[Primary pulmonary myxoid sarcoma with EWSR1-CREB1 fusion]. / Pervichnaya legochnaya miksoidnaya sarkoma so sliyaniem genov EWSR1-CREB1.

Primary pulmonary myxoid sarcoma with EWSR1-CREB1 fusion is an extremely rare tumor. Its clinical manifestation is unspecific and only molecular genetic method can proof this diagnosis. This paper describes an unusual clinical presentation of primary pulmonary myxoid sarcoma in a 68-year-old patient with involvement of both lungs.

FLI1 induces erythroleukemia through opposing effects on UBASH3A and UBASH3B expression.

BACKGROUND: FLI1 is an oncogenic transcription factor that promotes diverse malignancies through mechanisms that are not fully understood. Herein, FLI1 is shown to regulate the expression of Ubiquitin Associated and SH3 Domain Containing A/B (UBASH3A/B) genes. UBASH3B and UBASH3A are found to act as an oncogene and tumor suppressor, respectively, and their combined effect determines erythroleukemia progression downstream of FLI1. METHODS: Promoter analysis combined with luciferase assays and chromatin immunoprecipitation (ChIP) analysis were applied on the UBASH3A/B promoters. RNAseq analysis combined with bioinformatic was used to determine the effect of knocking-down UBASH3A and UBASH3B in leukemic cells. Downstream targets of UBASH3A/B were inhibited in leukemic cells either via lentivirus-shRNAs or small molecule inhibitors. Western blotting and RT-qPCR were used to determine transcription levels, MTT assays to assess proliferation rate, and flow cytometry to examine apoptotic index. RESULTS: Knockdown of FLI1 in erythroleukemic cells identified the UBASH3A/B genes as potential downstream targets. Herein, we show that FLI1 directly binds to the UBASH3B promoter, leading to its activation and leukemic cell proliferation. In contrast, FLI1 indirectly inhibits UBASH3A transcription via GATA2, thereby antagonizing leukemic growth. These results suggest oncogenic and tumor suppressor roles for UBASH3B and UBASH3A in erythroleukemia, respectively. Mechanistically, we show that UBASH3B indirectly inhibits AP1 (FOS and JUN) expression, and that its loss leads to inhibition of apoptosis and acceleration of proliferation. UBASH3B also positively regulates the SYK gene expression and its inhibition suppresses leukemia progression. High expression of UBASH3B in diverse tumors was associated with worse prognosis. In contrast, UBASH3A knockdown in erythroleukemic cells increased proliferation; and this was associated with a dramatic induction of the HSP70 gene, HSPA1B. Accordingly, knockdown of HSPA1B in erythroleukemia cells significantly accelerated leukemic cell proliferation. Accordingly, overexpression of UBASH3A in different cancers was predominantly associated with good prognosis. These results suggest for the first time that UBASH3A plays a tumor suppressor role in part through activation of HSPA1B. CONCLUSIONS: FLI1 promotes erythroleukemia progression in part by modulating expression of the oncogenic UBASH3B and tumor suppressor UBASH3A.

A Comparison of Clear Cell Sarcoma to Jaw and Salivary Tumors Bearing EWS Fusions.

OBJECTIVE: To review tumors identified as "clear cell sarcoma" in order to determine similarities to the rare EWS fusion positive jaw and salivary gland tumors clear cell odontogenic carcinoma (CCOC) and clear cell carcinoma of the salivary gland (CCC). METHODS: PubMed was used to collect all reports of clear cell sarcoma (CCS). Search parameters were "clear cell sarcoma" and "CCS." References in the publications were screened and cross-referenced. Data extracted included demographic characteristics, presenting signs and symptoms, radiographic findings, histological and immunohistochemical features and known molecular/genetic aberrations. RESULTS: Clear cell sarcoma has several similarities to CCOC and CCC. All three tumor types have similar histologic appearances including the presence of clear cells, as well as similar genetic profiles in that all harbor an EWSR1-CREB family fusions. Additionally, these tumors appear in soft tissue as well as bone, and can have a prolonged clinical course. CCS can appear anywhere in the body, including the head and neck region. All three tumors appear to have a predilection to women, although CCS may have a slight younger age of onset as compared to CCOC and CCC (3rd vs 5th decade of life, respectively). CONCLUSION: Gaining a better understanding of the similarities and differences between these three tumors may lead to a better understanding of each one.

Endogenous EWSR1 Exists in Two Visual Modalities That Reflect Its Associations with Nucleic Acids and Concentration at Sites of Active Transcription.

EWSR1 is a member of the FET family of nucleic acid binding proteins that includes FUS and TAF15. Here, we report the systematic analysis of endogenous EWSR1's cellular organization in human cells. We demonstrate that EWSR1, which contains low complexity and nucleic acid binding domains, is present in cells in faster and slower-recovering fractions, indicative of a protein undergoing both rapid exchange and longer-term interactions. The employment of complementary high-resolution imaging approaches shows EWSR1 exists in two visual modalities, a distributed state which is present throughout the nucleoplasm, and a concentrated state consistent with the formation of foci. Both EWSR1 visual modalities localize with nascent RNA. EWSR1 foci concentrate in regions of euchromatin, adjacent to protein markers of transcriptional activation, and significantly colocalize with phosphorylated RNA polymerase II. Our results contribute to bridging the gap between our understanding of the biophysical and biochemical properties of FET proteins, including EWSR1, their functions as transcriptional regulators, and the participation of these proteins in tumorigenesis and neurodegenerative disease.

The diagnostic utility of cytology specimen in a case of EWSR1::NFATC2 sarcoma.

EWSR1::NFATC2 sarcoma, a rare round cell sarcoma constituting the majority of EWSR1::non-ETS sarcomas, has recently been defined in the latest WHO classification. To date, the cytological findings of EWSR1::NFATC2 sarcoma remain undocumented. We present the case of a 25-year-old man with a history of polyostotic fibrous dysplasia in the right leg, referred to our hospital with left thigh pain. Cytological findings included metachromasia, minimally pleomorphic round cells, and eosinophilic infiltration. There was no precursor fibrous dysplasia and the initial diagnosis was undifferentiated pleomorphic sarcoma. Following histologic review, we successfully performed immunocytochemistry and fluorescence in situ hybridization (FISH) on archival cytology specimens. The tumor cells were positive for NKX2-2, NKX3-1, and PAX7 and showed amplified 5' single signals of EWSR1 gene. Reverse transcriptase-polymerase chain reaction revealed an in-frame fusion of EWSR1 and NFATC2. This report describes the cytological features of EWSR1::NFATC2 sarcoma and highlights the diagnostic utility of archival cytology specimens.

Primary pulmonary myxoid sarcoma with EWSR1::CREB1 fusion: a literature review.

PURPOSE: This review primarily aims to review the epidemiology, clinical characteristics, imaging, pathology, immunohistochemistry, diagnosis, differential diagnosis, treatment, and prognosis of Primary pulmonary myxoid sarcoma (PPMS) with EWS RNA binding protein 1::cAMP response element binding protein 1 (EWSR1::CREB1) fusion. It provides reference for the diagnosis and treatment of this disease. METHODS: Retrospectively collected the literature about PPMS with EWSR1::CREB1 fusion, its clinical, radiology, histology, molecular characteristics and current treatment strategies were collated and analyzed. This review provides a detailed differential diagnosis of the disease. RESULTS: PPMS is an exceptionally rare, low-grade malignant tumor of the lung. This tumor commonly infiltrates lung tissue and develops within bronchial passages. It is identified by a genetic rearrangement involving the EWSR1 gene and a distinct chromosomal translocation t(2; 22)(q33; q12). Variants include EWSR1::CREB1 fusion and EWS RNA binding protein 1::activating transcription factors (EWSR1::ATF1) fusion. PPMS with EWSR1::CREB1 fusion is more prevalent among middle-aged individuals and affects both sexes almost equally. Clinical symptoms are relatively non-specific, primarily including cough, hemoptysis, and weight loss. Most patients undergo surgery and experience a favorable prognosis. Further research is required to validate the effectiveness of alternative treatments for PPMS with EWSR1::CREB1 fusion. CONCLUSION: EWSR1 rearrangement and EWSR1::CREB1 fusion are crucial genetic features of PPMS and serve as important diagnostic markers. Immunohistochemically, PPMS tests positive for EMA. In terms of treatment, surgery has been the primary approach in recent years. Therefore, the efficacy of other treatments still requires further investigation.

Identification of Novel/Rare EWSR1 Fusion Partners in Undifferentiated Mesenchymal Neoplasms.

Recurrent gene fusions (GFs) in translocated sarcomas are recognized as major oncogenic drivers of the disease, as well as diagnostic markers whose identification is necessary for differential diagnosis. EWSR1 is a 'promiscuous' gene that can fuse with many different partner genes, defining different entities among a broad range of mesenchymal neoplasms. Molecular testing of EWSR1 translocation traditionally relies on FISH assays with break-apart probes, which are unable to identify the fusion partner. Therefore, other ancillary molecular diagnostic modalities are being increasingly adopted for accurate classification of these neoplasms. Herein, we report three cases with rare GFs involving EWSR1 in undifferentiated mesenchymal neoplasms with uncertain differential diagnoses, using targeted RNA-seq and confirming with RT-PCR and Sanger sequencing. Two GFs involved hormone nuclear receptors as 3' partners, NR4A2 and RORB, which have not been previously reported. NR4A2 may functionally replace NR4A3, the usual 3' partner in extraskeletal myxoid chondrosarcoma. The third GF, EWSR1::BEND2, has previously been reported in a subtype of astroblastoma and other rare entities, including a single case of a soft-tissue tumor that we discuss in this work. In conclusion, our findings indicate that the catalogue of mesenchymal neoplasm-bearing EWSR1 fusions continues to grow, underscoring the value of using molecular ancillary techniques with higher diagnostic abilities in the routine clinical setting.

Report and literature review of four cases of EWSR1::NFATC2 round cell sarcoma.

BACKGROUND: EWSR1::NFATC2 rearranged sarcomas are a group of rare round, undifferentiated sarcomas with clinicopathological features different from those of Ewing's sarcoma (ES) family and other non-ES sarcomas. We report 4 cases of this rare sarcoma and review their features. MATERIALS AND METHODS: Four cases of EWSR1::NFATC2 rearranged round cell sarcoma of the bone from the Pathology Department of Peking University People's Hospital were retrospectively studied. Clinical and pathological data were summarized, and immunohistochemical staining, fluorescence in situ hybridization (FISH), and Next-generation sequencing (NGS) were performed. Relevant literature reports were also reviewed. RESULTS: Among the four cases of EWSR1::NFATC2 rearranged round cell sarcoma, three were male, and one was female, with the age ranged from 14 to 34 years old at diagnosis (mean age: 27.5 years). All tumors were located in the femur and ranged in size from 4 to 8cm (mean 6cm), involving the surrounding soft tissues. All four patients underwent surgical treatment, and three received chemotherapy and radiotherapy postoperatively. Follow-up results showed that all four patients were alive. Histologically, the tumors exhibited small round cell sarcoma phenotype, with the stroma rich in mucin or exhibiting a glassy appearance. The tumor cells diffusely expressed CD99, NKX2.2, NKX3.1 and focal expression of CK and EMA was observed. FISH analysis showed that EWSR1 gene rearrangement was detected in all 4 cases, accompanied by 5' locus amplification. EWSR1::NFATC2 fusion probe demonstrated multi yellow fusion signals. NGS identified EWSR1::NFATC2 breakpoints in exon 9 and exon 3 in all 4 cases. The average follow-up duration of the study group was 88 months (range from 26-180 months). One case experienced both local recurrence and metastasis to the lung and chest wall. One case presented with local recurrence. The remaining two cases did not have the recurrence or metastasis. CONCLUSION: Although the disease can locally recur and metastasize to the lungs, its mortality rate is significantly lower than that of Ewing sarcoma and other high-grade small round cell undifferentiated sarcomas. Therefore, it supports to classify this tumor as a separate subtype of small round cell sarcoma.

[Clinicopathological study of epithelioid and spindle cell rhabdomysarcoma with EWSR1/FUS-TFCP2 fusion].

Objective: To investigate the clinicopathological and genetic features of epithelioid and spindle cell rhabdomysarcoma with EWSR1-TFCP2 or FUS-TFCP2 fusion. Methods: The clinical, morphological and immunohistochemical features of 14 cases of epithelioid and spindle cell rhabdomysarcoma with EWSR1-TFCP2 or FUS-TFCP2 fusion diagnosed from January 2019 to December 2022 in the Department of Pathology, Foshan Traditional Chinese Medicine Hospital, Foshan, China were retrospectively analyzed. The cases were all subject to FISH or next generation sequencing for analysis of molecular genetic features. The literature was reviewed. Results: There were 5 males and 9 females, with the age at presentation ranging from 6 to 36 years (mean, 22 years). Tumors occurred in the head and neck (9 cases), pelvic region (2 cases), bladder (one case), right humerus (one case), and the abdominal wall, humerus and pubic at the same time (one case). Presenting symptoms varied by location but often included pain or discomfort. Most of the patients showed aggressive radiographic features with soft tissue extension. The tumors had a median size of 6.6 cm (range, 2-23 cm). The tumors were poorly defined and irregularly shaped. Microscopic examination showed diffuse proliferation of spindle or epithelioid cells. While morphologically high-grade tumors displayed obvious cytological atypia, a high mitotic count and tumor necrosis, low-grade tumors grew in sheets and fascicles composed of spindle, epithelioid cells with moderate or abundant amounts of eosinophilic cytoplasm, without pronounced cytological atypia. The tumor cells expressed Desmin, MyoD1, and Myogenin, as well as ALK, EMA, and CKpan. EWSR1/FUS-TFCP2 gene fusion was detected in 14 cases with next generation sequencing and confirmed by FISH. Six cases had EWSR1-TFCP2 fusions and 8 cases showed FUS-TFCP2 fusions. Follow-up information was available in 13 patients, ranged from 5 to 37 months. At the end of follow-up period, 7 patients died of the disease. Six patients were alive:two cases had local recurrences and metastases, two cases of recurrences, one case of metastasis and one case without recurrences and metastasis. Conclusions: Epithelioid and spindle cell rhabdomysarcomas with EWSR1-TFCP2 or FUS-TFCP2 fusion show a very aggressive clinical course, and more commonly occur in the head and neck. Their genetic hallmark is the presence of EWSR1/FUS-TFCP2 fusions. Familiarity with its clinicopathological characteristics is helpful in avoiding misdiagnoses.

Multi-omic and functional analysis for classification and treatment of sarcomas with FUS-TFCP2 or EWSR1-TFCP2 fusions.

Linking clinical multi-omics with mechanistic studies may improve the understanding of rare cancers. We leverage two precision oncology programs to investigate rhabdomyosarcoma with FUS/EWSR1-TFCP2 fusions, an orphan malignancy without effective therapies. All tumors exhibit outlier ALK expression, partly accompanied by intragenic deletions and aberrant splicing resulting in ALK variants that are oncogenic and sensitive to ALK inhibitors. Additionally, recurrent CKDN2A/MTAP co-deletions provide a rationale for PRMT5-targeted therapies. Functional studies show that FUS-TFCP2 blocks myogenic differentiation, induces transcription of ALK and truncated TERT, and inhibits DNA repair. Unlike other fusion-driven sarcomas, TFCP2-rearranged tumors exhibit genomic instability and signs of defective homologous recombination. DNA methylation profiling demonstrates a close relationship with undifferentiated sarcomas. In two patients, sarcoma was preceded by benign lesions carrying FUS-TFCP2, indicating stepwise sarcomagenesis. This study illustrates the potential of linking precision oncology with preclinical research to gain insight into the classification, pathogenesis, and therapeutic vulnerabilities of rare cancers.

Sarcomas with EWSR1::Non-ETS Fusion (EWSR1::NFATC2 and EWSR1::PATZ1).

The wide application of increasingly advanced molecular studies in routine clinical practice has allowed a detailed, albeit still incomplete, genetic subclassification of undifferentiated round cell sarcomas. The WHO classification continues to include provisional molecular entities, whose clinicopathologic features are in the early stages of evolution. This review focuses on the clinicopathologic, molecular, and prognostic features of undifferentiated round cell sarcomas with EWSR1/FUS::NFATC2 or EWSR1::PATZ1 fusions. Classic histopathologic findings, uncommon variations, and diagnostic pitfalls are addressed, along with the utility of recently developed immunohistochemical and molecular markers.

Sarcomas Harboring EWSR1::PATZ1 Fusions: A Clinicopathologic Study of 17 Cases.

Soft tissue sarcomas harboring EWSR1::PATZ1 are a recently recognized entity with variable morphology and a heterogeneous immunohistochemical profile. We studied 17 such tumors. The tumors occurred in 12 men and 5 women (median age, 50 years; range, 15-71 years), involved the thoracoabdominal soft tissues (14 cases; 82%), lower extremities (2 cases; 12%), and tongue (1 case; 6%), and ranged from 0.7 to 11.3 cm (median, 4.7 cm). All but 1 patient received complete surgical resection; 7 were also treated with neoadjuvant chemo/radiotherapy. All cases showed typical features of EWSR1::PATZ1 sarcoma, including uniform round to spindled cells, fibromyxoid matrix, fibrous bands, hyalinized vessels, and pseudoalveolar/microcystic spaces. Unusual features, seen in a subset of cases, included degenerative-appearing nuclear atypia, epithelioid cytomorphology, mature fat, abundant rhabdomyoblasts, high mitotic activity, and foci with increased cellularity and nuclear atypia. Positive immunohistochemical results were desmin (16/17, 94%), MyoD1 (13/14, 93%), myogenin (6/14, 43%), GFAP (10/10, 100%), S100 protein (15/17, 88%), SOX10 (7/13, 54%), keratin (10/17, 59%), CD99 (4/11, 36%), H3K27me3 (retained expression 9/9, 100%), p16 (absent expression 1/4, 25%), and p53 (wild type 3/3, 100%). Fusion events included EWSR1 exon 8::PATZ1 exon 1 (14/17, 82%), EWSR1 exon 9::PATZ1 exon 1 (2/17, 12%), and EWSR1 exon 7::PATZ1 exon 1 (1/17, 6%). No evaluated tumor had alterations of CDKN2A/B and/or TP53, or MDM2 amplification. Clinical follow-up (16 patients: median, 13.5 months; range, 1-77 months) showed distant metastases in 3 patients (1/3 at time of presentation) and no local recurrences. At the time of last follow-up, 14 patients were disease free, 1 was alive with disease, 1 was dead of disease (at 13 months), and 1 had an indeterminant pulmonary nodule. We conclude that the morphologic spectrum of EWSR1::PATZ1 is broader than has been previously appreciated. Although more long-term follow-up is needed, the prognosis of these very rare sarcomas may be more favorable than previously reported.

Gastrointestinal Ewing Sarcoma: A Clinicopathological and Molecular Genetic Analysis of 25 Cases.

Occurrence of extraskeletal Ewing sarcoma (ES) in the gastrointestinal (GI) tract is extremely rare. Here, we report 25 cases of ES arising primarily in the GI tract with a focus on the clinicopathological and molecular features, differential diagnosis, and biological behavior. Thirteen patients (52%) were male, and 12 (48%) were female with age ranging from 9 to 59 years (mean: 36.2 years; median: 38 years). Twenty-one tumors (84%) occurred in the small intestine, 3 (12%) in the stomach, and 1 (4%) in the anal canal. At operation, 8/18 (44.4%) patients presented with abdominopelvic disseminated disease. Tumor size measured from 2 to 25 cm (mean: 8.2 cm; median: 6 cm) in maximum size. Microscopically, the tumors were composed of infiltrative small round, ovoid, or short spindle cells arranged mostly in lobular and solid sheet-like patterns with a rich capillary vasculature. Focal formation of Homer Wright-type rosettes and pseudoalveolar architecture was noted each in 2 (8%) cases and 3 (12%) cases. Besides CD99 (25/25; 100%), Fli-1 (15/15, 100%), and NKX2.2 (14/16; 87.5%), the tumor cells also showed variable staining of CD117 (14/17; 82.4%). Of 25 cases, 23 (92%) demonstrated EWSR1 rearrangement by fluorescence in situ hybridization analysis. The 2 cases with negative fluorescence in situ hybridization results were found to harbor EWSR1::ERG and EWSR1::FLI1 fusion by further RNA sequencing, respectively, with a median follow-up of 12 months (range: 1 to 42 months), 5/19 (26.3%) patients developed visceral metastasis and 12/19 (63.2%) patients died of the disease (range:1 to 33 months; median: 9 months). This study showed that GI ES had a predilection for the small intestine, although other sites of the GI tract could also be involved. GI ES had a poor prognosis with a high rate of mortality, particularly in patients with abdominopelvic disseminated disease. In light of appropriate therapeutic strategies and prognostic considerations, it is essential not to misdiagnose GI ES as gastrointestinal stromal tumor owing to the expression of aberrant CD117.

Expanding the molecular landscape of undifferentiated sarcomas of bone with a novel EWSR1-SSX3 gene fusion.

Undifferentiated sarcomas characterized by a primitive monomorphic round to spindle cell phenotype and often non-specific immunoprofile remain difficult to subclassify outside molecular analysis. The increased application of RNA sequencing in clinical practice led to significant advances and discoveries of novel gene fusions that furthered our understanding and refined classification of otherwise undifferentiated neoplasms. In this study, we report an undifferentiated round to spindle cell sarcoma arising in the femur of a 34-year-old female. The round to spindle tumor cells were arranged in short fascicles, with focal rosette formation, within a hyalinized stroma. The tumor immunoprofile included diffuse reactivity for CD99, SATB2, and TLE1 and patchy positivity for Cyclin D1, Keratin AE1/AE3, synaptophysin, and chromogranin. Other markers, such as EMA, SMA, desmin, S100, ERG, and WT1, were negative. Fluorescence in situ hybridization analysis for EWSR1 gene alterations showed a break-apart signal and targeted RNA sequencing revealed an EWSR1::SSX3 gene fusion. The patient received neoadjuvant chemotherapy followed by surgery and subsequently relapsed in less than a year with lung metastasis. Larger series are needed to determine if this fusion defines a novel subset of undifferentiated tumors or represents a genomic variant of already existing primitive round cell sarcoma categories, such as Ewing sarcoma or synovial sarcoma.

PATZ1-Rearranged Tumors of the Central Nervous System: Characterization of a Pediatric Series of Seven Cases.

PATZ1-rearranged sarcomas are well-recognized tumors as part of the family of round cell sarcoma with EWSR1-non-ETS fusions. Whether PATZ1-rearranged central nervous system (CNS) tumors are a distinct tumor type is debatable. We thoroughly characterized a pediatric series of PATZ1-rearranged CNS tumors by chromosome microarray analysis (CMA), DNA methylation analysis, gene expression profiling and, when frozen tissue is available, optical genome mapping (OGM). The series consisted of 7 cases (M:F=1.3:1, 1-17 years, median 12). On MRI, the tumors were supratentorial in close relation to the lateral ventricles (intraventricular or iuxtaventricular), preferentially located in the occipital lobe. Two major histologic groups were identified: one (4 cases) with an overall glial appearance, indicated as "neuroepithelial" (NET) by analogy with the corresponding methylation class (MC); the other (3 cases) with a predominant spindle cell sarcoma morphology, indicated as "sarcomatous" (SM). A single distinct methylation cluster encompassing both groups was identified by multidimensional scaling analysis. Despite the epigenetic homogeneity, unsupervised clustering analysis of gene expression profiles revealed 2 distinct transcriptional subgroups correlating with the histologic phenotypes. Interestingly, genes implicated in epithelial-mesenchymal transition and extracellular matrix composition were enriched in the subgroup associated to the SM phenotype. The combined use of CMA and OGM enabled the identification of chromosome 22 chromothripsis in all cases suitable for the analyses, explaining the physical association of PATZ1 to EWSR1 or MN1. Six patients are currently disease-free (median follow-up 30 months, range 12-92). One patient of the SM group developed spinal metastases at 26 months from diagnosis and is currently receiving multimodal therapy (42 months). Our data suggest that PATZ1-CNS tumors are defined by chromosome 22 chromothripsis as causative of PATZ1 fusion, show peculiar MRI features (eg, relation to lateral ventricles, supratentorial frequently posterior site), and, although epigenetically homogenous, encompass 2 distinct histologic and transcriptional subgroups.

Expanding the Spectrum of EWSR1::CREM Fusion Tumors: An Unusual Pediatric Intranasal Myxoid Tumor.

EWSR1::CREM gene fusions are increasingly being recognized in a diverse number of soft tissue tumors, including well-defined entities such as angiomatoid fibrous histiocytoma or clear cell sarcoma, and other unclassifiable tumors. As a group, EWSR1::CREM fused tumors often demonstrate primitive spindle or epithelioid cells, myxoid stroma, and a broad immunophenotype. Herein we present an unusual case of a child diagnosed with an intranasal malignant myxoid tumor harboring an EWSR1::CREM gene fusion. To the best of our knowledge, this is the first case of intranasal myxoid tumor with this particular fusion. Diagnosis and management of the case is discussed.

The DNA/RNA helicase DHX9 orchestrates the KDM2B-mediated transcriptional regulation of YAP1 in Ewing sarcoma.

Ewing sarcomas (ES) are aggressive paediatric tumours of bone and soft tissues. Resistance to chemotherapy and high propensity to metastasize remain the main causes of treatment failure. Thus, identifying novel targets for alternative therapeutic approaches is urgently needed. DNA/RNA helicases are emerging as crucial regulators of many cellular processes often deregulated in cancer. Among them, DHX9 is up-regulated in ES and collaborates with EWS-FLI1 in ES transformation. We report that DHX9 silencing profoundly impacts on the oncogenic properties of ES cells. Transcriptome profiling combined to bioinformatic analyses disclosed a gene signature commonly regulated by DHX9 and the Lysine Demethylase KDM2B, with the Hippo pathway regulator YAP1 as a prominent target. Mechanistically, we found that DHX9 enhances H3K9 chromatin demethylation by KDM2B and favours RNA Polymerase II recruitment, thus promoting YAP1 expression. Conversely, EWS-FLI1 binding to the promoter represses YAP1 expression. These findings identify the DHX9/KDM2B complex as a new druggable target to counteract ES malignancy.

EWSR1::WT1 Fusions in Neoplasms Other Than Conventional Desmoplastic Small Round Cell Tumor: Three Tumors Occurring Outside the Female Genital Tract.

Desmoplastic small round cell tumor (DSRCT) is a high-grade, primitive round cell sarcoma classically associated with prominent desmoplastic stroma, coexpression of keratin and desmin, and a characteristic EWSR1::WT1 gene fusion. DSRCT typically arises in the abdominopelvic cavity of young males with diffuse peritoneal spread and poor overall survival. Although originally considered to be pathognomonic for DSRCT, EWSR1::WT1 gene fusions have recently been detected in rare tumors lacking the characteristic morphologic and immunohistochemical features of DSRCT. Here, we report 3 additional cases of neoplasms other than conventional DSCRCT with EWSR1::WT1 gene fusions that occurred outside the female genital tract. Two occurred in the abdominopelvic cavities of a 27-year-old man and a 12-year-old girl, whereas the third arose in the axillary soft tissue of an 85-year-old man. All cases lacked prominent desmoplastic stroma and were instead solid and cystic with peripheral fibrous pseudocapsules and occasional intervening fibrous septa. Necrosis was either absent (1/3) or rare (2/3), and mitotic activity was low (<1 to 3 per 10 hpf). In immunohistochemical studies, there was expression of smooth muscle actin (3/3) and desmin (3/3), rare to focal reactivity for EMA (2/3), and variable expression of CK AE1/AE3 (1/3). Myogenin and MyoD1 were negative, and C-terminus-specific WT1 was positive in both cases tested (2/2). All 3 tumors followed a more indolent clinical course with 2 cases demonstrating no evidence of disease at 20 and 44 months after resection. The patient from case 3 died of other causes at 14 months with no evidence of recurrence. DNA methylation profiling showed that the 3 cases clustered with DSRCT; however, they demonstrated fewer copy number variations with 2 cases having a flat profile (0% copy number variation). Differential methylation analysis with hierarchical clustering further showed variation between the 3 cases and conventional DSRCT. Although further study is needed, our results, in addition to previous reports, suggest that EWSR1::WT1 gene fusions occur in rare and seemingly distinctive tumors other than conventional DSRCT with indolent behavior. Proper classification of these unusual soft tissue tumors with EWSR1::WT1 gene fusions requires direct correlation with tumor morphology and clinical behavior, which is essential to avoid overtreatment with aggressive chemotherapy.

Spindle cell rhabdomyosarcomas: With TFCP2 rearrangements, and novel EWSR1::ZBTB41 and PLOD2::RBM6 gene fusions. A study of five cases and review of the literature.

AIMS: Spindle-cell/sclerosing rhabdomyosarcomas (SS-RMS) are clinically and genetically heterogeneous. They include three well-defined molecular subtypes, of which those with EWSR1/FUS::TFCP2 rearrangements were described only recently. This study aimed to evaluate five new cases of SS-RMS and to perform a clinicopathological and statistical analysis of all TFCP2-rearranged SS-RMS described in the English literature to more comprehensively characterize this rare tumour type. METHODS AND RESULTS: Cases were retrospectively selected and studied by immunohistochemistry, fluorescence in situ hybridization with EWSR1/FUS and TFCP2 break-apart probes, next-generation sequencing (Archer FusionPlex Sarcoma kit and TruSight RNA Pan-Cancer Panel). The PubMed database was searched for relevant peer-reviewed English reports. Five cases of SS-RMS were found. Three cases were TFCP2 rearranged SS-RMS, having FUSex6::TFCP2ex2 gene fusion in two cases and triple gene fusion EWSR1ex5::TFCP2ex2, VAX2ex2::ALKex2 and VAX2intron2::ALKex2 in one case. Two cases showed rhabdomyoblastic differentiation and spindle-round cell/sclerosing morphology, but were characterized by novel genetic fusions including EWSR1ex8::ZBTB41ex7 and PLOD2ex8::RBM6ex7, respectively. In the statistical analysis of all published cases, CDKN2A or ALK alterations, the use of standard chemotherapy and age at presentation in the range of 18-24 years were negatively correlated to overall survival. CONCLUSION: EWSR1/FUS::TFCP2-rearranged SS-RMS is a rare rhabdomyosarcoma subtype, affecting predominantly young adults with average age at presentation 34 years (median 29.5 years; age range 7-86 years), with a predilection for craniofacial bones, rapid clinical course with frequent bone and lung metastases, and poor prognosis (3-year overall survival rate 28%).