FISH was performed on interphase nuclei from FFPE tissue sections of three undifferentiated uterine sarcomas and all leiomyosarcomas with TrkA or pan-Trk expression. Custom probes using bacterial artificial chromosomes (BAC) flanking NTRK1, NTRK3, LMNA, TPR, and TPM3 genes were chosen according to the UCSC genome browser (http://genome.ucsc.edu). BAC clones were retrieved from BACPAC sources of the Children’s Hospital of Oakland Research Institute (CHORI, Oakland, CA) (http://bacpac.chori.org) (Supplementary Table 1). The DNA from individual BACs were isolated according to the manufacturer’s recommendations, labeled with nick translation, and validated on normal metaphase chromosomes. Whole tissue 5 μm tumor sections mounted on charged slides were deparaffinized, pretreated, and hybridized with denatured probes overnight, followed by post-hybridization washes and counterstaining with DAPI. Slides were examined on a Zeiss fluorescence microscope (Zeiss Axioplan, Oberkochen, Germany) using Isis 5 software (Metasystems). Two hundred tumor nuclei were counted, and gene rearrangement was confirmed if >20% of tumor nuclei demonstrated break-apart signals. As the NTRK1 gene on 1q25 is located in close proximity to its gene partners (LMNA, TPR and TPM3) on 1q21-23, we also applied a two color-fusion assay which can document the fusion with more confidence, as the standard break-apart assay may not be definitive due to the small gaps detected. Thus NTRK1 telomeric portion was labeled in green, while centromeric LMNA, TPR and TPM3 were labeled in red. A come-together signal (yellow), seen in >20% of tumor nuclei, was considered a positive result 14 (link).