Cytogenetic studies of the pediatric solid tumor alveolar rhabdomyosarcoma have demonstrated the presence of a consistent chromosomal translocation, t(2 I3)(q35;q 14). We recently identified PAX3 and FKHR as the genes on chromosomes 2 and I 3, respectively, that are juxtaposed by this translocation. As one means of detecting the t ( 2 I 3) translocation in clinical specimens, we have developed a fluorescence in situ hybridization (FISH) assay that may be used for both interphase and metaphase cells.
Translocation of the 5' region of the FKHR gene t o the derivative chromosome 2, and retention of the 3' region of FKHR on the derivative chromosome I 3 [(der( I3)], were demonstrated in metaphase cells from a rhabdomyosarcoma cell line with a previously identified t(2; 13) translocation. A 5' PAX3 cosmid probe was shown t o localize t o 2q35 in normal cells, and to translocate t o the der( 13) in the rhabdomyosarcoma cell line. In order t o detect the der( 13) in interphase nuclei, we labeled the 3'FKHR and the S'PAX3 cosmid probes with digoxigenin and biotin, respectively, and used these in a two-color FISH assay.
The presence of the der( 13) was visualized as juxtaposed or overlapping red and green signals in metaphase and interphase tumor cells. The PAX3-FKHR FISH assay was then applied t o a series of cytogenetically characterized pediatric sarcoma cell lines.
The presence of the der( ) was demonstrated by FISH in all cases containing a cytogenetically detectable t(2 13). The FISH assay was then applied t o a series of 20 embryonal and alveolar rhabdomyosarcoma samples. All 10 of the alveolar rhabdomyosarcoma specimens demonstrated a der( I 3) with the FISH assay. We did not detect a fAX3-FKHR fusion in I0 embryonal rhabdomyosarcoma cases. Thus, the two-color FISH assay is a sensitive and rapid means of identifying the t(2; I 3) in rhabdomyosarcoma specimens, and it will be a useful adjunct for the diagnosis of pediatric small round cell tumors. The cosmid probes for the 5' and 3' regions of FKHR, as well as the probe for PAX3, will be useful for molecular cytogenetic studies of variant translocations in rhabdomyosarcoma, such as the t ( 1;13)(p36;q14). Genes Chromosom Cancer /2:/86-/92 (I995).