p25pter (60%), 5p15.1p15.3 (60%), 7 (60%), 13q22q31 (60%), and 22 (60%), which were detected as minimal common regions in all five tumor samples. Chromosome 22 gain, which had not been reported in neuronal tumors before, and novel site 13q22q31 may be considered to play an important role in progression and differentiation of ganglioneuroblastoma. © 2002 Elsevier Science Inc. All rights reserved.

1. Introduction The major tumors of the central nervous system (CNS) with neuronal origin are neuroblastomas, ganglioneuromas, and gangliogliomas [1]. There are many similarities between these tumors. All are rare and are usually found in children or young adults [2]. Neuroblastoma differentiation occurs in varying levels. The designation of ganglioneuroblastoma is reserved for rare lesions with widespread neuronal differentiation between neuroblasts and mature ganglion cells [3]. Comparative genomic hybridization (CGH) has been shown to be useful in detecting losses and gains of whole chromosomes and of chromosomal regions in solid tumors, which have complex karyotypes [4]. Comparative genomic hybridization can detect chromosomal copy number changes larger than approximately 10 Mb in the genome in a single hybridization [5]. Although chromosomal rearrangements were reported using conventional cytogenetic technique and CGH in neuroblastoma [6–12], a translocation (1;13)(q22; q12) was described in a patient with ganglioneuroblastoma [13], with no CGH data. Chromosome 1p rearrangements were found to be the most frequent karyotypic changes reported in neuroblastoma [8,10]. Rearrangements of chromosomes 11, 14, 17, double minutes (dmin), and homoge* Corresponding author. Tel.: 90-242-2274343, ext: 44356; fax: 90-242-2274495. E-mail address: [email protected] (I. Keser).

neously staining regions (hsrs) and 2p23p25 (harbors N-myc) amplification have been reported in neuroblastoma [9]. The whole chromosome losses of 11, 14, X, and partial losses of 1p and 11q, gains of chromosomes 6, 7, 17, 18, and amplification of N-myc region were detected by CGH analysis [12]. Glioma, which is a neuronal tumor, and glioma cell lines were found to have gains of chromosomes 7, 11, 14, 17, and 20, losses of different chromosomes [14,15]. Polysomy 7, amplification of 7p, 11q, 12q, and deletions of 9p, 10, 13 were found in another neuronal tumor glioblastoma multiforme using CGH [16,17]. We applied CGH to screen genetic imbalances in five ganglioneuroblastoma samples. 2. Materials and methods 2.1. Tumor samples Five selected archival formalin-fixed, paraffin-embedded ganglioneuroblastoma tumor samples from children (age range 0.5–13 years) were obtained from Pathology Departments of Akdeniz University Medical School and Çukurova University Medical School. Hematoxylin-eosin (H&E) stained tissue sections (5 m) were prepared from each tumor and the histological diagnosis was confirmed in all cases. Genomic DNA was extracted by proteinase K digestion and phenol-chloroform extraction as described previously [18].

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A.D. Toraman et al. / Cancer Genetics and Cytogenetics 132 (2002) 36–40

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Metaphase chromosome spreads were prepared from peripheral blood leukocytes from healthy individuals. Blood cells were grown for 72 h in RPMI medium supplemented with 16% fetal bovine serum, penicillin-streptomycin (100 U/ml), L-glutamine, and phytohemagglutinin (5 g/ml). Cells were harvested using standard procedures. Slides were freshly equilibrated in 2 standard saline citrate solution (pH 7.0) at 37C and dehydrated in ethanol series of 70, 90, and 100%.

denatured in 70% formamide, 2SSC pH 7.0 for 1 min and 15 s at 68C, dehydrated through ice-cold ethanol series (70, 90, 100%), treated with proteinase K (10 g/ml in 20 mM Tris-HCI, 2 mM CaC12, pH 7.5) at 37C for 7.5 min and dehydrated again. Hybridization was done under sealed coverslips for 2–3 days at 37C in a moist chamber. After hybridization, the slides were washed according to previously described protocols [20] and chromosomes were counterstained with 4,6-diamidino 2-phenylindole (DAPI) in an antifade solution.

2.3. Comparative genomic hybridization

2.4. Comparative genomic hybridization analysis

Comparative genomic hybridization was performed with modifications according to standard procedures [4,19,20]. Reference DNA was prepared from peripheral blood lymphocytes of healthy female and male donors, and labeled with spectrumRed-dUTP (Vysis Inc., Downers Grove, IL, USA). DNA extracted from tumor samples was labeled with spectrumGreen-dUTP (Vysis Inc.). Both reference and tumor DNA were labeled using degenerate oligonucleotide primed-PCR (DOP-PCR) as described by Telenius et al. [21]. DOP-PCR product was run on 1.2% agarose gel with a proper marker DNA in order to obtain the yield of 400– 2000 bp probe fragment size for uniform and intense hybridization. Five-hundred nanograms of each labeled probe and 50 g unlabeled Cot-1 DNA was precipitated with ethanol. The DNAs were dissolved in 15 l hybridization buffer [50% formamide/10% dextrane sulfate/2 standard saline citrate (2SSC), pH 7.0] and denatured at 70C immediately before applying onto the slides. Metaphase slides were

The hybridization was analyzed by using PSI (Perceptive Scientific International Ltd.) digital image software Mac Probe version 4.0 that was based on a Zeiss Axiophot-2 epifluorecence microscope equipped with a cooled CCD camera (Photometrics Ltd., Tucson, AZ, USA) and a filter system (LEP, USA) consisting of six position computerized Ludl filter wheel. Excitation of each fluorochrome was accomplished by using these filters in a computer-controlled filter wheel. Three fluorochrome images (DAPI, SpectrumGreen, and SpectrumRed) were properly registered and processed with PSI workstation using a Mac Probe Program Version 4.0 software for pseudo-color display. Three color images were used to visualize the color changes along the metaphase chromosomes. For each case, 10 metaphases were analyzed for the chromosomal locations of DNA sequence gains and losses. These regions were determined by using green-to-red fluorescence intensity ratio profiles. Defining the gains and losses of DNA-sequence copy number in tumors were based

2.2. Metaphase preparation

Fig. 1. DNA copy number profiles that were obtained from case 1.

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A.D. Toraman et al. / Cancer Genetics and Cytogenetics 132 (2002) 36–40

Table 1 Chromosomal gains detected by comparative genomic hybridization (CGH) in 5 ganglioneuroblastoma samples Case no

CGH gains

1 2

4q34qter,7,13,22 2q37.1qter,2p25.1pter,5p15.1pter,13, 18p11.31pter,20,22 1p35,5p15.1pter,6p23,12q12q13.2,12q24.2qter, 13q22q31,17p12p13,Xq21.1q21.3 2p,4p15.1,4q21.2q22,6q26,7,8q13, 10q11.2q21.2,Xq12q13 1p35pter,2p24pter,4p,5p15.1p15.3,6,7, 8,9q34.1qter,10,12q24.32qter,14q23q24, 15,17,18,22,Xp11.4p21.2

3 4 5

on comparison of normal DNAs labeled with two different colors. The decision limits of the green-to-red ratios were 0.8 for the loss of DNA copy number and 1.50 for the gain of DNA copy number (Fig. 1). Control labeling and hybridizations were done by nick translation (Boehringer Mannheim) and cross red/green labeling of the normal and tumor DNA probes, respectively. Heterochromatic regions of chromosomes 1, 9, 16, 19, Y, and acrocentrics 13, 14, and 15 were excluded from analysis. Processing and evaluation were carried out as described previously [19]. 3. Results Five ganglioneuroblastoma samples were analyzed using CGH. The number of chromosomal aberrations per tumor

ranged from 4 to 16. The mean number of aberrations per tumor was 8.6 (gains) in the metaphases. Loci and frequencies of chromosomal gains in each ganglioneuroblastoma sample are summarized in Table 1 and Fig. 2. The minimal common regions of gains of chromosomes 2p25.1pter, 5p15.1p15.3, 7, 13q22q31, and 22 were observed in 3 of the 5 (60%) ganglioneuroblastoma samples. Also, chromosomes 1p35, 4p15.1, 10q11.2q21.2, 12q24.3qter, 13, 17p12p13, and 18p11.3pter were found in 2 of the 5 (40%) samples. Chromosomes 7, 13, and 22 were whole chromosome gains. There was no loss of chromosomes in any sample.

4. Discussion Ganglioneuroblastoma is reserved for rare tumors with widespread neuronal differentiation, which contain primitive neuroblasts and maturing ganglion cells [3]. Therefore, the development of useful prognostic parameters may help for therapeutic strategies in tumor clonality. In this study, we used CGH to evaluate chromosome copy number alterations in ganglioneuroblastoma samples, and compared them to other reported results on neuronal tumors [6,13]. The most frequent chromosomal aberrations found as minimal common regions by CGH in 5 ganglioneuroblastoma samples are shown in Table 2. An average of 8.6 alterations in chromosomal copy number was observed per tumor sample. Keser et al. [22] found an average of 8.1 alterations in chromosomal copy number in other 5 ganglioneurolastomas. Although

Fig. 2. Summary of chromosomal gains detected by comparative genomic hybridization in five ganglioneuroblastoma samples. The Y chromosome, acrocentric p-arms, and chromosome centromeric heterochromatin regions were excluded from analysis.

A.D. Toraman et al. / Cancer Genetics and Cytogenetics 132 (2002) 36–40 Table 2 Most frequent gains detected by CGH in 5 ganglioneuroblastoma samples Chromosome and arm

Most frequent region

Frequency (%) (n5)

2p 5p 7 13q 22 1p 4p 10q 12q 13 17p 18p

p25.1pter p15.1p15.3 7 q22q31 22 p35 p15.1 q11.2q21.2 q24.3qter 13 p12p13 p11.35pter

60 60 60 60 60 40 40 40 40 40 40 40

gains of the whole chromosomes 7, 13, and 22 were found in our study, Keser et al. [22] found gains of the whole chromosomes 18 and 22 using CGH. The difference could be the consequence of the number of cases used in this study. The results of both studies are compared in Table 3. In previous studies, various chromosome abnormalities including the gains and losses of partial and whole chromosomes have been reported in tumors with the neuronal origin [12,14–17,22]. The whole chromosome losses of 11, 14, X, and the partial losses of 1p and 11q, gains of chromosomes 6, 7, 17, 18, and amplification of the N-myc region (2p24p32) were found in neuroblastoma by CGH analysis [12]. Glioma and glioma cell lines were also found to have gains of chromosomes 7, 11, 14, 17, and 20, losses of different chromosomes [14,15]. The polysomy 7, amplification of 7p, 11q, 12q, and deletions of 9p, 10, and 13 were found in glioblostoma multiforme that have neuronal origin [17]. In our study, we found gains of chromosome 7 in 3 of 5 ganglioneuroblastomas using CGH. Keser et al. [22] observed 7p gain in 1 case of 10 metaphases analyzed by CGH. Therefore, chromosomes 7 and 7p may be considered to play an important role in progression and differentiation of ganglioneuroblastoma as well as other neuronal originated tumors. While the N-myc amplification was the most frequent observed in neuroblastoma [9], here, we also found the amplification of N-myc region in 2 of 5 cases. These results suggest that 2p amplification may be important for progression from neuroblastoma to ganglioneuroblastoma. Chromosome 5p15.3 showed over-amplification in medulloblastoma by CGH [23,24]. In our study, chromosome 5p15.1p15.3 amplification was found as minimal common region in 3 of 5 samples. This result suggested that 5p15.3 site may play a role in both brain tumors and peripheric neuronal tumors. While chromosome 13 gains were detected in neuroblastoma using CGH analysis, the 13q22q31 restricted amplification region was identified in 3 of 5 ganglioneuroblastomas in present study. This restricted site may harbor an oncogene(s) for ganglioneuroblastoma. In our study, gains of chromosome 22 were observed in 3 of 5 ganglioneuroblastoma samples. Chromosome

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Table 3 Comparison of the CGH results reported by us and by Keser et al. [22] Present study (n5) Chromosome

Gain

(%)

Loss

1p 2p 4p15.1 5p15.1p15.3 7 7p 8p 9p 10p 10q11.2q21.2 12p 12q24.32qter 13 13q22q31 17p 18p11.35pter 18 18q 22

     — — — —  —        

40 60 40 60 60

— — — — — — — — — — — — — — — — — — —

40 40 40 60 40 40 20 20 60

Keser et al. (n5) (%)

Gain — — — — —     —  — —      

(%)

20 80 60 60 80

20 60 20 80 100 20

Loss

(%)

 — — — — — — — — — — — — — — — — — —

40

22 amplification had not been reported in neuronal tumors [6]. Also, Keser et al. [22] found gain of chromosome 22 in 1 of 5 ganglioneuroblastomas. Because chromosome 22 harbors the oncogenes of YESP, SIS, PDGB, and NRASL2 [25], amplification of chromosome 22 may be considered an important prognostic factor for identification of ganglioneuroblastoma in tumor monitoring. Keser et al. [22] found amplification of chromosome 18 and 18q in 5 of 5 cases of ganglioneuroblastoma, while we detected the same amplification in 1 of 5 cases. However, glioma cell lines were found to have loss of chromosome 18 [14]. On the basis of these results we can suggest that gains of chromosome 18 and 22 could be prognostic factors for identification of ganglioneuroblastoma. These results show a pattern of genetic changes in ganglioneuroblastomas that are different from other pattern observed in neuronal originated tumors. These genetic changes (gains) detected may play an important role in progression and transformation of ganglioneuroblastoma. We suggest that the identification of the transitional stages in tumor clonicity is important for treatment and following. Further investigations will focus on transitional stages of neuronal tumors. The data obtained from investigations will be used to classify the tumors according to their specific chromosome imbalances. Acknowledgments This study is supported by Research Foundation of Akdeniz University. Grant number: 99.01.0122.05. References [1] Burger PC, Scheithauer BW. Tumors of the central nervous system. In: Rosai J, Sobin LH, editors. Atlas of tumor pathology. Third Series, Fascicle 10. Washington, D.C.: Armed Forces Institute of Pathology, 1994. p. 196–200.

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