Molecular defects affecting tumor-suppressor genes are an important step in the genesis of sarcomas. For example, inheritance of a defective Rb or p53 gene predisposes the carrier to develop osteosarcoma, among other malignancies. In this study, we have assessed the occurrence of p53, Rb and MDM2 alterations in the same samples of osteosarcomas, along with representative samples of various other sarcomas. Point mutations of the p53 gene were found in 13 of 42 osteosarcomas and 1 of 8 leiomyosarcomas, and gross rearrangement of the p53 gene was demonstrated in 5 of 37 osteosarcomas. The retinoblastoma susceptibility gene (Rb) was either rearranged or deleted in 7 of 37 osteosarcomas, 1 of 7 soft-tissue sarcomas and 1 of 4 Ewing sarcomas. Remarkably, 5 of the osteosarcomas having Rb alterations also had p53 mutations. Amplification and overexpression of the MDM2 oncogene may lead to increased MDM2-p53 binding resulting in inactivation of p53 function. A two- to threefold increase in the copy number of MDM2 was detected in 7 of 37 samples, 5 of which were osteosarcomas. Amplification of the MDM2 gene occurred independently of p53 mutation; one sample having threefold amplification of MDM2 also had a p53 mutation. In summary, 34 alterations of the p53, Rb and MDM2 genes were found in 26 of 42 (62%) osteosarcomas.

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INK4D gene were found in samples from ®ve of 67 (7%) patients with osteosarcomas and none were found in other types of sarcomas or in lung cancers. Five osteosarcoma samples had Southern blot patterns consistent with gene rearrangement. These samples included a primary and recurrent osteosarcoma from the same patient; both with the same rearrangement. Four samples had SSCP patterns consistent with sequence alterations, sequencing determined that three were due to silent base changes and apparently polymorphisms. Sequencing the fourth shifted band revealed a one base insertion causing a frameshift beginning with codon 27. In summary, these studies found alterations aecting the p19 INK4D gene in a small but signi®cant number of osteosarcomas. Presumably, abnormalities of this gene contribute to the development of cancer of bone cells.

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