In the pediatric cancer alveolar rhabdomyosarcoma, characteristic t(2;13)(q35;q14) or variant t(1;13)(p36;q14) chromosomal translocations generate PAX3-FKHR or PAX7-FKHR fusion genes. Using fluorescence in situ hybridization, reverse transcriptase-polymerase chain reaction and quantitative Southern blot analyses, we demonstrate that these fusion genes are amplified in 20% of fusion-positive tumors. In particular, we found in vivo amplification of these fusions in one of 22 PAX3-FKHR-positive cases and five of seven PAX7-FKHR-positive cases. These findings indicate that translocation and amplification can occur sequentially in a cancer to alter both the structure and copy number of a gene and thereby activate oncogenic activity by complementary mechanisms.
Molecular assays for specific gene fusions provide a genetic approach to the differential diagnosis of soft tissue sarcomas. The genetic categories correspond closely to the standard histopathologic categories. The polymerase chain reaction assays for chimeric transcripts are useful tools for the rapid and objective assessment of pediatric soft tissue sarcomas.
Alveolar rhabdomyosarcoma (ARMS) cells often harbor one of two unique chromosomal translocations, either t(2;13)(q35;q14) or t(1;13)(p36;q14). The chimeric proteins expressed from these rearrangements, PAX3-FKHR and PAX7-FKHR, respectively, are potent transcriptional activators. In an effort to exploit these unique cancer-specific molecules to achieve ARMS-specific expression of therapeutic genes, we have studied the expression of a minimal promoter linked to six copies of a PAX3 DNA binding site, prs-9. In transient transfections, expression of the prs-9-regulated reporter genes was Ϸ250-fold higher than expression of genes lacking the prs-9 sequences in cell lines derived from ARMS, but remained at or below baseline levels in other cells. High expression of these prs-9-regulated genes was also observed in a cancer cell line that lacks t(2;13) but was stably transfected with a plasmid expressing PAX3-FKHR. Transfection of a plasmid containing the diphtheria toxin A chain gene regulated by prs-9 sequences (pA3-6PED) was selectively cytotoxic for PAX3-FKHR-expressing cells. This was shown by inhibition of gene expression from cotransfected plasmids and by direct cytotoxicity after transfected cells were isolated by cell sorting. Gene transfer of pA3-6PED may thus be useful as a cancer-specific treatment strategy for t(2;13)-or t(1;13)-positive ARMS. Furthermore, gene transfer of fusion proteinregulated toxin genes might also be applied to the treatment of other cancers that harbor cancer-specific chromosomal translocations involving transcription factors.Rhabdomyosarcoma is the most common soft tissue sarcoma of childhood. While implementation of surgery, radiation, and chemotherapy has improved the overall long-term survival rate, patients often suffer numerous acute and chronic sequelae (1). In addition, those with metastatic disease or an unfavorable histological subtype continue to have a poor prognosis (2). Identification of one of these high risk types, alveolar rhabdomyosarcoma (ARMS), has been facilitated by cytogenetic and molecular detection of the characteristic chromosomal translocations t(2;13)(q35;q14) or t(1;13)(p36;q14).The t(2;13)(q35;q14) or t(1;13)(p36;q14) fusion genes encode hybrid proteins derived from the DNA-binding domains of PAX3 or PAX7, respectively, and the transactivation domain of an unrelated transcription factor, FKHR (3-6) ( Fig.
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