Over the past several decades, molecular genetics has revolutionized the field of soft-tissue pathology and expanded our diagnostic abilities as cytopathologists. Many soft-tissue neoplasms are now known to harbor defining molecular genetic alterations, and at least one-third of tumor types have recurrent chromosomal translocations. Ewing sarcoma, the prototypical “small round blue cell tumor,” was the first sarcoma recognized to harbor a recurrent cytogenetic abnormality, most commonly the translocation t(11;22)(q24;q12).1 This translocation results in a fusion gene involving the Ewing sarcoma breakpoint region 1 gene (EWSR1) and the friend leukemia virus integration 1 gene (FLI1).2, 3 Since discovery of the EWSR1-FLI1 fusion in Ewing sarcoma, numerous studies have led to insights into the molecular mechanisms of tumorigenesis in Ewing sarcoma as well as the continued discovery of defining molecular alterations that have affected soft-tissue tumor classification, although much remains to be elucidated. The EWSR1 gene, encoded on chromosome 22q12, is a member of the TET family, a highly conserved group of multifunctional, RNA-binding proteins.4 EWSR1 has a 17-exon coding sequence, which encodes a 656-amino-acid protein that is highly conserved among TET family genes (including FUS and TAF15). Roles for EWSR1 have been implicated in transcription, DNA repair mechanisms, meiotic and mitotic cell division, and cellular aging. The N-terminus (exons 1-7) mediates transcriptional activation through degenerate repeats of the SYGQ motif, whereas the C-terminus contains an 87-amino-acid RNA-recognition motif encoded by exons 11 through 13.4 Although variable, the majority (80%) of the breakpoints in EWSR1 occur in intron 7 or 8, resulting in fusion of the EWSR1 N-terminus to heterologous DNA-binding domains of its partners.5, 6 EWSR1-FLI1 fusions have long been considered to result from a balanced translocation (ie, transposition of chromosomal material without loss of genetic material); however, recent studies have demonstrated that these fusions can arise in the context of chromoplexy in approximately 40% of Ewing sarcomas, which are associated with a poorer prognosis.7 Chromoplexy refers to the disruption of multiple, geographically distant genomic regions through multiple breakpoints, resulting in an accumulation of linked translocations that involve multiple chromosomes.8 In these cases of Ewing sarcoma that exhibit chromoplexy, looped chromosomal rearrangements are nearly always centered on EWSR1-FLI1 and simultaneously “weave” together additional genes (up to 18) with the fusion.7 Regardless of the mechanism, EWSR1-FLI1 fusion is considered an early and necessary pathogenetic event in Ewing sarcoma, and this is supported by studies showing that EWSR1-FLI is crucial for the growth and survival of Ewing sarcoma cells and sufficient for oncogenic transformation of primary mesenchymal stem cells.9, 10 Ewing sarcoma has served as an important paradigm in our study of fusion oncogenes, although the normal function of wild-type EWSR1 protein remains unknown. The EWSR1-FLI1 fusion protein acts as an aberrant transcription factor (EWSR1 providing the transcriptional activation domain, and FLI1 contributing the DNA-binding domain) that deregulates genes involved in tumorigenesis through a complex transcriptional program involving both gene activation and repression. In cell lines, EWSR1-FLI1 directly induces or represses enhancers through divergent chromatin-modeling mechanisms; EWSR1-FLI1–induced activation through opening chromatin and recruiting chromatin-modifying complexes occurs by binding at sites of GGAA repeat motifs, whereas repression occurs by the displacement of wild-type ETS transcription factors.6 EWSR1-FLI1 can bind and recruit the BRG1/BRM-associated factor chromatin-remodeling complex, which is an ATP-dependent chromatin remodeler known to be mutated in greater than 20% of human malignancies.11 Dysregulated transcription mediated by EWSR1-FLI1 is implicated in the sensitivity of Ewing sarcoma to genotoxic chemotherapies (etoposide being a first-line treatment); in Ewing sarcoma cell lines, gene transcription mediated by EWSR1-FLI1 leads to the frequent formation and accumulation of R-loops, which are 3-stranded nucleic acid structures composed of a DNA-RNA hybrid and nontemplated, single-stranded DNA. These R-loops are deleterious, inducing replication stress by impacting transcription and leading to DNA damage, possibly in part by the functional impairment of BRCA1.12 Studying EWSR1 in Ewing sarcoma has also led to insights into “fusion gene promiscuity.” FLI1 is a member of the ETS family of transcription factors, which are involved in many human cancers through diverse mechanisms of activation.13 Although most cases (approximately 90%) of Ewing sarcoma have EWSR1-FLI fusions, a small subset has variant EWSR1 fusions involving other members of the ETS family. The EWRSR1-ERG fusion is the second most common fusion in Ewing sarcoma (approximately 5%), and the ETS family members FEV, ETV1, ETV4, and ZSG substitute for FLI1 in rare cases.14 Given the homology between the TET family genes, it is also not surprising that rare cases of Ewing sarcoma harbor variant fusions of FUS in lieu of EWSR1. Furthermore, a broader group referred to as “Ewing sarcoma family tumors” have noncanonical EWSR1 fusions to non-ETS partners, including NFATC2, PATZ1, SP3ER, and SMARCA514; such tumors are morphologically indistinguishable from Ewing sarcoma and are currently treated on Ewing-specific regimens. Beyond Ewing sarcoma, EWSR1 rearrangements are now known to be the most frequent among all soft-tissue neoplasms that have recurrent chromosomal translocations. EWSR1 forms fusion genes with a wide spectrum of partner genes among clinicopathologically diverse soft-tissue tumors, as well as specific carcinomas and a subset of mesothelioma (Fig 1), and the list of tumor types with EWSR1 rearrangements and fusion partners continues to grow. Desmoplastic small round cell tumor has EWSR1-WT1 fusion,15 and extraskeletal myxoid chondrosarcoma harbors EWSR1-NR4A3 fusion.16 At least one-half of all soft-tissue myoepithelioma and myoepithelial carcinomas have EWSR1 rearrangements, with a broad range of partners including POU5F1, PBX1, ZNF444, KLF17, ATF1, and PBX3.17-22 One interesting aspect of this “promiscuity” is that identical fusion genes are present in tumor types that have entirely different histologic features and biologic potential, ranging from indolent to aggressive, such as EWSR1-ATF1 and EWSR1-CREB fusions, which are characteristic of clear cell sarcoma,23 clear cell sarcoma-like tumor of the gastrointestinal tract,24 primary pulmonary myxoid sarcoma,25 and angiomatoid fibrous histiocytoma.26 As stated above, EWSR1 and FUS can substitute for one another (although one usually predominates within a specific tumor type). Among neoplasms with FUS fusions, variant EWSR1 fusions occur in myxoid liposarcoma (EWSR1-DDIT3) and in low-grade fibromyxoid sarcoma and sclerosing epithelioid fibrosarcoma (EWSR1-CREB3L1/2).14 EWSR1 fusions have also been identified in nonmesenchymal tumors, including salivary clear cell carcinoma (EWSR1-ATF1),27 clear cell odontogenic tumor (EWSR1-ATF1),28 and mesothelioma (EWSR1-YY1 and EWSR1-ATF1).29, 30 Interestingly, the recently described entity “adamantinoma-like Ewing sarcoma” is characterized by EWSR1-FLI1 fusions; it is unclear whether this entity truly represents a true variant of Ewing sarcoma or is a carcinoma or sarcoma, and the debate brings into question whether this fusion is truly specific to Ewing sarcoma. It is still unclear why identical fusion genes can be present in diverse tumor types. Studies have suggested that tumorigenesis depends on the differentiation state of a cell and its cellular compartment31; additional genetic events likely also are a factor. The broad range of entities that are characterized by EWSR1 rearrangements has implications for the diagnostic role of molecular testing. Accurate classification of sarcomas is important, given that the results dictate appropriate clinical management, such as specific treatment regimens. Furthermore, many clinical trials require molecular confirmation in the diagnostic workup for patient enrollment. Although the diagnosis of soft-tissue neoplasms has long relied on the detection of recurrent molecular genetic alterations, the issue of nonspecificity of EWSR1 fusions can pose diagnostic pitfalls. EWSR1 fluorescence in situ hybridization (FISH) has been the conventional diagnostic approach, typically using break-apart probes; however, a positive result is not necessarily specific for one entity. Sequencing-based methods (including next-generation sequencing assays) can be helpful in identifying fusion partners to EWSR1 and offer more specific diagnostic information; however, these tests are not widely implemented or available in many practice settings. In practice, EWSR1 FISH remains the mainstay, and test selection and interpretation should be informed by the clinical, morphologic, and immunohistochemical features of a given tumor. For instance, although Ewing sarcoma and desmoplastic round cell tumor both show “small round blue cell” morphology, immunohistochemistry is helpful in discriminating between the two: Ewing sarcoma shows membranous CD99 staining and nuclear positivity for FLI-1 and NKX2.2, whereas desmoplastic small round cell tumor is positive for keratin, EMA, desmin, and WT-1 (using antibodies for the C-terminus). It should also be noted that because EWSR1 and FUS can substitute for one another, testing for FUS rearrangement (also conventionally by FISH) should be considered if EWSR1 FISH is unexpectedly negative. Practical applications of ancillary testing in the differential diagnosis of round cell sarcomas and other soft-tissue tumor differentials have been extensively reviewed,32-35 and the practicing cytopathologist should adhere to the judicious selection of immunohistochemical panels and molecular assays that are guided by clinical context and morphology. With increased adoption of next-generation sequencing assays, the selection of single tests may no longer be relevant in the future. Nonetheless awareness of the specific clinicopathologic features of the tumor types that have EWSR1 rearrangements is important for the appropriate interpretation of molecular testing results. No specific funding was disclosed. The author made no disclosures. Vickie Y. Jo received a BA at Williams College in Williamstown, Massachusetts, and an MD at the University of Virginia in Charlottesville, Virginia, where she also completed her Anatomic and Clinical Pathology residency. She did her fellowship training in soft-tissue pathology and cytopathology at Brigham and Women's Hospital (BWH) in Boston, Massachusetts, and joined the BWH faculty in 2012 and is currently an Assistant Professor at Harvard Medical School. She is active in the head and neck, soft-tissue, and cytopathology services at BWH and Dana-Farber Cancer Institute, with research interests in the clinicopathologic characterization of soft-tissue and head and neck neoplasms and the development of diagnostic biomarkers in surgical pathology and cytopathology.