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Sarcomas are a diverse group of tumors, with >70 subtypes in the current World Health Organization classification, each with distinct biological behavior requiring specific clinical management. A significant portion of sarcomas are molecularly defined by expression of a driver fusion gene; identification of such fusions is the basis of molecular diagnostics in sarcomas, which is of increasing complexity due to the ongoing discovery of new gene fusions. Recently, a multiplex NanoString platform-based assay was developed and clinically implemented, with fusion junction-spanning probes that detect the majority of sarcoma fusion types, with high sensitivity and specificity, and with lower cost and shorter turnaround time than those of targeted next-generation sequencing-based alternatives. Despite the effectiveness of this assay, there are several entities for which fusion-junction probes are not suitable due to multiple possible gene partners or excessive variability at the exon junctions. Here, the development and evaluation of a companion assay are described that uses NanoString-based gene expression analysis to detect aberrant 3'/5' exon expression imbalance and/or total gene overexpression as a surrogate marker for fusion gene rearrangement. This assay evaluates exon imbalance in 23 genes involved in over 25 mesenchymal tumor types and five genes specific to sarcomas with CIC rearrangements. Based on evaluation of 115 retrospectively and 91 prospectively collected cases, an assay sensitivity of 92.8% and specificity of 93.5% are demonstrated., (Copyright © 2023 Association for Molecular Pathology and American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

The NanoString nCounter assay is a high-throughput hybridization technique using target-specific probes that can be customized to test for numerous fusion transcripts in a single assay using RNA from formalin-fixed, paraffin-embedded material. We designed a NanoString assay targeting 174 unique fusion junctions in 25 sarcoma types. The study cohort comprised 212 cases, 96 of which showed fusion gene expression by the NanoString assay, including all 20 Ewing sarcomas, 11 synovial sarcomas, and 5 myxoid liposarcomas tested. Among these 96 cases, 15 showed fusion expression not identified by standard clinical assay, including EWSR1-FLI1, EWSR1-ERG, BCOR-CCNB3, ZC3H7B-BCOR, HEY1-NCOA2, CIC-DUX4, COL1A1-PDGFB, MYH9-USP6, YAP1-TFE3, and IRF2BP2-CDX1 fusions. There were no false-positive results; however, four cases were false negative when compared with clinically available fluorescence in situ hybridization or RT-PCR testing. When batched as six cases, the per-sample reagent cost was less than conventional techniques, such as fluorescence in situ hybridization, with technologist hands-on time of 1.2 hours per case and assay time of 36 hours. In summary, the NanoString nCounter Sarcoma Fusion CodeSet reliably and cost-effectively identifies fusion genes in sarcomas using formalin-fixed, paraffin-embedded material, including many fusions missed by standard clinical assays, and can serve as a first-line clinical diagnostic test for sarcoma fusion gene identification, replacing multiple individual clinical assays., (Copyright © 2018 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.)

Ewing family tumors are molecularly characterized by expression of chimeric transcripts generated by specific chromosomal translocations, most commonly involving fusion of the EWS gene to a member of the ETS family of transcription factors (including FLI1, ERG, ETV1, E1AF, and FEV). Approximately 85% of reported cases of Ewing sarcoma bear an EWS-FLI1 fusion. In rare cases, FUS can substitute for EWS, with translocation t(16;21)(p11;q24) producing a FUS-ERG fusion with no EWS rearrangement. We report a case of Ewing sarcoma, presenting as a pathological fracture of the distal clavicle in a 33-year-old male, in which cytogenetic analysis revealed a single t(2;16)(q35;p11) balanced translocation. Fluorescence in situ hybridization using a commercially available diagnostic probe was negative for an EWS gene rearrangement; instead, break-apart fluorescence in situ hybridization probes for FUS and FEV were positive for a translocation involving these genes. Cloning and sequencing of the breakpoint region demonstrated an in-frame fusion of FUS to FEV. In conclusion, this represents the first reported case of Ewing family tumors demonstrating a variant translocation involving FUS and FEV and highlights the need to consider alternative permutations of fusion partners for molecular diagnosis of sarcomas.