FISH on interphase nuclei from paraffin embedded 4-micron sections was performed applying custom probes using bacterial artificial chromosomes (BAC), covering and flanking EWSR1 in 22q12, FUS in 16p11, PBX1 in 1q23, ZNF444 in 19q13 and POU5F1 in 6p21 (Fig. 1 ). BAC clones were chosen according to USCS genome browser (http://genome.uscs.edu ). The BAC clones were obtained from BACPAC sources of Children's Hospital of Oakland Research Institute (CHORI) (Oakland, CA) (http://bacpac.chori.org ). DNA from individual BACs was isolated according to the manufacturer’s instructions, labeled with different fluorochromes in a nick translation reaction, denatured, and hybridized to pretreated slides. Slides were then incubated, washed, and mounted with DAPI in an antifade solution, as previously described (Agaram et al., 2008 (link)). The genomic location of each BAC set was verified by hybridizing them to normal metaphase chromosomes. Two hundred successive nuclei were examined using a Zeiss fluorescence microscope (Zeiss Axioplan, Oberkochen, Germany), controlled by Isis 5 software (Metasystems). A positive score was interpreted when at least 20% of the nuclei showed a break-apart signal. Nuclei with incomplete set of signals were omitted from the score.
All cases were first tested with an EWSR1 probe. The EWSR1-rearranged tumors were then evaluated for break-apart signals using probes for PBX1, ZNF444, and POU5F1. The EWSR1 negative tumors were then tested for FUS break-apart, since FUS may substitute for the EWSR1 gene in certain translocation-associated sarcomas. In selective cases, two-color FISH was applied using probe-sets centromerically flanking one gene and telomerically flanking the partner gene, in order to confirm the fusion between EWSR1 and the partner genes. In one case a G-banded karyotype was obtained after short term culture.
All cases were first tested with an EWSR1 probe. The EWSR1-rearranged tumors were then evaluated for break-apart signals using probes for PBX1, ZNF444, and POU5F1. The EWSR1 negative tumors were then tested for FUS break-apart, since FUS may substitute for the EWSR1 gene in certain translocation-associated sarcomas. In selective cases, two-color FISH was applied using probe-sets centromerically flanking one gene and telomerically flanking the partner gene, in order to confirm the fusion between EWSR1 and the partner genes. In one case a G-banded karyotype was obtained after short term culture.
