CHROMOSOMAL IMBALANCES OF OSTEOSARCOMA DETECTED BY COMPARATIVE GENOME HYBRIDIZATION (CGH).

BM04

Angel Mauricio Castro-Gamero (1); María Sol Brassesco (2); Luciano Neder (3); Edgard Engel (4) and Luiz Gonzaga Tone (2).

(1) Departamento de Genética, (2) Departamento de Puericultura e Pediatria, (3) Departamento de Patologia, (4) Departamento de Biomecânica, Medicina e Reabilitação do Aparelho Locomotor, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo – Brazil

Osteosarcoma (OS) is the most frequent aggressive bone malignancy affecting children and young adults with an event-free survival of 50-70% after 3 years. The incidence peak occurs during the second decade of life, suggesting a relationship between rapid bone growth and the development of this tumor. The knowledge of the genetic basis behind tumor progression is still limited. Conventional cytogenetic studies have demonstrated that OS exhibits high karyotipic heterogeneity, with different degrees of aneuploidy and complex structural rearrangements. The CGH is an important tool for studying the genomic profiles of solid tumors, and has confirmed the complexity of karyotipic alterations in OS. However, previous studies have shown divergent results and few have correlated them with tumor progression. The objective of present study was to identify chromosome imbalances in nine samples of OS by CGH. 3 biopsies, 5 resections before chemotherapy and 1 metastasis were analyzed. The experiments were performed accordingly with Kallioniemi et al. (1994). Briefly, test and control genomic DNAs were labeled by nick-translation using the commercial Biotin-Nick translation Mix and Dig-Nick translation Mix kits (Roche, Mannheim, Germany), respectively, according to the manufacturer’s instructions, to obtain DNA fragments ranging from 300 to 1000 pb. Hybridization was performed at 37°C for 72 h. 15 metaphase images were analyzed by using an epiflourescence microscope (Olympus BX-40) equipped with a cooled charge-coupled device camera. Ratio profiles were calculated using the CGHView^TMEXPO software (Applied Spectral Imaging®, Carlsbad, CA, USA). The thresholds for identification of imbalances were established through control experiments. Chromosome or chromosomal regions outside the calculated interval were considered to be over- or underrepresented. Telomeric and heterochromatic regions were excluded from the analysis, according to Kalliomeni et al. (1994). CGH detected chromosomal imbalances in all samples. Gains were more frequent than losses. Many chromosomal alterations were observed, especially gains at 1q, 2, 3p, 4, 5p, 6, 7, 8, 11p, 14q, 16, 21q and X; and losses at 1p, 2q, 3q, 5q, 9q, 11q and 17q. The minimal regions of superposition were gains of 2p13-p14, 2q36-q37, 4q21 and 8p22, and losses of 1p43.2, 3q22-q23 and 3q24. Three patients had consecutive samples, and the chromosomal alterations varied, reflecting the chromosomal heterogeneity for each case. The highest clonal divergence among the consecutive samples was observed between resection and the corresponding metastatic sample, showing the chromosomal complexity acquired during the progression and dissemination in this case. Additional investigations for the characterization of genes at these regions are necessary.