Chromosomal alterations in osteosarcoma cell lines revealed by comparative genomic hybridization and multicolor karyotyping

Cancer Genetics and Cytogenetics 140 (2003) 145–152

Chromosomal alterations in osteosarcoma cell lines revealed by comparative genomic hybridization and multicolor karyotyping Toshifumi Ozakia,b,*, Thomas Neumannc, Daniel Waid, Karl-Ludwig Schäferd,e, Franz van Valenf, Norbert Lindnera, Christina Scheelf, Werner Böckerd, Winfried Winkelmanna, Barbara Dockhorn-Dworniczakd,e, Jürgen Horstc, Christopher Porembad,e a Department of Orthopaedics, Westfälische Wilhelms-University, Münster, Germany Department of Orthopaedic Surgery, Okayama University Medical School, Okayama, Japan c Gerhard-Domagk-Institute of Pathology, Westfälische Wilhelms-University, Münster, Germany c Institute of Human Genetics, Westfälische Wilhelms-University, Münster, Germany d Gerhard-Domagk-Institute of Pathology, Westfälische Wilhelms-University, Münster, Germany e Institute of Pathology, Heinrìch-Heìne-University, Düsseldorf, Germany f Laboratory of Experimental Orthopaedics, Westfälische Wilhelms-University, Münster, Germany Received 25 February 2002; received in revised form 16 July 2002; accepted 17 July 2002 b

Abstract

We characterized the chromosomal alterations in eight osteosarcoma cell lines (OST, HOS, U-2 OS, ZK58, MG-63, SJSA-1, Saos-2, and MNNG) by comparative genomic hybridization (CGH); gains and losses of DNA sequences were defined as chromosomal regions with a fluorescence ratio, wherein all of the 95% confidence interval was above 1.25 and below 0.75, respectively. In four of 8 cell lines, multicolor karyotyping (MK) was added. CGH revealed the average number of aberrations per cell line was 20.8 (range: 10–31); the average numbers of gains and losses were 11.1 and 9.6, respectively. The frequent gains were identified on 1p21q24, 1q25q31, 7p21, 7q31, 8q23q24, and 14q21; frequent losses were at 18q21q22, 18q12, 19p, and 3p12p14. High-level gains were observed on 8q23 q24, 5p, and 1p21p22. MK revealed the most common translocations in the four cell lines were t(8;9), t(1;3), t(3;5), t(1;13), t(2;6), t(3;17), t(1;15), t(10;20), and t(6;20). Chromosomes 1, 3, 8, 9, and 20 were most frequently involved in translocation events. The concordance rate of aberrations in CGH and translocations in MK was 76%. MK was useful to identify the chromosomal alterations and as a supplement to the CGH results in three of four chromosomes. © 2003 Elsevier Science Inc. All rights reserved.

1. Introduction Osteosarcoma is the most frequent bone sarcoma affecting children and adolescents. Despite of the large number of reports on molecular genetic analysis, the genetic background of osteosarcoma is incomplete. Loss of heterozygosity (LOH) studies have shown a frequent LOH at chromosome arms 3q, 13q, 17p, and 18q [1]; LOH at 13q and 17p may reflect inactivation of RB1 and TP53 [2,3] and LOH at 18q may mean inactivation of DCC [4]. Amplification of genes at 12q13q15 is a frequent event; the 12q13q15 region contains MDM2, CDK4, and SAS [5]. MDM2 amplifi-

* Corresponding author. Department of Orthopaedic Surgery, Okayama University Medical School, Okayama 700–8558, Japan. Tel.: 81-86-2357273; fax: 81-86-223-9727. E-mail address: [email protected] (T. Ozaki).

cation was reported in 30% of recurrent and metastatic osteosarcomas [6]. ERBB2 (17q12q21) expression has been detected in 42% of osteosarcomas [7]. Amplification of MYC (8q24.12q24.13) was detected in 7% to 12% of osteosarcomas [8,9]. In recent years, comparative genomic hybridization (CGH) has been widely performed for a genome-wide screening of DNA sequence copy number changes. CGH studies revealed a high incidence of genetic aberrations in osteosarcomas; there have been five series of CGH analysis in osteosarcoma [10–14]. However, there are few reports of CGH on osteosarcoma cell lines, which are commonly used in the experimental model [15]. The majority of osteosarcomas often show complex karyotypes [16]. Recent technologic developments allow detailed analysis of these complicated chromosomal aberrations [17,18]. There are two new techniques for multicolor karyo-

0165-4608/03/$ – see front matter © 2003 Elsevier Science Inc. All rights reserved. PII: S0165-4608(02)00 6 8 5 - 4

146

T. Ozaki et al. / Cancer Genetics and Cytogenetics 140 (2003) 145–152

typing (MK): multiplex fluorescence in situ hybridization (M-FISH) [19,20] and spectral karyotyping (SKY) [21,22]. M-FISH excites and detects each of the five employed fluorochromes separately with narrow band-pass excitation/ emission filters [23]. SKY is based on the simultaneous hybridization of 24 chromosome-specific painting probes labeled with different fluorochromes or fluorochrome combinations, allowing the simultaneous visualization of all human chromosomes in different pseudo colors [18]. Both SKY and M-FISH identify chromosomal material by comparing the spectral information obtained with the labeling scheme of the probe set used [23]. Although several CGH studies have been published on osteosarcomas, the correlation between gains and losses by CGH and the cytogenetic findings is not performed. In this study, we investigated molecular cytogenetic aberrations by CGH in eight osteosarcoma cell lines. Furthermore, the aberrations detected by CGH were analyzed by MK in four cell lines for which MK was available.

2. Materials and methods 2.1. Tumor samples The eight human osteosarcoma cell lines used for the analysis were OST, HOS, U-2OS, ZK-58, MG63, SJSA-1, Saos-2, and MNNG/HOS. All eight osteosarcoma cell lines were cultured in RPMI-1640 medium supplemented with 2 mM L-glutamine, 100 U/mL penicillin G, 100 g/mL streptomycin, 0.25 g/mL amphotericin B, and 10% fetal calf serum in a humidified atmosphere of 5% CO2 at 37C. The cells were passaged twice a week for 4 weeks. 2.2. Labeling procedures, CGH, and detection DNA was isolated by phenol-chloroform extraction according to standard protocols. The hybridization was performed as described by Kallioniemi et al. [24] with some modifications [25]. Briefly, tumor DNA was labeled with biotin-16-dUTP (Boehringer Mannheim, Mannheim, Germany) and reference DNA from a healthy male donor with digoxigenin-11-dUTP (Boehringer Mannheim) in a standard nick translation reaction. The DNase concentration in the labeling reaction was adjusted individually. The labeled DNA fragments were purified from remaining nucleotides by column chromatography. The hybridization procedure was carried out as previously described. Separate digitized gray level images of DAPI, FITC, and rhodamine fluorescence were taken with a CCD camera connected to a Leica DMRBE microscope. The image processing was carried out using Applied Imaging Software (Cytovision 3.1). Average green-to-red ratios were calculated for each chromosome in at least 10 metaphases. In the designation of this study, gains and losses of DNA sequences were defined as chromosomal regions with a fluorescence ratio wherein all of the 95% confidence interval was above 1.25 and below 0.75,

respectively. For the assignment of these gains to chromosomal bands, the signal intensities were compared to the DAPI banding on individual chromosomes. As tumor specimens and normal DNA were not sex matched, X and Y chromosomes were excluded. Judgment was based on a consensus of at least two of three authors in all cases without reference to information on cell lines. 2.3. M-FISH Metaphase spreads from the osteosarcoma cell lines MNNG, Saos-2, and ZK-58 were prepared according to standard protocols. M-FISH was performed as described by Eils et al. and Speicher et al. [19,20] with minor modifications. Briefly, five pools of whole chromosome painting probes (kindly provided by M Ferguson-Smith, Cambridge, UK) were amplified and labeled by degenerate oligonucleotide primer-polymerase chain reaction using five different fluorochromes (FITC, Cy3, Cy3.5, Cy5, and Cy5.5), respectively. About 100 ng of each chromosome painting probe was precipitated in the presence of 30 g Cot-1 DNA, resolved in 10 L hybridization mixture (15% dextraane sulfate, 2 SSC) and hybridized for 48 hours. Multiscopic evaluation was performed using a Leica DMRXA-RF8 microscope (Leica, Wetzlar, Germany) equipped with a Sensys CCD camera (Photometrics, Tucson, AZ, USA) with a Kodak KAF 1400 chip. Images for each fluorochrome were acquired separately using highly specific filter sets (Chroma Technology Corp., Brattleboro, VT, USA) and processed using the Leica MCK software (Leica Microsystems Imaging Solutions Ltd., Cambridge, UK). 2.4. SKY Metaphase spreads from the osteosarcoma cell lines OST were prepared according to standard protocols. SKY was performed as described elsewhere [21,22]. The SKT® KIT (Applied Spectral Imaging, Ltd., Migdal HaEmek, Israel) was used for painting probes. Hybridization took place for 2 days at 37C. Repetitive sequences were blocked by Cot-1 DNA. The probe DNA was labeled with five fluorochromes (SpectrumOrange, TexasRed, Cy5, SpectrumGreen, and Cy5.5) and their combinations and counterstained with 4,6dia-mino-2-phenylindole (DAPI). Image acquisitions were performed using a SD200 Spectra cube system (ASI) mounted on a Zeiss Axioskop microscope with a custom-designed optical filter (SKY-1; Chroma Technology). Emission spectra were converted to the display colors by assigning blue, green, and red colors to specific sections of the emission spectrum.

3. Results 3.1. CGH Chromosomal instabilities were identified in all eight osteosarcoma cell lines. The numbers of aberrations of gains

T. Ozaki et al. / Cancer Genetics and Cytogenetics 140 (2003) 145–152

and losses were different according to the level of definition. OST had 9 gains and 16 losses, HOS had 6 gains and 4 losses, U-2 OS had 15 gains and 9 losses, ZK-58 had 13 gains and 8 losses, MG-63 had 6 gains and 7 losses, SJSA-1 had 11 gains and 6 losses, Saos-2 had 15 gains and 16 losses, and MNNG had 14 gains and 11 losses (Table 1). The results are shown on the chromosome ideograms (Fig. 1). All osteosarcoma cell lines had gains, high-level gains, and losses of chromosomal materials. Gains (N  89) were more frequent than losses (N  77) at a ration of 1.2:1. The average number of aberrations per cell line was 20.8 (range: 10–31); the average numbers of gains and losses were 11.1 (range: 6–15) and 9.6 (range: 4–16), respectively. The frequent gains were observed on 1p21q24 (6 cases), 1q25 q31 (5 cases), 7p21 (5 cases), 7q31 (5 cases), 8q23q24 (5 cases), 14q21 (5 cases), 7p22 (4 cases), 9q21 (4 cases), 12p12 (4 cases), 14q22qter (4 cases), 1p22p31 (4 cases), 5p (4 cases), 6p21.2p22 (4 cases), and 6q22 (4 cases). The frequent losses were observed at 18q21q22 (7 cases), 18q12 (5 cases), 19p (5 cases), 3p12p14 (5 cases), and 7q33qter (4 cases). High-level gains were detected 32 times and included 8q23q24 (4 cases), 5p (4 cases), 1p21p22 (4 cases), and 6q12q15 (3 cases). 3.2. MK MK detected numerous structural abnormalities of chromosomes in four cell lines (Table 1). The chromosome numbers ranged from 59 to 63 in OST (hypotriploid) (Fig. 2a), from 53 to 54 in ZK-58 (hyperdiploid) (Fig. 2b), from 56 to 60 in MG-63 (hypotriploid), and from 48 to 52 in Saos-2 (hyperdiploid). In 59 chromosomes of OST (Fig. 2a), there were 15 translocations including 4 complex rearrangements composed of 3 to 5 different chromosomes. In 53 chromosomes of ZK-58 (Fig. 2b), there were 24 translocations including 9 complex rearrangements. In 60 chromosomes of MG-63 there were 14 translocations including 3 complex rearrangements. In 52 chromosomes of Saos-2, there were 29 translocations including 14 complex rearrangements. Two identical derivative chromosomes were found in chromosome 1 of OST; chromosomes 1, 6, 7, and 8 of ZK-58; chromosome 9 of MG-63; and chromosomes 5 and 14 of Saos-2. The most common translocations in the four cell lines were t(8;9) (5 in 3 cases), t(1;3) (4 in two cases), t(3;5) (4 in one case), t(1;13) (3 in one case), t(2;6) (3 in one case), t(3;17) (3 in one case), t(1;15) (3 in two cases), t(10;20) (3 in two cases), and t(6;20) (3 in one case). When analyzed independently, chromosomes 1, 3, 8, 9, and 20 were the most frequently involved in translocation events. When the number of translocation events was adjusted by the size of the chromosome reported by Morton [26], chromosomes 20, 17, 8, 22, 19, and 9 were most frequently involved. As for the chromosomal breakpoints, it is sometimes difficult to identify them exactly by MK. The most frequently

147

involved in structural rearrangements (2 or more specimens) were 1p31, 1q22, 1q31, 1q41, 4q32, 6q22q24, 8q24, 9q22, 9q33, 10p12p13, 11p13p14, 11q23q24, 12p12, 13q12q13, 15q13q14, 17p11p12, 17q21q22, 21q11, and 22q12q13. 3.3. Comparison of CGH and MK We assessed the concordance between gain or loss detected by CGH and translocation shown by MK in 22 different chromosomes in four cell lines (Table 2). In each cell line, the rate of CGH results, which could be explained by MK was 76% at an average: 68% (15 chromosomes) in OST; 91% (20 chromosomes) in ZK-58; 73% (16 chromosomes) in MG-67; and 73% (16 chromosomes) in Saos-2.

4. Discussion Unbalanced chromosomal rearrangements are predominant and essential for the pathogenesis of solid tumors [27]; CGH is an ideal method for analysis of chromosomal aberrations in osteosarcomas. CGH analyses of high-grade osteosarcomas have been published by several separate groups [11–13]. One group reported the CGH results in 31 osteosarcoma cases [12]; the average aberrations were 9.6 per tumor and the ratio of gain to loss was 1.9:1 [12]. Another study included four osteosarcomas and 10 independent xenografts [11]. This study reported that the average aberration was 3.1 per tumor. The other study included 16 osteosarcomas and five corresponding cell lines [13]; the average number of gain and loss was 10 and 0.8 in five cell lines and that of gains and loss was 5.5 and 0.56 in 16 tumors. Summarizing these reports, gains of chromosomes 1q21, 8q23, 8q21.3, and 5p14 occurred frequently. In the current study, the average number of gains was 11.1 and that of losses was 9.6; the gain to loss ratio was 1.2 to 1. The average numbers of gains in the current study was similar to the results in five cell lines reported by Stock et al. [13], but the losses were more frequent in the current study. In chromosome 1, six cell lines showed gain of copy number and three of four cell lines showed complex rearrangements. The gene c-JUN (1p31) in chromosome 1 may be related to the development or progression of sarcomas [28,29]. High-grade osteosarcoma expressed the highest levels of c-FOS (14q21q31) and c-JUN [30]. Chromosome 7 gain was detected in several cell lines. c-MET (7q31) may be a candidate of gene aberration [31]. In 23% of osteosarcomas, c-MET expression was noted by immunohistochemical studies [32]. Chromosome 8 frequently had increase of copy number with rearrangements in the karyotype. The most frequent changes in 8q were copy number increases at 8q23q24 (5 cell lines). Copy number increase at chromosome 8 often covered the whole chromosome or whole 8q, affecting the MYC locus (8q24) [33]; however, amplification of MYC is an uncommon event in osteosar-

8

1p31q31, 2p21, 2q36qter, 4p15.1q12/p13 p14, 6p22qter/ 6q, 7p14, 7q31, 8q, 10p14p15,14q/q22q23, 15q21q22, 16q12q23, 17p, 21q22 21q22 1q32q41, 2q12q22, 4q24qter, 5p14pter, 8p, 9p22pter, 9q31q32, 11pterq22, 12q23qter, 15q25qter, 18q

1p31q32/p2, 2p12p22, 2q32q33, 4p13p15.1, 4q28q12, 6q/q12q15, 7p15p21, 7q22q31, 8q23q24, 9p2, 9q21, 12p,13q/q22, 14q12q21, 17q22qter 1q42qter, 2p24pter, 3p12p14, 8p23, 8q12, 9q33qter, 10, 11q,12q23qter, 14q23qter, 15q11q24, 16p, 17p, 18q22qter, 19p, 22q

2q24qter, 5p, 5q33qter, 6p, 7p, 9q13q21, 11p11.2p14, 11q23, 14q21qter, 15q21q23, 20q 4p15.3pter, 7q32qter, 16, 18q, 19, 22q

1p21qter, 3q, 4q31, 8/q2324, 9p22, 12p12 3p, 7q, 10, 13q 14q, 16q22qter, 18q

1p32-q31/p22-p31,1q23q25, 2q3133/q32, 3p24pter, 5q13q22/q14q21, 6q22, 7p2, 7q11.232/q21q31, 8q21.2qter, 9q22, 12pterq23, 13q12q14, 14q22qter, 20q11.2q12 2q33qter, 3p12p21, 4q31.2qter, 6q10q16, 11p, 13q22q31, 18q21.3qter, 19

1p31q24/p1331,q2123, 4p12p15.1, 4q28qter/q3, 5p, 6p12qter/q12q14, 7p2, 7q22q31, 8q23qter, 9p21pter, 12p, 12q14q15, 13q21q31, 14q12q21, 15q25qter, 17q22qter 2p23pter, 3p14q26, 4q13-q25, 8pter-q21.1, 11q23-qter, 12q23-q24.2, 16, 17p, 19p

5p, 6p10p22, 7p, 11, 14q21qter/q22, 18p 4p15.3pter, 7q32qter, 17q25, 18q

1p21q31/1p13p21, 5pter5q21/5p, 6p21pter, 7q22q31, 9q21, 10q21, 11pq14, 12q10q21, 15q24-qter 1p32p35, 2q33qter, 3p11p13, 4q32qter, 5q32qter, 6q, 7p12p14, 7q33qter, 8q12q23, 9p2,10p, 11q23qter, 13q13, 18, 19, 22q11.2q12

CGH

Boldface represents high-level gains.

Loss 11

MNNG Gain 14

Loss 16

Loss 6 Saos-2 Gain 15

SJSA-1 Gain 11

6

7

MG-63 Gain 6 Loss 7

Loss 8

ZK-58 Gain 13

Loss 9

HOS Gain 6 Loss 4 U-2 OS Gain 15

5

4

3

2

1

Loss 16

No. of aberrations

OST Gain 9

Case no.

Table 1 Summary of CGH and multicolor karyotyping

52,X,add(X),der(1)()t(1;1;9),1ph(),der(1)t(13;1;15;8;20;15),der(1)t(9;13;1;20),der(1)t(1;1;3;1;3),1ph() der(1)t(15;1),1ph(),der(1)t(13;1;12;4),der(2),der(2)t(2;6;7;12;7;6),der(3)t(1;3),3,der(4)t(15;4), der(4)t(4;20),der(4),der(5)t(5;19),der(5)2,der(6)t(15;1;6;2),der(6)t(2;6;1),der(7)t(7;20), der(8)t(8;17),der(8)t(6;17;X;8),der(9),der(9)t(9;19),der(9)t(21;9;19),der(10)t(10;20), der(10)t(20;6;20;10),der(11)t(11;4;20;6),der(12)t(3;12),der(12)t(20;12),der(13)t(13;14),13,der(14), der(14),der(15),16,der(17)t(16;17),der(17)t(11;17),der(18),der(18)t(X;18),19,20,20,21,21, der(22)t(22;21;1;6)

60,XY,der(Y)t(Y;12),der(1)t(1;12),der(3)t(3;21),4,der(4)t(3;4),der(6)t(6;8;20),der(7)t(7;20),der(8), der(8)t(21;8;4),der(8)t(17;8),der(9)t(8;9)2,9,10,der(11)t(11;12),der(12)t(12;13),12,13,13, 14,der(15)t(17;9;15),der(16),der(17)t(9;17),der(17),17,18,der(19),20,21,21

53,XY,der(1)t(1;14)2,der(1)t(1;19),der(1)t(1;9;15;20),ins(2;1),der(3)t(3;8),der(3)t(7;14;3;10;3), der(4)t(4;14),der(4),der(5),der(6),der(6),der(7)t(12;13;2;7),der(7)t(7;19),del(7q)2, der(8)t(8;9),der(8)t(8;22),der(8)t(6;8),der(8)t(8;19)2, der(9)t(18;9;8;18),del(11q), der(11)t(18;14;11),del(11p),der(12),der(12)t(12;17;22;3),der(13)t(13;18),der(13),14, der(15),der(17)t(3;5;3;17;4),der(17)t(3;5;3;17;3),del(18p),der(18)t(10;18;12),19,der(19), der(20)t(20;21),der(20)t(10;20),der(20),21,22,22

59,XX,der(X)t(X;5)(q21;?q?),der(1)?inv(1)(??)t(1;3)(p?31;?)t(1;?22)(q14;q?)2,der(1)t(1;9) (q1;q1),der(2)t(2;?7)(q3;?),3,3,der(4)?dup(4)(?;?)t(4;18)(q?;q21),4,del(5)(q?),?i(5p), der(6)t(6;15)(p23;?), der(6)del(6)(??)dup(6)(??)t(6;?7 or 15)(?;?),der(6)?del(6)(q10)?dup(6)(??), 7,der(8)t(8;9)(q10;q2),9,9,der(10)t(10;12;10),der(11)dup(11)(??)t(?1;11)(?p34;q),13, del(13)(q2?2),14[5],14[2],18, del(18)(q21),19,19,22,der(22)t(8;22)(?;q11.2 or 12), mar(?)t(13;11;7;1;6)(?;p10q10;q1q22;?)

Multicolor karyotyping

148 T. Ozaki et al. / Cancer Genetics and Cytogenetics 140 (2003) 145–152

T. Ozaki et al. / Cancer Genetics and Cytogenetics 140 (2003) 145–152

149

Fig. 1. Chromosomal aberrations of osteosarcoma cell lines by comparative genomic hybridization. Lines on the left side of chromosome indicate losses and lines on the right side of chromosome indicate gains. The broad lines indicate high-level gains. The number on the lines indicates the number of cell lines (see Table 1).

coma, detected in 7–12% of cases [8,9]. The loss of 17p in two cell lines may affect the tumor suppressor gene TP53 on 17p13.1. However, TP53 may also be involved through other mechanisms than physical loss, in that the losses are beyond the resolution capacity of CGH, or that copy number increases in the same chromosome arm might prevent the detection of losses [12]. In addition, several investigators reported that 60% to 70% of the osteosarcomas showed loss of heterozygosity (LOH) at the RB1 locus [13]. We found two cases with losses of 13q14 by CGH. CGH can detect chromosome copy number changes larger than 10 megabases in the genome, whereas balanced translocations and ploidy shifts are undetectable by CGH [18]. The conventional chromosomal banding techniques in the assessment of the karyotype are important; however, it is often difficult to karyotype solid tumors because they have complex chromosome patterns [17]. MK can improve analysis of these changes [17], so the combination of CGH and MK allows for comprehensive assessment of genomic alterations in tumors. The sensitivity limitation of spectral karyotyping was reported to be more than 1,500 kbp [21]. In four cell lines, CGH revealed that the following aberrations were common: gains of 1p21q31, 7q22q31, and 8q2324, and losses of 3p12p13, 18q22q23, 10p, and 19p. Chromosomal translocation is one mechanism of oncogene activation in malignant tumors [17]. The high aberra-

tion rate of chromosomes 1, 3, and 8 could be explained by instabilities due to translocation, which was shown by MK; however, chromosomes 10, 18, and 19 had a lower rate of translocations. These chromosome aberrations were probably related to instabilities, deletions, or other mechanisms. About 76% of CGH results have been explained by MK in the current study; it was lower than the previous report on breast cancer cell lines with 95% of matching rate between CGH and SKY [18]. A positive correlation between increasing numbers of CGH alterations and increasing numbers of translocation events in the individual breast cancer cell lines was demonstrated [18]. In osteosarcoma cases, chromosomal aberrations are more complicated than other sarcomas such as Ewing sarcomas [34] or synovial sarcomas [35]. In the current study, the number of translocation and number of CGH were similar in three cell lines (ZK-58, MG-63, and Saos-2). Bridge et al. [16] reported that the most frequently involved translocations involved 1p11p13, 1q10q12, 1q21q22, 1q42q44, 2p22p25, 3p25p26, 3q10q12, 4p10p13, 11p15, 12p11p13, 14p11, 16q23q24, 17p11, 17p12p13, 19p13, 19q13, and 22q11q13. Comparing the current study and the report by Bridge et al. [16], breakpoints located on 1q22, 12p12, 17p11p12, and 22q12q13 were noted in both studies. CGH analysis is useful for scanning a tumor total genome for gain, loss, and amplification. However, there is a

150

T. Ozaki et al. / Cancer Genetics and Cytogenetics 140 (2003) 145–152

Fig. 2. Metaphase of osteosarcoma cell lines (A) OST (SKY) and (B) ZK-58 (M-FISH).

limit for CGH detection due to the size of chromosomal area, and balanced rearrangements are undetectable. Chromosomal aberrations of osteosarcomas were complex, and it was difficult to recognize them precisely only by CGH.

MK was useful to identify each chromosomal alteration and as a supplement to the CGH results in three of four chromosomes. This study sheds light on typical derivative chromosomes.

T. Ozaki et al. / Cancer Genetics and Cytogenetics 140 (2003) 145–152

151

Table 2 Correlation between chromosomal aberrations in CGH and MK Chromosome Cell line 1 OST ZK–58 MG–63 SaOS–2

   

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

Correlation rate (%)

   

   

   

   

   

   

   

   

   

   

   

   

   

   

   

   

   

   

   

   

   

68 91 73 73

Abbreviations: , correlated; , not correlated.

Acknowledgments Supported by the Alexander von Humboldt Foundation and the German Research Foundation DFG (grant no. PO 529/5-1). [15]

References [1] Yamaguchi T, Toguchida J, Yamamuro T, Kotoura Y, Takada N, Kawaguchi N, Kaneko Y, Nakamura Y, Sasaki MS, Ishizaki K. Allelotype analysis in osteosarcomas: frequent allele loss on 3q, 13q, 17p, and 18q. Cancer Res 1992;52:2419–23. [2] Toguchida J, Ishizaki K, Sasaki MS, Ikenaga M, Sugimoto M, Kotoura Y, Yamamuro T. Chromosomal reorganization for the expression of recessive mutation of retinoblastoma susceptibility gene in the development of osteosarcoma. Cancer Res 1988;48:3939–43. [3] Masuda H, Miller C, Koeffler HP, Battifora H, Cline MJ. Rearrangement of the p53 gene in human osteogenic sarcomas. Proc Natl Acad Sci USA 1987;84:7716–9. [4] Horstmann MA, Posl M, Scholz RB, Anderegg B, Simon P, Baumgaertl K, Delling G, Kabisch H. Frequent reduction or loss of DCC gene expression in human osteosarcoma. Br J Cancer 1997;75:1309–17. [5] Tarkkanen M, Bohling T, Gamberi G, Ragazzini P, Benassi MS, Kivioja A, Kallio P, Elomaa I, Picci P, Knuutila S. Comparative genomic hybridization of low-grade central osteosarcoma. Mod Pathol 1998;11:421–6. [6] Ladanyi M, Cha C, Lewis R, Jhanwar SC, Huvos AG, Healey JH. MDM2 gene amplification in metastatic osteosarcoma. Cancer Res 1993;53:16–8. [7] Onda M, Matsuda S, Higaki S, Iijima T, Fukushima J, Yokokura A, Kojima T, Horiuchi H, Kurokawa T, Yamamoto T. ErbB-2 expression is correlated with poor prognosis for patients with osteosarcoma. Cancer 1996;77:71–8. [8] Pompetti F, Rizzo P, Simon RM, Freidlin B, Mew DJ, Pass HI, Picci P, Levine AS, Carbone M. Oncogene alterations in primary, recurrent, and metastatic human bone tumors. J Cell Biochem 1996;63:37–50. [9] Ladanyi M, Park CK, Lewis R, Jhanwar SC, Healey JH, Huvos AG. Sporadic amplification of the MYC gene in human osteosarcomas. Diagn Mol Pathol 1993;2:163–7. [10] Tarkkanen M, Karhu R, Kallioniemi A, Elomaa I, Kivioja AH, Nevalainen J, Bohling T, Karaharju E, Hyytinen E, Knuutila S, Kallioniemi O-P. Gains and losses of DNA sequences in osteosarcomas by comparative genomic hybridization. Cancer Res 1995;55:1334–8. [11] Forus A, Weghuis DO, Smeets D, Fodstad O, Myklebost O, Geurts van Kessel A. Comparative genomic hybridization analysis of human sarcomas: II. Identification of novel amplicons at 6p and 17p in osteosarcomas. Genes Chromosomes Cancer 1995;14:15–21. [12] Tarkkanen M, Elomaa I, Blomqvist C, Kivioja AH, KellokumpuLehtinen P, Bohling T, Valle J, Knuutila S. DNA sequence copy number increase at 8q: a potential new prognostic marker in high-grade osteosarcoma. Int J Cancer 1999;84:114–21. [13] Stock C, Kager L, Fink FM, Gadner H, Ambros PF. Chromosomal regions involved in the pathogenesis of osteosarcomas. Genes Chromosomes Cancer 2000;28:329–36. [14] Zielenska M, Bayani J, Pandita A, Toledo S, Marrano P, Andrade J,

[16]

[17]

[18]

[19] [20]

[21]

[22]

[23]

[24]

[25]

[26] [27]

[28] [29]

Petrilli A, Thorner P, Sorensen P, Squire JA. Comparative genomic hybridization analysis identifies gains of 1p35 approximately p36 and chromosome 19 in osteosarcoma. Cancer Genet Cytogenet 2001;130:14–21. Scheel C, Schaefer KL, Jauch A, Keller M, Wai D, Brinkschmidt C, van Valen F, Boecker W, Dockhorn-Dworniczak B, Poremba C. Alternative lengthening of telomeres is associated with chromosomal instability in osteosarcomas. Oncogene 2001;20:3835–44. Bridge JA, Nelson M, McComb E, McGuire MH, Rosenthal H, Vergara G, Maale GE, Spanier S, Neff JR. Cytogenetic findings in 73 osteosarcoma specimens and a review of the literature. Cancer Genet Cytogenet 1997;95:74–87. Kubota H, Nishizaki T, Harada K, Oga A, Ito H, Suzuki M, Sasaki K. Identification of recurrent chromosomal rearrangements and the unique relationship between low-level amplification and translocation in glioblastoma. Genes Chromosomes Cancer 2001;31:125–33. Kytola S, Rummukainen J, Nordgren A, Karhu R, Farnebo F, Isola J, Larsson C. Chromosomal alterations in 15 breast cancer cell lines by comparative genomic hybridization and spectral karyotyping. Genes Chromosomes Cancer 2000;28:308–17. Speicher MR, Gwyn Ballard S, Ward DC. Karyotyping human chromosomes by combinatorial multi-fluor FISH. Nat Genet 1996;12:368–75. Eils R, Uhrig S, Saracoglu K, Satzler K, Bolzer A, Petersen I, Chassery J, Ganser M, Speicher MR. An optimized, fully automated system for fast and accurate identification of chromosomal rearrangements by multiplex-FISH (M-FISH). Cytogenet Cell Genet 1998;82:160–71. Schrock E, du Manoir S, Veldman T, Schoell B, Wienberg J, Ferguson-Smith MA, Ning Y, Ledbetter DH, Bar-Am I, Soenksen D, Garini Y, Ried T. Multicolor spectral karyotyping of human chromosomes. Science 1996;273:494–7. Liyanage M, Coleman A, du Manoir S, Veldman T, McCormack S, Dickson RB, Barlow C, Wynshaw-Boris A, Janz S, Wienberg J, Ferguson-Smith MA, Schrock E, Ried T. Multicolour spectral karyotyping of mouse chromosomes. Nat Genet 1996;14:312–5. Lee C, Gisselsson D, Jin C, Nordgren A, Ferguson DO, Blennow E, Fletcher JA, Morton CC. Limitations of chromosome classification by multicolor karyotyping. Am J Hum Genet 2001;68:1043–7. Kallioniemi OP, Kallioniemi A, Piper J, Isola J, Waldman FM, Gray JW, Pinkel D. Optimizing comparative genomic hybridization for analysis of DNA sequence copy number changes in solid tumors. Genes Chromosomes Cancer 1994;10:231–43. Brinkschmidt C, Poremba C, Christiansen H, Simon R, Schafer KL, Terpe HJ, Lampert F, Boecker W, Dockhorn-Dworniczak B. Comparative genomic hybridization and telomerase activity analysis identify two biologically different groups of 4s neuroblastomas. Br J Cancer 1998;77:2223–9. Morton NE. Gene maps and location databases. Ann Hum Genet 1991;55:235–41. Mitelman F, Mertens F, Johansson B. A breakpoint map of recurrent chromosomal rearrangements in human neoplasia. Nat Genet 1997; 15:417–74. Curran T, Franza Jr BR. Fos and Jun: the AP-1 connection. Cell 1988;55:395–7. Franza Jr. BR, Rauscher 3rd FJ, Josephs SF, Curran T. The Fos com-

152

[30]

[31]

[32]

[33]

T. Ozaki et al. / Cancer Genetics and Cytogenetics 140 (2003) 145–152 plex and Fos-related antigens recognize sequence elements that contain AP-1 binding sites. Science 1988;239:1150–3. Franchi A, Calzolari A, Zampi G. Immunohistochemical detection of c-fos and c-jun expression in osseous and cartilaginous tumors of the skeleton. Virchows Arch 1998;432:515–9. Rao UN, Gollin SM, Beaves S, Cieply K, Nalesnik M, Michalopoulos GK. Comparative genomic hybridization of hepatocellular carcinoma: correlation with fluorescence in situ hybridization in paraffinembedded tissue. Mol Diagn 2001;6:27–37. Naka T, Iwamoto Y, Shinohara N, Ushijima M, Chuman H, Tsuneyoshi M. Expression of c-met proto-oncogene product (c-MET) in benign and malignant bone tumors. Mod Pathol 1997;10:832–8. Knuutila S, Bjorkqvist AM, Autio K, Tarkkanen M, Wolf M, Monni O, Szymanska J, Larramendy ML, Tapper J, Pere H, El-Rifai W,

Hemmer S, Wasenius VM, Vidgren V, Zhu Y. DNA copy number amplifications in human neoplasms: review of comparative genomic hybridization studies. Am J Pathol 1998;152:1107–23. [34] Ozaki T, Paulussen M, Poremba C, Brinkschmidt C, Rerin J, Ahrens S, Hoffmann C, Hillmann A, Wai D, Schaefer KL, Boecker W, Juergens H, Winkelmann W, Dockhorn-Dworniczak B. Genetic Imbalances Revealed by Comparative Genomic Hybridization in Ewing Tumors. Genes Chromosomes Cancer 2001;32:164–71. [35] Szymanska J, Serra M, Skytting B, Larsson O, Virolainen M, Akerman M, Tarkkanen M, Huuhtanen R, Picci P, Bacchini P, AskoSeljavaara S, Elomaa I, Knuutila S. Genetic imbalances in 67 synovial sarcomas evaluated by comparative genomic hybridization. Genes Chromosomes Cancer 1998;23:213–9.