Elomaa, Inkeri

Karaharju, Erkki

Kivioja, Aarne

Knuutila, Sakari

Larramendy, Marcelo Luis

Salmivalli, Tarja

Tarkkanen, Maija

Valle, Julio

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No DNA Copy Number Changes in Osteochondromas - A Comparative Genomic Hybridization Study

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ELSEVIER No DNA Copy Number Changes in Osteochondromas: A Comparative Genomic Hybridization Study Marcelo L. Larramendy, Julio Valle, Maija Tarkkanen, Aarne H. Kivioja, Erkki Karaharju, Tarja Salmivalli, Inkeri Elomaa, and Sakari Knuutila ABSTRACT: Cytogenetic hanges in osteochondroma samples were studied by comparative genomic hybridization and by chromosome banding. No DNA copy number changes (15 patients) or chromo- somal aberrations (9 patients) were observed in any of the patients. © Elsevier Science Inc., 1997 INTRODUCTION Osteochondroma (OC) or osteocartilaginous exostosis is a benign cartilaginous tumor with a controversial pathogen- esis [1-3]. About 90% of osteochondromas rise in the long bones of the limbs in the vicinity of metaphyses. Although most of the OCs are sporadic and solitary, multi- ple lesions are seen in 2 rare autosomal dominant disor- ders: hereditary multiple exostoses syndrome and Langer- Giedion syndrome, also called tricho-rhino-phalangeal syndrome type II [4]. The OCs of these syndromes are his- topathologically indistinguishable from sporadic tumors, but are more prone to transform into chondrosarcomas [5]. Cytogenetic data on OC is sparse, but a limited number of OCs have a deletion in 8q [6, 7]. Patients with heredi- tary multiple exostoses syndrome or Langer-Giedion syn- drome have constitutional chromosomal rearrangements at 8q24, a region linked to hereditary multiple exostoses [8, 9]. Linkage studies have identified 3 chromosomal lo- cations for the hereditary multiple exostoses syndrome at 8q24.1 (EXT1), the pericentromeric egion of chromosome 11 (EXT2), and at 19p11-p13 (EXT3) [9-12]. Loss of het- erozygosity has been reported with markers linked to EXT1 and EXT2 in both sporadic chondrosarcomas nd From the Department of Medical Genetics, Haartman Insti- tute, University of He]sinki, Finland [M. L. L., M. T., S. K.]; the Laboratory of Cytogenetics and Cytology, Faculty of Natural Sci- ences, National University of La Plata, Argentina [M. L. L.]; Department of Pathology, Haartman Institute, University of Hel- sinki, Finland [J. V.]; Department of Orthopaedics and Trauma- tology, Helsinki University Central Hospital, Helsinki, Finland [A. H. K., E. K.]; Department of Radiology, Helsinki University Central Hospital, Helsinki, Finland [T. S.]; Department of Oncol- ogy, Hel-sinki University Central Hospital, Helsinki, Finland [I. E.]. Address reprint requests to: Sakari Knuutila, Ph.D., Depart- ment of Medical Genetics, Haartman Institute, P.O. Box 21 (Haartmaninkatu 3), FIN-O0014 University of Helsinki, Finland. Received July 2, 1996; September 20, 1996. Cancer Genet Cytogenet 97:76-78 (1997) © Elsevier Science Inc., 1997 655 Avenue of the Americas, New York, NY 10010 chondrosarcomas derived from multiple exostoses [9-13]. Thus, it has been suggested that EXT genes have tumor- suppressor properties or are indeed tumor-suppressor genes [12, 14]. CGH offers the possibility to detect DNA sequence copy number changes in tumors without specific probes and/or previous knowledge of the chromosomal rearrangements [15]. In the present study we have adopted CGH to detect gains and losses of DNA sequences in 15 osteochondro- mas. Chromosome banding analysis also was performed on 9 tumors. MATERIAL AND METHODS The material for CHG comprised 15 primary OC samples obtained from 15 patients treated at the Department of Orthopaedics and Traumatology, Helsinki University Central Hospital (Table 1). The maximum thickness of the cartilag- inous cap was measured in perpendicular hematoxylin and eosin-stained sections. The tumor specimens, used for the DNA extraction according to standard methods, contained in general tissue from the cap and the stalk. For tumor specimens of patients 1, 2, 4-8, 12, and 13, conventional chromosome banding analysis was performed after short- term (1-21 days) culture [16]. CGH was performed using direct fluorochrome-conju- gated DNAs for all samples according to the protocol de- scribed elsewhere [15, 17] with minor modifications. The hybridizations were analyzed using an Olympus fluorescence microscope and the ISIS digital image analy- sis system (MetaSystems GmbH, Altlussheim, Germany) based on an integrated high-sensitivity monochrome CCD camera nd automated CGH analysis oftware. Three-color images (red [Texas red] for the reference DNA, green [FITC] for the tumor DNA, and blue [DAPI] for the chromosome counterstain) were obtained from 12 metaphases from each sample. DNA copy number changes were confirmed using 0165-4608/97/$17.00 PII S0165-4608(96)00332-9 CGH Analys is  in Osteochondromas 77 Table I Cl inical  characterist ics of the 15 osteochondromas nalyzed by comparat ive genomic  hybr id izat ion a Thickness of the Sample number Sex/age b Tumor location Tumor size cartilage cap (cm) 1 M, 21 Fibula 4.6 x 4.4 x 5.7 0.4 2 M, 24 Tibia 4.0 x 2.0 x 1.0 0.2 3 M, 14 Femur NA 0.4 4 M, 17 Tibia 5.0 x 3.0 x 3.0 0.4 5 M, 46 Hand 1.3 x 1.0 x 1.0 0.1 6 M, 15 Femur NA 0.3 7 M, 17 Femur 1.7 x 0.7 x 0.5 0.9 8 F, 14 Humerus 8.0 X 2.5 x 2.5 0.7 9 F, 28 c Scapula NA 0.5 10 M, 57 c Femur 12.0 X 7.5 x 8.5 2.0 11 M, 32 Femur 6.0 x 8.0 x 5.0 1.5 12 M, 19 Femur NA 0.3 13 M, 28 c Femur 10.0 X 6.0 x 6.0 0.5 14 M, 31 Scapula 6.0 x 5.0 x 2.5 0.6 15 M, 37 Tibia NA 0.5 Abbreviations: F, female; M, male. a Tumors 1, 2, 4-8, 12, and 13 had b Age in years at diagnosis. c Hereditary multiple xostoses. normal karyotypes after conventional G-banding analysis. a conf idence interval  of 99% with a 1% error probabil i ty.  In each CGH exper iment,  negat ive (peripheral  b lood DNA from normal  donors) and posit ive (tumor DNA wi th  known copy number  changes) controls were inc luded and run s imul taneous ly  wi th  the tumor  samples. RESULTS AND DISCUSSION The present study is the first report of CGH per formed on osteochondromas.  None of the 15 OC samples showed any ampl i f icat ions or losses of DNA sequences by CGH. These negative CGH f indings are wel l  in l ine wi th  our normal  results from karyotype analysis. Earl ier karyotype analyses by others have demonstrated delet ions of the distal part of 8q, but only a very l imited number  of tumor cases have been reported so far [6, 7]. In the present study no changes were detected at 8q24. As both CGH and chromosome banding are rough techniques that reveal  only genetic changes of megabases, it still is possible that 8q24 or other chromosomal  areas contain loss of heterozygosity or gene mutat ions,  such as the frameshift  mutat ion reported by Ahn  et al. [14]. This study was supported by the Sigrid Jus~lius Foundation, the Clinical Research Institute of the Helsinki University Central Hos- pital, the Finnish Cancer Society, and the Foundation of Ortho- paedics and Tranmatology, all from Finland, and from the National Council of Scientific and Technological Research (CONICET) and the National University of La Plata, from Argentina. REFERENCES 1. Milgran JR (1983): The origins of osteochondromas nd enchondromas. Clin Orthop Rel Res 174:264-284. 2. Langenskiold A (1947): Normal and pathological bone growth in the light of the development of cartilaginous loci in chon- drodysplasia. Acta Chir Scand 95:367-372. 3. Klnmper A (1977): Intraosseous angiography in the investiga- tion of the pathogenesis of osteochondromas. Fortschr Geb R6ntgenstr Nuklearmed Erg~inzungsband 127:142-145. 4. McKusick VA (1994): Mendelian Inheritance in Man, ed 11, Johns Hopkins University Press, Baltimore. 5. Campanacci M (1990): Bone and Soft Tissue Tumors, Springer Verlag, New York. 6. Bridge JA, Bhatia PS, Anderson JR, Neff JR (1993): Biologic and clinical significance of cytogenetic and molecular cyto- genetic abnormalities in benign and malignant cartilaginous lesions. Cancer Genet Cytogenet 69:79-90. 7. Mertens F, Rydholm A, Kreicbergs A, Will6n H, Jonsson K, Heim S, Mitelman F, Mandahl N (1994): Loss of chromosome band 8q24 in sporadic osteocartilaginous exostoses. Genes Chromosom Cancer 9:8-12. 8. Lfidecke H-J, Johnson C, Wagner MJ, Wells DE, Turleau C, Tommeup N, Latos-Bielenska A, Sandig K-R, Meinecke P, Zabel B, Horsthemke B (1991): Molecular definition of the shortest region of deletion overlap in the Langer-Giedion syndrome. Am J Hum Genet 49:1197-1206. 9. Cook A, Raskind W, Halloran-Blanton S, Pauli RM, Gregg RG, Francomano CA, Puffenberger E, Conrad EU, Schmale G, Schellenberg G, Wijsman E, Hecht JT, Wells D, Wagner MJ (1993): Genetic heterogeneity in families with hereditary multiple exostoses. Am J Hum Genet 53:71-79. 10. Wu Y-Q, Heutink P, de Vries BBA, Sandkuijl LA, van den Ouweland AMW, Niermeijer MF, Galjaard H, Reyniers E, Willems P], Halley DJJ (1994): Assignment of a second locus for multiple exostoses to the pericentromeric region of chro- mosome 11. Hum Mol Genet 3:167-171. 11. Le Merrer M, Legeai-Mallet L, Jeannin PM, Horsthemke B, Schinzel A, Plauchu H, Toutain A, Achard F, Munnich A, Maroteaux P (1994): A gene for hereditary multiple exostoses maps to chromosome 19p. Hum Mol Genet 3:717-722. 12. Hecht JT, Hogue D, Strong LC, Hansen MF, Blanton SH, Wag- ner M (1995): Hereditary multiple exostosis and chondrosar- 78 M.L .  Lar ramendy et al. coma: Linkage to chromosome 11 and loss of heterozygosity for EXT-linked markers on chromosomes 11 and 8. Am J Hum Genet 56:1125-1131. 13. Raskind WH, Conrad EU, Chansky H, Matsushita M (1995): Loss of heterozygosity in chondrosarcomas for markers linked to hereditary multiple exostoses loci on chromosomes 8 and 11. Am J Hum Genet 56:1132-1139. 14. Ahn J, L~decke H-J, Lindow S, Horton WA, Lee B, Wagner MJ, Horsthemke B, Wells DE (1995): Cloning of the putative tumour suppressor gene for hereditary multiple exostoses (EXT1). Nature Genet 11:137-143. 15. Kallioniemi A, Kallioniemi O-P, Sudar D, Rutovitz D, Gray 16. 17. JW, Waldman F, Pinkel D (1992): Comparative genomic hybridization for molecular cytogenetic analysis of solid tumors. Science 258:818-821. Tarkkanen M, Kaipainen A, Karaharju E, B6hling T, Szyman- ska J, Hell6 H, Kivioja A, Elomaa I, Knuutila S (1993): Cyto- genetic study of 249 consecutive patients examined for a bone tumor. Cancer Genet Cytogenet 68:1-21. Larramendy ML, Tarkkanen M, Valle J, Kivioja AH, Ervasti H, Karaharju E, Salmivalli T, Elomaa I, Knuutila S (1997): Gains, losses, and amplifications of DNA sequences evalu- ated by comparative genomic hybridization in chondrosarco- mas. Am J Pathol 150:685-691. 

No DNA Copy Number Changes in Osteochondromas - A Comparative Genomic Hybridization Study

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