We have studied 107 bone and soft-tissue sarcomas and 8 lipomas for amplification of the MDM2 gene. This gene was amplified in 3 out of 67 osteosarcomas, 3 out of 20 malignant fibrous histiocytomas, 4 out of 20 liposarcomas, and 4 out of 8 lipomas. The amplification was associated with overexpression of mRNA. In osteosarcomas, contrary to previous findings, all amplifications were observed in primary lesions. In liposarcomas, the amplification was seen exclusively in well-differentiated tumors with high frequency (4/5) but not in other subtypes (0/15). In addition, MDM2 amplification was also frequently found in deep-seated intra- or intermuscular lipomas (4/5). Hence, it is suggested that MDM2 amplification plays a significant role in the development of differentiated adipose-tissue tumors. Three well-differentiated liposarcomas with MDM2 amplification coexisted with high-grade dedifferentiated sarcomas, in which MDM2 amplification was also observed. Interestingly, in 2 of these cases, the grades of amplification correlated with the histological grades, indicating an important role of MDM2 overexpression in tumor progression.

Fusion of TLS/FUS and CHOP gene by reciprocal translocation t(12;16)(q32;q16) is a common genetic event found in myxoid and round-cell liposarcomas. Characterization of this genetic event was performed by three methods, Southern blot, RT ± PCR, and genomic long-distance PCR in nine myxoid and three round-cell liposarcomas. All but one tumors showed genetic alternations indicating the fusion of TLS/FUS and CHOP gene. Two novel types of fusion transcripts were found, of which one lacked exon 2 sequence of CHOP gene, and the other lacked 3' half of exon 5 of TLS gene. The latter case was caused by a cryptic splicing site which was created by the genomic fusion. Detailed analyses genomic fusion points revealed several sequence characteristics surrounding the fusion points. Homology analyses of breakpoint sequences with known sequence motifs possibly involve in the process of translocation uncovered Translin binding sequences at both of TLS/ FUS and CHOP breakpoints in two cases. Translocations were always associated with other genetic alterations, such as deletions, duplications, or insertions. Short direct repeats were almost always found at both ends of deleted or duplicated fragments some of which had apparently been created by joining of sequences that ank the rearrangement. Finally, consensus topoisomerase II cleavage sites were found at breakpoints in all cases analysed, suggesting a role of this enzyme in creating staggered ends at the breakpoint. These data suggested that sequence characteristics may play an important role to recruit several factors such as Translin and topoisomerase II in the process of chromosomal translation in liposarcomas.

Myxoid and round-cell liposarcomas share the translocation t(12;16)(q13;p11) creating the TLS-CHOP fusion gene as a common genetic alteration. We previously reported several unique characteristics of genomic sequences around the breakpoints in the TLS and CHOP loci, and among them was the presence of consensus recognition motifs of Translin, a protein that associates with chromosomal translocations of lymphoid neoplasms. We further extended our search for Translin binding motifs in sequences adjacent to breakpoints and investigated whether Translin binds to these sequences in vitro by mobility-shift assay. Computer-assisted search found sequences highly homologous (470%) with Translin binding motifs adjacent to the breakpoints in 10 out of 11 liposarcomas with the TLS-CHOP fusion genes. All of 13 oligonucleotides corresponding to the putative binding sequences in these cases bind to Hela cell extract and also recombinant Translin protein, although the binding a nity of each motif showed considerable di erences. The DNA-protein complex formation was inhibited by non-labeled competitor or anti-Translin antibody, suggesting the speci®city of the complex formation. Considering the high incidence and speci®c binding property, the presence of Translin binding motif may be one of the important determinants for the location of breakpoints in the TLS and CHOP genes in liposarcomas. Oncogene (2000) 19, 5821 ± 5825.