Genetic imbalances revealed by comparative genomic hybridization in Ewing tumors

GENETIC IMBALANCES REVEALED BY COMPARATIVE GENOMICHYBRIDIZATION IN OSTEOSARCOMASToshifumi OZAKI

1, Karl-Ludwig SCHAEFER2,6, Daniel WAI

2, Horst BUERGER2, Silke FLEGE

3, Norbert LINDNER1, Matthias KEVRIC

3,Raihanatou DIALLO

2,6, Agnes BANKFALVI2, Christian BRINKSCHMIDT

4, Heribert JUERGENS3, Winfried WINKELMANN

1,Barbara DOCKHORN-DWORNICZAK

5, Stefan S. BIELACK3 and Christopher POREMBA

2,6*1Department of Orthopaedics, Westfalische Wilhelms-University, Munster, Germany2Gerhard-Domagk-Institute of Pathology, Westfalische Wilhelms-University, Munster, Germany3Pediatric Hematology and Oncology, Westfalische Wilhelms-University, Munster, Germany4Institute of Pathology, Starnberg, Germany5Institute of Pathology, Kempten, Germany6Institute of Pathology, Heinrich-Heine-University, Dusseldorf, Germany

Osteosarcomas are the most frequent bone sarcomas. Themolecular chromosomal aberrations in osteosarcomas wereanalyzed by comparative genomic hybridization (CGH). Westudied 47 frozen tumors (41 primary samples, 6 relapses) inosteosarcoma patients registered in the Cooperative Osteo-sarcoma Study (COSS) protocol. Genomic imbalances weredetected in 40 of 41 primary tumors and 6 of 6 relapsedtumors. Gains were more frequent than losses (ratio of 1.3:1). The median number of changes was 16 and 12 in primaryand relapsed osteosarcomas, respectively. The median num-ber of aberrations in primary high-grade osteosarcomas(17.0) was significantly higher than in low- or intermediate-grade osteosarcoma subtypes (3.0) (p � 0.038). The mostfrequent gains included 8q, 1p21-p31 and 1q21-q24, and themost frequent losses were 10q, 5q and 13q. High-level gainswere observed on 8q23-q24, 17p13 and 1q21-q24. A gain of19p (p < 0.001) or loss of 9p (p � 0.027) was more frequentin poor responders than in good responders. Univariate anal-ysis revealed that patients with primary metastases (p �0.002), poor histologic responses (p � 0.005), high-level gainsof 19p (p � 0.012) or losses of 13q14 (p � 0.042) had signifi-cantly lower event-free survival (EFS), whereas patients witha loss of 5q (p � 0.007) or a loss of 10q21-22 (p � 0.017) hadsignificantly higher EFS than patients without these aberra-tions. Multivariate analysis demonstrated that primary me-tastasis, loss of 13q14 and loss of 5q were independent prog-nostic factors. The findings of our study seem to be useful forevaluating the prognosis of patients and may finally lead totreatment strategies based on genetic background of osteo-sarcoma.© 2002 Wiley-Liss, Inc.

Key words: osteosarcoma; chromosomal aberrations; CGH; progno-sis; genetic pathways

Osteosarcoma is the most common primary malignant tumor ofbone. In the 1980s and 1990s, the development of more effectiveimaging techniques,1,2 chemotherapy3–6 and surgical techniques7,8

have improved the prognosis of patients with osteosarcoma. How-ever, the genetic background of osteosarcoma remains poorlyunderstood.

Several recent studies attempted to analyze the molecular ge-netics of osteosarcoma. Loss of heterozygosity (LOH) studies haveshown a frequent LOH on chromosome arms 3q, 13q, 17p and18q.9 LOH of retinoblastoma 1 (RB1; 13q14) has been shown toindicate tumors with poor prognoses.10,11 Amplification of thehuman homolog of mouse double minute 2 (MDM2; 12q13-q15) isreported to occur in 30% of recurrent or metastatic osteosarco-mas.12 Amplification of DNA-damage-inducible transcript 3(DDIT3; 12q13.1-q13-2) has also been detected in 12% of osteo-sarcomas,13 and SAS (12q13-q14) amplification has been detectedmainly in surface osteosarcomas.14 EBRBB2 (17q21.1) expressionhas been detected in 42% of osteosarcomas15 and was correlatedwith both histologic response to preoperative chemotherapy andpoor prognosis.16

Comparative genomic hybridization (CGH) is a useful tech-nique for a genome-wide screening of DNA sequence copy num-ber changes.17,18 Up to now, there have been 5 series of CGHanalyses on osteosarcomas.19–23 These studies revealed that ge-netic aberrations in osteosarcomas were extensive.21 However, thepatient numbers in previous studies were low or the treatmentmodality, including chemotherapy or extent of the surgical margin,was different between patients. Therefore, it was difficult to esti-mate the clinical relevance of the aberrations identified. Moreover,the frequencies of the combinations of each aberration, and theirinfluence on clinical course, were not evaluated. In our study, weanalyzed the molecular chromosomal aberrations by CGH in 47osteosarcomas. In 41 primary tumors with detailed follow-upinformation, the CGH data were statistically compared to clinicaldata.

MATERIAL AND METHODS

PatientsOf 52 osteosarcoma tissues obtained from 51 patients who were

treated from 1996–2000, 47 biopsy tissues were selected for ourstudy (Table I). Two tissues (one primary tumor and one relapsedtumor) were obtained from one patient with extraskeletal osteo-sarcoma. All samples were obtained from frozen tissues, whichwere taken before preoperative treatment and preserved at �80°C.Tumor tissues were taken from typical and viable tumor areas with�80% tumor-cell content. The diagnosis was histologically con-firmed as high-grade osteosarcoma. Our study was conducted afterhuman experimentation review by the ethics committee.

Of 47 tumors, 41 were primary osteosarcomas (15 osteoblasticincluding 1 postradiation osteoblastic, 7 fibroblastic, 3 chondro-blastic, 2 parosteal, 2 teleangiectatic, 1 periosteal and 11 unclas-sified) that did not undergo any pretreatment. Two parosteal os-teosarcomas were intermediate grade and 1 periosteal

Grant sponsor: Alexander von Humboldt Foundation; Grant sponsor:German Research Foundation DFG; Grant number: PO 529/5-1.

The first three authors contributed equally to this work.

*Correspondence to: Institute of Pathology, Heinrich-Heine-University,Moorenstrasse 5, 40225 Dusseldorf, Germany. Fax: �49-201-81-18353.E-mail: [email protected]

Received 21 November 2001; Revised 15 April, 23 July 2002; Accepted25 July 2002

DOI 10.1002/ijc.10709Published online 7 October 2002 in Wiley InterScience (www.

interscience.wiley.com).

Int. J. Cancer: 102, 355–365 (2002)© 2002 Wiley-Liss, Inc.

Publication of the International Union Against Cancer

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356 OZAKI ET AL.

osteosarcoma was a low-grade tumor; however, all 3 tumors ex-hibited intramedullary infiltration). The 1 postradiation osteoblas-tic osteosarcoma developed in the femur 2 years after 45-Grayirradiation to a Ewing sarcoma that originated in the ilium. Sixrelapsed tumors (3 osteoblastic, 1 chondroblastic, 1 extraskeletaland 1 unclassified osteosarcoma) included 4 metastases and 2 localrecurrences. The relapsed tumors developed from 8–141 months(median � 27 months) after surgical excision of the primarytumors.

Among the 41 patients with primary tumors, the male to femaleratio was 23 to 18 (1.3) and their ages ranged from 5–53 years(median age 16 years). Twelve tumors were proximally located (5proximal humerus, 3 pelvis, 3 proximal femur and 1 proximalthigh) and 29 were distally located (14 distal femur, 11 tibia, 2fibula, 1 radius and 1 ulna). In 21 of 41 patients with primarytumors, tumor volume could be evaluated according to the methodby Gobel et al.24 Tumor volume ranged from 6–734 mL; themedian volume was 90 mL. Eight patients had tumors with largevolumes (�100 mL) and 13 patients had tumors with small vol-umes (�100 mL). Eight of 41 patients with primary tumors hadmetastases at diagnosis.

In 41 patients with primary tumors, 40 patients were registeredin the Cooperative Osteosarcoma Study (COSS); however, all 41patients were reported to have received chemotherapy according tothe current COSS 96 protocol. In this protocol, both pre- andpostoperative chemotherapy were scheduled. All patients weredesignated to receive high-dose methotraxate with leucovorin res-cue, doxorubicin, cisplatinum and ifosfamide. In a high-risk group,etoposide and carboplatin were added as a risk-stratified therapy.

As a local treatment, 40 patients underwent tumor excision andno information was available for one patient. The surgical marginwas classified according to the method by Enneking et al.7:1radical, 38 wide, 0 marginal, and 1 intralesional. The surgicalspecimens were examined histologically and classified into 6 cat-egories of regression grades according to the criteria published bySalzer-Kuntschik et al.25 Grades 1–3 were classified as goodresponses and grades 4–6 were designated poor responses. Infor-mation on the histologic effect of preoperative chemotherapy wasavailable for 35 patients: 20 were classified as good (grades 1–3)and 15 as poor responders (grades 4–6). The follow-up periodranged from 8–43 months (median 24 months).

Comparative genomic hybridization (CGH)Reference DNA from healthy blood donors and tumor DNA

were labeled by the nick translation method with digoxigenin-11-dUTP (Boehringer Mannheim, Mannheim, Germany) and biotin-14-dATP (Gibco BRL, Gaithersburg, MD), respectively. The hy-bridization was performed as described by Kallioniemi et al.26

with some modifications.27,28

Normal lymphocyte metaphase preparations were denatured at73°C for 5 min in formamide sodium (70% formamide/2� SSC,pH 7), dehydrated and treated with proteinase K (0.1 �gml-1 in 20mM tris-HCl/2 mM calcium chloride, pH 7) at 37°C for 6 min anddehydrated again. The probe mixture, after ethanol precipitationand resuspension in 10 �l of 50% formamide/10% dextran sul-phate/2� saline sodium citrate (SSC), was denatured at 75°C for5 min, applied to the slides and hybridized for 3 days at 37°C. Thehybridization was analyzed using an Olympus fluorescence micro-scope and the ISIS digital image analysis system (MetaSystems,Altlussheim, Germany) based on a high-sensitivity interratingmonochrome CCD camera and an automated CGH analysis soft-ware package. Ratio profiles were averaged from 10 metaphasesper sample (up to 20 chromosome homologues). Gains of DNAsequences were defined as chromosomal regions with a fluores-cence ratio above 1.25 and losses as regions with a ratio below0.75. A positive control with known aberrations and a negativecontrol were included in each CGH experiment as quality controls.Overrepresentations were considered to be high-level gains whenthe fluorescence ratio exceeded 1.5. Heterochromatic regions near

the centromeres and the entire X and Y chromosomes were ex-cluded from the analysis. Judgement was based on a consensus ofat least 2 of 3 authors in all cases without reference to the patient’sclinical information.

Interphase cytogenetics by 2-color FISH analysisParaffin-embedded osteosarcoma cell line pellets (cell lines:

OST, MG-63, U2OS) and paraffin-embedded specimens from 2primary high-grade osteosarcomas with losses of chromosome 13qand 19p in CGH analysis were analyzed by 2-color FISH with achromosome13q32-q33-specific DNA probe in combination with achromosome 2 �-satellite probe, a chromosome 13q14-specificDNA probe in combination with a chromosome 13,21 satelliteprobe and a TEL 19p DNA probe in combination with a chromo-some 1,15,19 satellite probe. In brief, slides were pretreated with2� SSC, pH 7.0 at 37°C for 30 min, dehydrated in 70%, 80% and95% ethanol for 2 min each and denatured in 70% formamide/2�SSC, pH 7.0 at 70°C for 2 min, followed by dehydration in cold(�20°C) 70%, 80% and 95% ethanol for 2 min each. For hybrid-ization, the prewarmed probes were incubated o/n at 37°C in ahumidified chamber. After posthybridization washes according tothe manufacturer’s description, detection was carried out as fol-lows: for the chromosome 13q probe (Quint-Essential 13-specificDNA probe, human chromosome assignment: 13q32-q33, locus:D13S585; Appligene Oncor, Illkirch, France) 60 �l of FITC-labeled anti-Digoxenin were used for incubation at 37°C. For thealpha-satellite chromosome 2 probe (chromosome 2 �-satellite[D2Z], Appligene Oncor) 30 �l of Texas Red labeled Avidin wereused for incubation at 37°C. The chromosome 13q14 (13S272)probe and the chromosome 13,21 satellite probe (QBiogene, Hei-delberg, Germany) are each direct-labeled with Rhodamine (Exc.max. 565 nm, Em. max. 590 nm); therefore, hybridization wascarried out on separate slides of consecutive tissue cuttings fromthe paraffin blocks. The TEL 19p DNA probe is direct-labeled(green, FITC spectrum) (QBiogene). The chromosome 1,5,19 sat-ellite probe is direct-labeled with Fluorescein (Exc. 495 nm, Em.520 nm) (QBiogene). For microscopy, the cells were counter-stained with DAPI (final concentration 0.1 �g/ml in Antifade).

Immunohistochemistry for p53-protein accumulationA mouse monoclonal antibody directed against the p53 nuclear

phosphoprotein (Clone Bp53-11; Ventana Medical Systems, Tuc-son, AZ) was used for immunohistochemistry of formalin-fixedand paraffin-embedded osteosarcoma tissues on a Ventana auto-mated slide-staining device. In brief, deparaffinized 4 �m sectionson poly-L-lysine compound coated slides were incubated with theanti-p53 primary antibody for 25 min at 37°C. Afterwards, abiotin-conjugated secondary antibody was first added followed byan avidin/streptavidin-enzyme conjugate. The specific antibody-secondary antibody-avidin/streptavidin-enzyme complex was vi-sualized using a precipitating enzyme reaction product readilydetected by light microscopy. For verification of the immunohis-tochemical results, all samples were incubated with anti-p53 pro-tein monoclonal antibody, clone DO7; (Novocastra Laboratories,Newcastle, UK). The presence of nuclear staining was counted aspositive.

Statistical analysisSignificance of differences in the ratios between or among

groups was evaluated by the 2 test with/without Fisher’s correc-tion. The Mann-Whitney U-test evaluated differences of the meanrank between 2 groups. Event-free survival (EFS) was defined asthe time from diagnosis until the first occurrence of an event(death, relapse or disease progression). One patient treated byintralesional surgery (residual tumor tissue) was counted as arelapsed case in further follow-up. The cumulative probability ofEFS was calculated by univariate analysis with the Kaplan-Meiermethod. Tests of the difference between survival curves werecarried out using the log-rank test. All values, which were signif-icant in the univariate analysis, were entered and reanalyzed in the

357CHROMOSOMAL INSTABILITIES IN OSTEOSARCOMAS

TABLE II – CHROMOSOMAL ABERRATIONS DETECTED BY CGH IN 47 OSTEOSARCOMAS

Case Material CGH results1

1 Primary G 11 �1p31-q25/��1p31-p22/��1q22-q25, ��4p15.3-q32, �5p, �5q21-5q22, �6p21.3-6q16/��6pcent-p21.2, �8q12-q22, ��9p22-pter, �10pter-10q11.2, �10q24-q25, �13q14-q22/��q21, �20p12

L 16 �1p33-pter, �1q32-qter, �2qcent-q21, �3p14-p21, �3q21, �6q24-qter, �7q34-qter, �11p15-pter,�11q13, �12q23-qter, �13q33, �15q23, �16, �17, �19, �22q

2 Primary G 9 �1p22-p31, �4qcent-q22/��q12, �6p, ��8q, ��11p11.1-p12, �14q23-qter, ��17p,��19q13.1,�20q12-qter

L 7 �1q41-qter, �2q34-qter, �3p22-pter, �5q, �6q16-21, �8p, �13q3 Primary G 6 �1p,�5pter-q21, ��8q, �9q21, �17q22-qter, �21q

L 4 �3q1, �10q21, �12, �17p4 Primary G 4 ��6p12-21.3, �8q, ��12q13-15, �14q

L 1 �2q24-qter5 Primary G 11 �1p31-p32, �1q21-25, �2p24-pter, �2q31, �3p22-pter, �3q24-qter, �5p, �8q11.2-q21.3, �9q21-31,

�18, �21qL 7 �5q32-qter, �6, �7pcent-p15, �10, �13q, �16, �19p

6 Primary G 12 �1p31-q25/��1p22-p31, �2pcent-p13, �4q12-q24, �5p, �6p21, �6q23-qter, �7q11.2-q21, �8q, �12p,�13q12, �16q, �17p

L 7 �4p15.3-p16, �5q32-qter, �7p12-p15, �9q31-qter, �10q, �11q23-qter, �16p7 Primary G 18 �1, �2, �3q13.1-qter, �4, ��5p, �6p, �7q11.2-q21, �8, �11q, �12, �13q21-q31, �14q23-qter,

��15q23-qter, �16q22-qter, �17p12, �19, �20, ��21q22,L 10 �3p21-23, �5q12-q23, �6q12-q24, �7p, �9, �10, �16p, �17q, �18qcent-q21.3, �22q

8 Primary G 7 �2q22-qter, �5, �7, �8, �19q, �20, �21qL 5 �3qcent-q13.2, �12p, �13q12-q13, �14q, �18q

9 Primary G 14 �2p14-q32/��q12-q31, �4, ��6p2, �6q23-qter, �7q/��7q11.2-32, �8, �9q21-33, �11pter-q13,��12p, �12q15-qter, �14q, �17, ��20p, �22q13

L 11 �2p23-pter, �3, �5, �6q12-16, �7p, �9p22-pter, �10, �11q21-qter, �13, �15q, �1810 Primary G 13 �1, �2, �4q13-31.2, �6p, ��7p21-pter, ��7q22-q31, �8p, �9p, ��11q14-qter, �12p, ��13q13,

��17p, �20L 10 �3pter-p14, �5q, �6q16-qter, �7p14-q11.2, �10, �11p12-p13, �12q, �13q21-q22, �16p, �17q

11 Primary G 12 �3q22-qter, �4q, �5pter-q15-/��p14, �6p12-p21.2, �7p, �8q, �9q21-qter, ��17p, �18q, ��19p,��20, �21q

L 10 �1q41-qter, �3p, �4p, �6q, �9p21-pter, �10, �12q, �13q, �16, �19q13.212 Primary G 5 �8q, ��12p13, �13q31-qter, ��19p13, �21q

L 2 �8p12-pter, �1013 Primary G 9 �1p34-pter, �1q41-qter, �2qcent-q32, �8q, �12/��12q15, �14q, �17, ��19p, �20p

L 8 �1p13-p22, �3p21-pter, �4qcent-q24, �5q, �10q, �13q, �15qcent-q15, �16q22-qter14 Primary G 3 �5q31, �20q12-13.2, �22q12

L 015 Primary G 10 �1p32-q24/p21-p31, �3q, �4/��4q12-21/��4q32-qter, ��6p12-p21.3, �8q, �13q31 33, �14q, �17p,

�18q, �21q22L 10 �1q32-qter, �2q, �3q13.1-qter, �5q3, �8p, �10, �11p15-pter, �15q21-22, �16q, �19q

16 Primary G 0L 0

17 Primary G 3 �12q12-q22/��12q14, ��18p, �19pL 0

18 Primary G 7 �1p31-q24, �6, �7q22, �8/��q12-21, �9p23-pter, �12p, �13q21-qterL 9 �1q31-32, �2q34-qter, �5q11.2-q33, �9q, �10cent-q23, �1ip, �16, �19, �22q

19 Primary G 11 �1p22-qter, �2p, �5p13, ��7p15-pter, ��8q, ��9p22-pter, �12p, �13q14-31, �14q11.2-q2 2,��17p, ��19qcent-q13.3

L 8 �2q34-qter, �3, �5, �6q21-qter, �10, �11p, �16p, �17q12-2120 Primary G 4 �1p31-qter, �4q13-q27, �5p, �8q22

L 6 �2q34-qter, �5q32-qter, �6q24-qter, �9q33-34, �16p, �19p21 Primary G 5 �1p31-q31, �2q14.1-q32, �5q21, �8q22-qter/��8q23, �17q22-q23

L 5 �10q24-qter, �16, �17p, �19, �22q22 Primary G 5 �1p31-pter, �4q, ��8q, ��11q13-22, ��12q12-q15

L 5 �1p13-p22, �3p, �8p, �13q, �15qcent-q2423 Primary G 13 �1pter-q25/��1q22-q24, �2p, �2q24, �3pcent-p14, �4q, �7, �8qcent-q23, ��9p21-p23, �11q13-22,

�12p11.2-12, ��17p, �19p, �20q12-qterL 8 �1q43-qter, �2q34-qter, �3p22-pter, �4p15.1-pter, �6qcent-q22, �10q23-qter, �13q, �19q13.3-q13.4

24 Primary G 10 �1p33-pter, �1q22-qter, �7p15-p21, �7q32-qter, �9q, �12q13, �12q23-q24.1, �19p, �20q,�22qL 8 �2q21-qter, �5q, �6pter-q16, �10qcent-q22, �11, �12p12-pter, �13q, �18q22-q23

25 Primary G 2 �1p33-pter, �8qL 1 �10pter-q21

26 Primary G 10 �1p33-q25/��q21-24, �3p23-pter, �4qcent-q22, �6p12-p21.3, �7p21-pter, �8q, ��9p, �16q22-q23,�20, �22q11.2-q12

L 7 �2q14.3-qter, �6q14-23, �7q21-qter, �11q13-q22, �12q, �13qcent-q22, �18q21.1-qter27 Primary G 10 �1pter-q21/��1p22, �3q25, �4q, �5p13-p14, �6p21.1-p21.2, �7, �8q/��q21.3-qter, �13q31-qter,

�17p, ��17q23-q24L 6 �1q32-qter, �2q33-qter, �3q13.3-22, �4p15.3-pter, �10, �20p12-pter

28 Primary G 11 �1pter-q25, �2pter-q34, �3q24-qter, �4, �5p, ��8q11.2, �9p21-pter, ��13q14, �14q, ��17p,��21qcent-q22

L 11 �1q32-qter, �5q, �6p22-pter, �8p, �9q31-qter, �10q, �12p, �13q31-qter, �16q12.2-qter, �19q13.2-q13.3, �22q

29 Primary G 1 �20q12-13.2

358 OZAKI ET AL.

multivariate method with a stepwise regression test. Only p-values�0.05 are given in the text. The statistical software used was StatView version 5.0.

RESULTS

Incidence of genomic imbalances in 47 casesGenomic imbalances were detected in 40 of 41 (98%) primary

tumors and 6 of 6 (100%) relapsed tumors (Table II). Gains(median 9.0, range 0–18) were more frequent than losses (median7.1, range 0–16). Many chromosomal aberrations were observed,especially gains of chromosome 1, 4q, 5p, 6p, 7, 8q, 12p, 17, 20qor 21q and losses of chromosome 2q, 5q, 6q, 10, 13q or 16.High-level gains were frequently noted in 1p21-p31, 1q22-q24,8q23-q24, 17p13 or 19p (Fig. 1).

To confirm the CGH data by interphase cytogenetics, FISH forcentromere 2 and 13q32-q33 as well as 13q14 and chromosomes13,21 satellite and 19pter and chromosome 1,5,19 satellite wereperformed on 2 primary osteosarcomas with loss of 13q and gain

of 19p in CGH analysis: both tumors revealed aneuploidy com-bined with loss of 13q32-q33 and 13q14 and gain of 19pter. Basedon 50 tumor cell nuclei in each case, the following results wereobtained: case 11, centromere 2 signals: 2.6 1.0573 (range 0–5),13q32-q33 signals: 1.075 0.7642 (range 0–3), chromosomes13,21 satellite signals: 5.7 1.943 (range 1–9), chromosome13q14 signals: 1.167 0.617 (range 0–3), chromosome 1,5,19satellite signals: 7.977 4.014 (range 3–15), chromosome 19ptersignals: 8.741 2.322 (range 2–12); case 13: centromere 2 sig-nals: 3.0 1.2609 (range 1–7), 13q32-q33 signals: 1.225 0.6597 (range 0–2), chromosomes 13,21 satellite signals: 6.1 1.893 (range 2–9), chromosome 13q14 signals: 1.404 0.433(range 0–3), chromosome 1,5,19 satellite signals: 8.235 3.873(range 0–13), chromosome 19pter signals: �10 with high unspe-cific background in 2 independent hybridizations. The results werefurther confirmed by FISH in 3 osteosarcoma cell lines (OST,MG-63, U2OS) for which we previously obtained data from CGH,24-color multiplex fluorescence in situ hybridization (M-FISH)and conventional cytogenetics (data not shown).

TABLE II – CHROMOSOMAL ABERRATIONS DETECTED BY CGH IN 47 OSTEOSARCOMAS (CONTINUED)

Case Material CGH results1

30 Primary G 4 �12p, �17p, �19p, �22q12L 3 �2q32-34, �6, �9p21-pter

31 Primary G 17 �1pter-1q25/��p34/��q21-24, ��2p, �4pter-q12/��pcent-p14, ��4q28-31.2, �6p21, ��7p21-pter,�7q31, �9q22-qter, ��10p12-15, �11p, �13q12-13, ��15q22-24, �17q/��q24-25, �18p, �19q,�20q, �21q

L 16 �1q32, �2q22-qter, �3p, �5q15-qter, �6p23-pter, �6q16-21, �7p14-q11.2, �8p, �9p, �10q, �11q,�13q21-qter, �14q21-qter, �16p13, �16q12.2-qter, �17p

32 Primary G 9 ��1pter-q23, �3p13-p21, �5pter-p14, �6p2, �9pter-q22/��pter-p22/��cent-q22, ��12p13-pter,��13q31-qter, �19q31-32,�22q12

L 9 �1q31-qter, �2q31-qter, �5q22-qter, �9q31-qter, �10q23-qter, �11q23-qter, �13q12-q21, �17p, �20p33 Primary G 9 �1pter-q24, �5p13, �6p21, �8q/��q21.3-qter, �9q13-qter, �18p, �19q, �20q, �22q

L 5 �2q32-qter, �5q14-qter, �6q22, �13q13-qter, �18q234 Primary G 12 �1pter-q24, �3p, ��5p, �8, �9, �11q, �12p, �14q24-qter, �17/��p/��q24-qter, �18p,

��19p,��21qL 9 �1q31-qter, �2q24-q36, �3q22-qter, �5q, �6, �10, �1ip, �13q12-q33, �16q12.2-qter

35 Primary G 4 �1q21-22, �8q/��q22-qter, ��17p, ��19pL 4 �1q25-qter, �3p13-pter, �13q, �15qcent-q23

36 Primary G 15 �1p36.1-q25/��1q21-23, �3q, �4qcent-27, ��6p, �7/��q22-qter, �8q, �11pter-q22, �12, ��13q31-qter, �14q, �17/��17p, �18q, �19q, �20q, �21q

L 10 �2, �3p21-pter, �4p, �4q31.2-qter, �5, �6q, �8p, �9q31-33, �10, �13qcent-1437 Primary G 12 �1pter-q25/��p21/��q23-24, �5pter-q12, �6p, �7p, �8pter-q21, ��9q21-31, ��13q32-qter,

��15q25-qter, �17p, �19p, �20p, �21qL 9 �2q, �3p21-23, �4p15.1-pter, �5q32-qter, �6q, �10q, �12q, �13q12-14, �22q

38 Primary G 9 �1p31-q31, �3q24, �5p, �6q, �7p, ��8q, �15q25-qter, �17q22-qter, �21qL 8 �1q41-qter, �2q, �4p, �5q, �10, �13q, �16, �22q

39 Primary G 15 �1p32-q25/��p31-22/��q21-q25, �2p, �3p, �3q13, �3q26.3-qter, 4q/��q24-28, �5p �6p, �6q24-qter, �7pter-q22, �8pter-q23, �12q21, �14q, �17q, �18q

L 12 �1q32-qter, �2q, �3q23, �5q, �6q14-21, �7q35-36, �10, �11, �12p13, �13q, �15, �1640 Primary G 15 �1, �2q31-q32, �4q, �5q2, �6p, �7p, ��8q23-q24.1, �10qcent-q11.2, �11,��17p, �17q22-qter,

�18, ��19pter-q13.1, �20q, �21q,L 15 �2p22-pter, �3pter-q13.1, �5q12-q14, �5q31-qter, �6q, �7q, �8pter-q13, �9p22-pter, �10p15,

�10q23-qter, �12q23-qter, �13q, �15q15-21, �16, �22q1341 Primary G 13 �1p13-q23, �3p/��p21-pter, �4q, �5p, �7q, ��8q, �9q13-33, �12qcent-q22, �14q11.2-13, �15q,

�18p, �21q21-qter, �22qL 8 �3q13-q21, �5q32-qter, �6q15-21, �8p, �10q22-qter, �11p15, �11q22-qter, �16p

42 Metasasis G 3 �8q23-qter, �12/��12pter-12q21, �20pL 5 �10q, �11p, �13q, �15qcent-q15, �18q

43 Metastasis G 14 ��1p12-31, �4pter-q13/��4qpter-q12, �4q28-q31.3,��6p12-22, �6q23-24, �7pter-q21/��7p, �8q22-23, �12q12-15, ��14q, �16q22-qter, �17q22, �18p, ��20q �22q

L 13 �1q41-qter, �2q34-qter, �3p21-q24, �8p, �9p21-pter, �10pter-p13, �10q, �11q14-qter, �12q23-q24.1,�13q, �16p12-pter, �18q21.1-q21.3, �19q13.2-q13.3

44 Metastasis G 5 �4q21-33/��4q28-31, �7q21-qter, �12/��12q12-q21, �17q22-qter, �22q13L 4 �5q12-23, �6q15-qter, �8p22-pter, �13q21-qter

45 Recurrence G 7 �4, �6, �7p, ��8q23-q24.1, �16, �20,�22q12-qterL 6 �2, �5q, �8 pter-q21.3, �9p2, �10p11.2-qter, �14q

46 Metastasis G 7 �1p21-p22, �3p25-pter, �3q28-qter, �4p12-p14, �6p21-q16, �14q21, �20qL 4 �6p23-pter, �9q, �12q23-qter, �19q13.1-q13.2

47 Recurrence G 11 �1p33-pter, �1q22-24, �4q31, �6p21-pter, �10p, �15q24-qter, �16p13, �17, �19q, ��20q12-q13.2,�21q22

L 4 �2q22-32, �3p13-q26.1, �5q14-21, �6qcent-q231G, gain, number of aberrations; L, loss, number of abberations; �, gain of DNA sequence copy number; ��, high-level gain; �, loss.


Number of aberrations in 41 primary and 6 relapsed tumorsIn 41 primary tumors, 370 gains and 290 losses were noted. The

median number of changes was 16.1 per tumor (range 0–33, 9.0gains and 7.1 losses). In 6 relapsed tumors, 47 gains and 36 losseswere detected. The median number of changes was 12.0 per tumor(range 8–27), which was not significantly different from that inprimary tumors. The frequent aberrations that developed in 41primary osteosarcomas are shown in Table III. The most frequentgains in primary tumors involved 8q (33 cases, 80%) including8qcen-q13 (30 cases, 73%), 8q21.3-q22 (29 cases, 71%) or 8q23-q24 (28 cases, 68%). The most frequent losses in primary tumorsinvolved 10q (26 cases, 63%) including 10q21-23 (26 cases, 63%).A total of 107 high-level gains were noted in 32 of 41 primarytumors. The most frequent high-level gain included 8q23-q24 (10cases, 24%).

Comparison of number of aberrations between primary andrelapsed tumors

Table IV shows aberrations with at least a 20% difference inoccurrence rates between 41 primary and 6 relapsed tumors. Gainsof either 8qcen-q13 (p � 0.002) or 8q21.3-q22 (p � 0.013) weresignificantly more frequent in primary tumors than in relapsedtumors. However, there was no aberration that occurred at asignificantly higher rate in relapsed tumors vs. primary tumors.

p53-protein accumulationTen cases, 7 of which displayed high-level gains of 17p13 and

3 exhibited gains of 17p, were analyzed for accumulation of p53protein by immunohistochemistry (Fig. 2). Homogenous nuclearstaining in almost 100% of the tumor-cell nuclei was observed in6 cases (5 with high-level gains of 17p13 and 1 with gain of 17p).In 4 cases, no p53 immunostaining was detectable. Both antibodies(Clone Bp53-11, Clone DO7) revealed similar staining results. In

contrast to the 6 positive cases, however, these 4 negative speci-mens required more extensive decalcification for making the his-tologic sections. This raises the possibility that decalcification mayhave led to false-negative results. Furthermore, paraffin-embeddedosteosarcoma cell line pellets of which the p53 status was known28

were included as positive and negative controls. Both antibodiesrevealed the same staining results (�, nuclear staining; �, nonuclear staining: MNNG �, HOS�, ZK58-, OST- SAOS-).

Univariate analysis of clinical data

There were no significant differences regarding the mediannumber of aberrations between men (19.0) and women (16.5),adolescents (�16 years of age; 7.0) and adults (�16 years of age;7.5), proximal (14.5) and distal (18.0) tumors, large (20.0) andsmall (16.0) tumors or tumors with (18.0) and without (17.0)primary metastases. The median numbers of aberrations among thesubtypes were 18.0 in 15 osteoblastic osteosarcomas (including 1postradiation osteoblastic osteosarcoma), 17.0 in 7 fibroblasticosteosarcomas, 21.0 in 3 chondroblastic osteosarcomas, 6.5 in 2parosteal osteosarcomas, 2.5 in 2 teleangiectatic osteosarcoma, 1.0in 1 periosteal osteosarcomas. There was a significant differencebetween the median numbers of aberrations comparing 27 osteo-blastic, fibroblastic, chondroblastic, teleangiectatic osteosarcomas(17.0) against 3 parosteal or periosteal osteosarcomas (3.0) (p �0.038). Although there were no significant differences regardingthe median numbers of aberrations between good (grades 1–3;17.5) and poor (grades 4–6; 17.5) responders to chemotherapy, again of 19p (8 of 14 poor responders, 0 of 20 good responders; p �0.001) or a loss of 9p (6 of 14 poor responders, 1 of 20 goodresponders; p � 0.012) was more frequent in poor responders thanin good responders.

FIGURE 1 – Chromosomal aberrations of osteosarcomas by comparative genomic hybridization. Areas on the left side of the chromosomeindicate losses and areas on the right show gains. Dark areas highlight high-level gains.

360 OZAKI ET AL.

Combinations of chromosomal aberrations in 41 primary tumorsAmong the 41 primary tumors, 21 aberrations that appeared in

�13 cases were selected for combination analysis (Fig. 3) includ-ing loss of 5q, loss of 13q14 and gain of 4q13-q21 (as opposed toloss of 5q32-q33, loss of 13q and gain of 4q, respectively) and 3aberrations within the 8q region. Of 210 combinations, 65 com-binations occurred �13 times. Excluding combinations composedexclusively of 8q aberrations, the 3 most frequent combinationswere a gain of 1p21-p31 along with a gain of either 1q21-q24 (21cases) or 8qcen-q13 (21 cases) and a gain of 8qcen-q13 with a lossof 10q21-22 (21 cases).

Event-free survival (EFS) in 41 patients with primary tumorOur analysis of clinical factors revealed no difference in the

cumulative EFS according to tumor site or tumor size (Table III).Nevertheless, a lower EFS was observed in 8 patients with primarymetastasis at diagnosis (p � 0.002) or in 15 patients with poorhistologic response (p � 0.005). The event-free survival was notassociated with number of aberrations.

Chromosomal aberrations (gains or losses) that occurred in 13or more cases and high-level gains in 6 or more patients wereselected for univariate analysis. Patients with a loss of 13q14 (p �0.042) or a high-level gain of 19p (p � 0.012) had a significantlylower EFS than those without these aberrations. Surprisingly, a

loss of 10q21-22 (p � 0.017) or a loss of 5q (p � 0.007) wasassociated with a better prognosis (Fig. 4).

Furthermore, we analyzed the relationship between combina-tions of chromosomal aberrations and EFS in 41 primary tumors(Table V). A significantly higher EFS was observed for patientswith a loss of 5q32-q33 and either gains of 1p21-p31 (n � 17, p �0.009) or loss of 10q21-q22 (n � 16, p � 0.018). Moreover, ahigher EFS was noted for patients with a loss of 10q21-q22 andeither a gain of 1p21-p31 (n � 16, p � 0.001), a gain of 1q21-q24(n � 15, p � 0.002) or a loss of 2q34-qter (n � 12, p � 0.043).A significantly lower EFS was associated with combinations of aloss of 13q14 and a gain of either 8qcen-q13 (n � 14, p � 0.005),8q21.3-q22 (n � 12, p � 0.001), 8q23-q24 (n � 13, p � 0.001),17p (n � 9, p � 0.001) or 4q13-q21 (n � 8, p � 0.001) (Fig. 5).

The most important prognostic factors were identified by astepwise regression test among several covariates (including pri-mary metastasis; histologic response; gains of 1p21-p31, 1q21-q24, 8qcen-q13, 8q21.3-q22, 8q23-q24, 17p and 21q; high-levelgain of 19p; and losses of 5q, 10q21-q22 and 13q14) by multivar-iate analysis. By the Cox proportional hazard model (includingprimary metastasis), primary metastasis (relative risk [RR] �3.774; p � 0.017) and no loss of 5q (RR � 3.639; p � 0.029) wereidentified as being independent poor prognostic factors (Table VI).When applying the Cox proportional hazard model (and excludingprimary metastasis), no loss of 5q (RR �7.840; p � 0.001) andloss of 13q14 (RR � 5.988; p � 0.002) were shown to beindependent prognostic factors.

DISCUSSION

CGH analysis revealed a high number of unbalanced geneticalterations in nearly all our osteosarcoma cases. Despite this fact,we identified clear tendencies, or clustering, of aberrations. Up tonow, there have been 5 other reports on CGH on osteosarco-mas.19–23 In comparison with these studies (Table VII), we ob-served the highest number of aberrations per tumor (16.1). Ahigher frequency of gains over losses was reported by 4 groupsincluding our study, and gains of chromosome 1 or 8q werefrequently observed (Table VII). Tarkkanen et al.21 reported that

TABLE III – FREQUENT ABBERATIONS IN 41 PRIMARY TUMORS ANDEVENT-FREE SURVIVAL BY UNIVARIATE ANALYSIS

Variable Positive(n � 41)

Univariate analysis ofevent-free survival (p)

Gains (n � 13 cases)8q 33 NS2

8qcent-q13 30 NS8q21.3-q22 29 NS8q23-q24 28 NS

1p21-p31 26 NS1q21-q24 24 NS4q 19 NS

4q13-q21 18 NS6p12-p21 19 NS17p 18 NS5p13-p14 16 NS21q 15 NS7p21 14 NS20q 14 NS12p 13 NS

Losses (n � 13 cases)10q 26 NS10q21-221 23 0.0175q1 23 0.007

5q32-q331 22 0.01213q 23 NS

13q14 17 0.0422q34-qter 19 NS6q16-q22 18 NS10p 15 NS16p 15 NS16q 13 NS

High-level gains (n � 6 cases)8q23-q24 10 NS17p13 10 NS1q21-q24 8 NS1p21-31 6 NS19p 6 0.012

Clinical findingsTumor site (proximal) 12 NSSize (large) 8 NSPrimary metastasis (yes) 8 0.002Histologic response (poor) 15 0.005

Number of aberrations (CGH)�15 26 NS�20 15 NS

1Associated with a better prognosis.–2NS, not significant.

TABLE IV – FREQUENT ABERRATIONS IN 41 PRIMARY AND 6 RELAPSEDOSTEOSARCOMAS: ABERRATIONS WITH A DIFFERENCE OF �20% IN

OCCURRENCE RATES BETWEEN PRIMARY AND RELAPSED CASES

Aberrations Positive in primarycases (n � 41)

Positive in relapsedcases (n � 6)

2 test1

(p)

Frequent aberrations inprimary cases

Gains8qcent-q13 30 (73%) 0 (0%) 0.0018q21.3-q22 29 (71%) 1 (17%) 0.0188q23-24 28 (68%) 2 (33%) NS2

1p21-p31 26 (63%) 2 (33%) NS1q21-q24 24 (59%) 1 (17%) NS17p 18 (44%) 1 (17%) NS5p13-p14 16 (39%) 0 (0%) NS21q 15 (37%) 1 (17%) NS19p 12 (29%) 0 (0%) NS9q21-22 11 (27%) 0 (0%) NS2p 10 (24%) 0 (0%) NS2q31-32 9 (22%) 0 (0%) NS

Frequent aberrations inrelapsed cases

Gains4q13-q21 18(46%) 4 (67%) NS6p12-p21.2 19 (46%) 4 (67%) NS20q13 14 (34%) 4 (67%) NS12q13-q15 8 (20%) 3 (50%) NS17q22-q23 11 (27%) 3 (50%) NS

Losses8p2 9 (22%) 3 (50%) NS

1With Fischer’s correction.–2NS, not significant.


patients with copy number increase at either 1q21, 8cen-q13 or8q21.3-q22 had a poorer prognosis than patients without theseaberrations. Although the reported chromosomal losses variedamong studies, losses of 10q, 10p and 2q were frequently observedby Tarkkanen et al.19,21 and Zielenska et al.23 Excluding 1 study byTarkkanen et al.,21 however, these previous reports do not includea statistical analysis correlating chromosomal aberrations withpatient outcome. In general, our data on the distribution pattern ofchromosomal aberrations (Fig. 1) was similar to that obtained byTarkkanen et al.;21 however, we observed differences in the fre-quencies of losses of 5q and 16 and gain of 11q. The number oflosses reported by Stock et al.22 is too small to be compared tothose from the other studies. Our observations of frequent high-level gains of chromosome 1p, 8q or 17p support those by Tark-kanen et al.21 and Zielenska et al.23 The most frequent combina-tions of chromosomal aberrations were a gain of 1p21-p31 alongwith a gain of either 1q21-q24 or 8qcen-q13 and a gain of 8qcen-q13 with a loss of 10q21-22. In our study, the relapsed tumors didnot exhibit more aberrations than the primary tumors. This may bedue to the small number of relapsed cases (n � 6) or the milderbiologic character associated with late relapse dates (average 58months). A similar observation was made by Forus et al.,20 whodescribe an average of 3.7 and 1.8 aberrations in 10 primary and 4relapsed tumors, respectively.

In relating the identified chromosomal aberrations to clinico-pathologic results, we found that 27 high grade osteosarcomas (15osteoblastic, including 1 postradiation osteoblastic, 7 fibroblastic,3 chondroblastic, 2 teleangiectatic) had a significantly higherfreqeuncy of abbreviations (17.0) than the 3 tumors of otherosteosarcoma subtypes (3.0 per tumor, including 2 parosteal os-teosarcomas and 1 periosteal osteosarcoma. Similarly, Tarkkanenet al.21 observed an average of 3.5 aberrations in 2 cases ofhigh-grade periosteal osteosarcoma, whereas Szymanska et al.29

described a mean of 6 aberrations per tumor (range 1–13) in theirprimary parosteal osteosarcomas. We also observed that a gain of19p or a loss of 9p was more frequent in poor responders than ingood responders. Moreover, a high-level gain of 19p was associ-ated with a lower EFS. There are several candidate targets on 19psuch as the RAS-oncogene family member RAB3A (19p13.2) andthe insulin-receptor gene (INSR; 19p13.3-p13.2). The cyclin-de-pendent kinase inhibitor 2A (CDKN2A) tumor-suppressor gene islocated at 9p21,18 and its deletion has been associated with a poorprognosis in osteosarcomas.30

Losses of 5q and losses of 13q14 were the most importantprognostic factors among chromosomal aberrations identified in

FIGURE 2 – p53 immunohistochemistry of a high-grade osteoblasticosteosarcoma with a gain of 17p on CGH analysis. Distinct nuclearimmunostaining indicates accumulation/overexpression of p53 nuclearphosphoprotein. Original magnification 200� (a), 630� (b). Anti-p53mouse monoclonal antibody, Clone Bp53-11 (Ventana Medical Sys-tems).

FIGURE 3 – Combinations of aberrations in 41 primary cases. Thenumbers indicate the incidence of each aberration combination, andnumbers in parentheses denote the incidence of each individual aber-ration. Among 210 combinations, 65 combinations occurred �13times. Five combinations (blue) were associated with a better progno-sis, while 5 other combinations (magenta) were associated with aworse prognosis.

FIGURE 4 – Event-free survival in 41 patients with primary osteo-sarcomas according to the presence or absence of a 5q loss (p �0.007).

362 OZAKI ET AL.

our study. We observed 17 cases (41%) with a loss of 13q14 andcould confirm this finding by FISH in selected cases. Stock et al.22

have reported an incidence of LOH at the RB1 locus from 60–70%in osteosarcomas, which has been associated with a poor progno-sis.10,11 13q14 contains LEU1 (leukemia associated gene 1) andLEU2 (leukemia associated gene 2), which are strong candidates astumor suppressor genes relevant to chronic lymphocytic leuke-mia.18,31 We determined that a loss of 13q14 was a strong poorprognostic factor both alone and in combinations with other aber-rations.

Unexpectedly, a loss of 5q was associated with a better prog-nosis. We hypothesize that osteosarcomas may be predisposed tocarrying extra copies of chromosome 5; therefore, losses of the 5qarm (and its associated oncogenes) may account for the observedbetter survival rate. Preliminary CGH and spectral karyotypingexperiments on osteosarcoma cell lines have, in fact, revealedchromosome 5 gains, although not in every case (data not shown).Whereas additional copies of chromosome 5 may result in gain ofgenetic material on 5q and thus amplification/overexpression ofoncogenes in this region, loss of 5q may reverse this effect.Recently, several malignant glioma cell lines (LN-18, T98G andU251MG) harboring a gain of proximal 5q, where cyclin B1(CCNB1; 5q12) and cyclin H (CCNH; 5q13.3-q14) reside, havebeen shown to exhibit increased growth rates.32 Other candidategenes on 5q include interleukin 3 (IL3), IL4, IL5 and IL9 (all at5q31.1), adhesion proteins such as alpha 1 catenin (CTNNA1;5q31), fibroblast growth factor 1 (FGF1; 5q31), FGF receptor 4(FGFR4; 5q35.1-qter), platelet-derived growth factor receptor beta(PDGFRB; 5q31-q32) and transforming growth factor, beta-in-

duced (TGFBI; 5q31). FGF1 has recently been demonstrated toenhance the expression of cytosolic phospholipase A2 (cPLA2)and (in the presence of IL1) both the expression of prostaglandin-endoperoxide synthase 2 (PTGS2) and the biosynthesis of prosta-glandin E2 in human osteosarcoma cell line, MG-63.33

Univariate analysis, but not multivariate analysis, indicated thata 10q21-23 loss was an important good prognostic factor. Wespeculate that the loss of parentally imprinted genes on the 10qarm may result in a better prognosis if the ubiquitously expressedallele is required for oncogenesis. In fact, imprinting is thought toplay a role in the etiology of Hirschsprung’s disease caused bymutations in the RET proto-oncogene (10q11.2).34 Up to now,genomic imprinting in sarcomas has been reported in insulin-likegrowth factor 2 (IGF2; 11p15.5) in rhabdomyosarcomas35 andEwing tumors.36 In the 10q region, genomic imprinting of FGFR2has been reported in Apert syndrome.37 Another candidate gene inthis region is FGF8 (10q25-26).

The most frequent aberration was a gain of 8q. Chromosome 8gains are also frequent in Ewing tumors.38,39 Although we ob-served a high-level gain of 8q23-q24 in 24% of cases, MYC(8q24.12-q24.13) amplification has been reported to occur in only7–12% of osteosarcomas.40,41 Tarkannen et al.21 reported that acopy-number increase at 8q represents a statistically significantpoor prognostic factor; we arrived at this same conclusion for 8qgains, but only when present in combination with losses of 13q14.

Other frequent gains included 1p21-p31 and 1q21-q22. The 1pgains may implicate the gene for v-jun avian sarcoma virus 17oncogene homolog (JUN; 1p31) or cysteine-rich, angiogenic in-ducer, 61 (CYR61, 1p22-p31), which mediates cell adhesion, mi-gration and angiogenesis.42,43 The 1q21-q22 region includes genesfor the S100 family of calcium-binding proteins (S100A1, A4 andA6 on 1q21), small proline-rich protein 3 (SPRR3; 1q21-q22)17 andcathepsins S and K (CTSS and CTSK, respectively; 1q21). Al-though patients with a 1q21 gain have been reported to exhibit apoor prognosis (p � 0.04),21 we associated good prognosis withthis aberration when it occurred simultaneously with a loss of 5q.Molecular analysis of the 1q amplification in human sarcomasrevealed that FLG, NTRK1 and SPRR3, located in 1q21, were themost frequently amplified genes.17,44 However, the frequent 1qgains appear to have little impact on prognosis.

Seventeen percent of osteosarcomas showed a loss of 17p, andthis may affect the TP53 tumor suppressor gene on 17p13.1.18 Onthe other hand, a 17p gain was seen in 44% of cases in our studyand was determined to be a poor prognostic factor in combinationwith a 13q14 loss. Previous studies have reported an amplificationrate of 20–30% at 17p11-12 in osteosarcomas.20,21 We hypothe-size that 17p gains and high-level gains of 17p13 may result in theoverexpression of mutant p53 protein. Experimental evidence forthis hypothesis comes from our analyses of p53 protein accumu-lation in the tumor-cell nuclei in such cases. As wild-type p53protein is usually not detectable because of its short half-life,alterations in the p53 tumor suppressor gene result in an overpro-duction and accumulation of this protein and permit its detectionby immunohistochemistry. Therefore, p53 accumulation indicatesunderlying mutations or other alterations of TP53.45,46 Overex-pression of mutant p53 protein with the loss of its function as atumor suppressor gene would explain our association of a signif-icantly lower EFS with a combination of a loss of 13q14 (RB1locus) and gain of 17p.

Twenty-two percent and 10% of cases showed gains and high-level gains, respectively, of 12q13-q15. This region is well knownfor the MDM2-SAS oncogene locus (12q12-q13),17 which is am-plified in 14–27% of osteosarcomas.12,47 Unfortunately, the rela-tively low aberration frequency we observed did not permit furtherstatistical analysis.

In summary, genomic imbalances were detected in the majorityof osteosarcomas. The most frequent gains involved 8q, chromo-some 1 or 4q, whereas the most frequent losses included 10q, 5qor 13q. A gain of 19p or loss of 9p was correlated with a poor

TABLE V – EVENT-FREE SURVIVAL BY UNIVARIATE ANALYSISACCORDING TO THE COMBINATIONS OF CHROMOSOMAL ABERRATIONS:

10 COMBINATIONS WERE ASSOCIATED WITH PROGNOSIS

Variable Positive(n � 41)

Univariate analysisof event-free survival

(p)

Good prognosis�5q32-q33 � �1p21-p31 17 0.009

� �10q21-q22 16 0.018�10q21-q22 � �1p21-p31 16 �0.001

� �1q21-q24 15 0.002� �2q34-qter 12 0.043

Poor prognosis�13q14 � �8qcent-q13 14 0.005

� �8q21.3-q22 12 �0.001� �8q23-q24 13 �0.001� �17p 9 0.001� �4q13-q21 8 �0.001

FIGURE 5 – Event-free survival in 41 patients with primary osteo-sarcomas according to the presence or absence of a combination of a13q14 loss and an 8q23-q24 gain (p � 0.001).


histologic response against chemotherapy. A lower EFS was ob-served for patients with primary metastasis, poor histologic re-sponse, high-level gain of 19p or loss of 13q14 than for patientswithout these aberrations. Losses of 10q21-22 or 5q were corre-lated with good prognosis. Multivariate analysis revealed thatprimary metastasis, loss of 5q or loss of 13q14 were independentprognostic factors. Although there were many genomic aberra-tions, a few chromosomal aberrations were closely related with theprognosis of osteosarcoma patients. These data were obtained froma large series of osteosarcomas, with 40 of 41 primary tumorstreated with the same protocol, and could help to clarify thebiologic role of certain genetic aberrations. We are aware thatthese findings need to be confirmed in prospective studies, but we

believe at the same time that they could turn out to be useful forevaluating prognosis, detecting additional target regions importantfor pathogenesis or biologic behavior and may finally lead totreatments based on the genetic background of osteosarcoma.

ACKNOWLEDGEMENTS

The authors thank Ms. P. Fischer, Ms. L. Grote, Ms. P. Meier,Ms. U. Neubert, Ms. F. Schmidt and Ms. A. Sommer for excellenttechnical support.

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TABLE VI – MULTIVARIATE ANALYSIS WITH THE COX MODEL, USING 2 SELECTED FACTORS, BY STEPWISE REGRESSION TESTING

Prognostic factor n2 factors including primary metastasis 2 factors excluding primary metastasis

Multivariate p Relative risk 95% Confidenceinterval Multivariate p Relative risk 95% Confidence

interval

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TABLE VII – SUMMARY OF THE REPORTS OF CGH IN OSTEOSARCOMA

Diagnosis n No. of caseswith aberration

Aberrationsper tumor Amplification Gain : loss

ratio Frequent gains Frequent losses Prognostic factors

Osteosarcoma1

(primary 9,metastasis, 1)

11 10 (91%) 11.0 8 (73%) 1 : 1.3 8q, Xp, 1q, 11q, Xq 2q, 6q, 8p, 10p –

Osteosarcoma2

(primary 10,metastasis 4)7

14 9 (64%) 3.18 7 (18%) – 1q, 6p, 8q, 17p – –

Osteosarcoma3

(high grade)31 30 (97%) 9.6 21 (68%) 1.9 : 1 1q, 8q, 14q, Xp 6q, 10p, 10q gain of 8q or 1q

Osteosarcoma4

(primary 15,relapse 1)

16 12 (75%) 6.1 – 9.9 : 1 8q, 4q, 7q, 5p, 1p 19q, 7p –

Osteosarcoma5

(primary 17,metastasis 1)

18 18 (100%) 4.7 4 (22%) 2.7 : 1 1p, 19, 17p, 5p, 16p, 8q 2q, 10, 13 –

Osteosarcoma6

(primary,current study)

41 40 (98%) 16.1 32 (78%) 1.3 : 1 8q, 1p, 1q, 4q, 6p 10q, 5q, 13q, 2q loss of 5q or 13q

1(19).–2(20).–3(21).–4(22).–5(23).–6Current study.–7Ten tumors were xenografts in nude mice and 4 were obtained from patients. Seven of 10xenografts were established from primary tumors and 3 of 4 tumors were obtained from primary tumors of patients. The other tumors weremetastases.–8Gains only.

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Genetic imbalances revealed by comparative genomic hybridization in Ewing tumors

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