In Vivo Amplification of the PAX3-FKHR and PAX7-FKHR Fusion Genes in Alveolar Rhabdomyosarcoma

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 situhybridization, reverse transcriptase-polymerase chain reaction and quantitative Southern blot analyses, wedemonstrate that these fusion genes are amplified in 20% of fusion-positive tumors. In particular, we found in vivoamplification of these fusions in one of 22 PAX3-FKHR-positive cases and five of seven PAX7-FKHR-positivecases. These findings indicate that translocation and amplification can occur sequentially in a cancer to alter boththe structure and copy number of a gene and thereby activate oncogenic activity by complementary mechanisms.INTRODUCTIONThe identification of nonrandom chromosomal alterations asso-ciated with specific neoplasms has resulted in the elucidation ofimportant steps in tumorigenesis and new strategies for tumordiagnosis and management. Two important cytogenetic cat-egories identified by these investigations are translocation andamplification. Molecular biology studies of translocations inleukemias and solid tumors have shown that these events activateoncogenes by one of two mechanisms (1). One class oftranslocations, which frequently occurs in lymphoid malig-nancies, juxtaposes the coding region of one gene into the vicinityof expression elements of another gene, resulting in increasedexpression and/or altered regulation. In the second category oftranslocations, a chimeric gene product is formed by the joiningof two coding regions. For amplification events, additional copiesof a cellular gene are generated in the form of double minutechromosomes or homogeneously staining regions (2). Thepresence of additional gene copies generally results in over-production of wild-type transcripts and associated proteinproducts.Cytogenetic investigations of the pediatric soft tissue canceralveolar rhabdomyosarcoma have identified the frequent presenceof chromosomal translocations and amplification (3). In a reviewof 28 published cases (4), a characteristic t(2;13)(q35;q14)translocation and a variant t(1;13)(p36;q14) translocation wereidentified in 64 and 18% of the cases respectively. Subsequentmolecular biology studies have demonstrated that these transloca-tions fuse the PAX3 gene on chromosome 2 or the PAX7 gene onchromosome 1 with the FKHR gene on chromosome 13 togenerate PAX3-FKHR or PAX7-FKHR fusion genes ( 5–8). Thesegenes encode chimeric transcription factors which, in the case ofPAX3-FKHR, have been shown to excessively activate transcrip-tion from binding targets of the wild-type PAX3 transcriptionfactor (9). Molecular assays for these fusion genes have identifiedadditional cases with cytogenetically undetectable fusions so thatthe overall frequency of PAX3/PAX7-FKHR fusions in alveolarrhabdomyosarcoma is >90% ( 10,11 , Barr et al ., unpublished data).Cytogenetic evidence of gene amplification was detected ineight of the 28 published cases (29%) of alveolar rhabdomyosar-coma (4). Two studies have used quantitative Southern blotassays to demonstrate amplification of the MYCN gene in four ofsix cases and three of seven cases of alveolar rhabdomyosarcoma(12,13). The wild-type MYCN protein product functions as atranscription factor regulating expression of growth-related genesand has previously been shown to be overproduced in neuroblas-toma as a result of MYCN gene amplification (14). Theamplification of other genomic loci in alveolar rhabdomyosarcomais indicated by the finding of one case with amplification of the12q region containing the GLI and CDK4 genes ( 15) and severalcases with double minute chromosomes without detectableamplification of MYCN or GLI (16).