[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.

EWSR1::YY1 fusion positive peritoneal epithelioid mesothelioma harbors mesothelioma epigenetic signature: Report of 3 cases in support of an emerging entity.

Mesothelioma is a rare, aggressive malignant neoplasm of mesothelial origin. A small subset of peritoneal mesothelioma is driven by recurrent gene fusions, mostly EWSR1/FUS::ATF1 fusions, with predilection for young adults. To date, only two cases of mesothelioma harboring EWSR1::YY1 fusions have been described. We present three additional cases of EWSR1::YY1-fused peritoneal mesotheliomas, two localized and one diffuse, all occurring in the peritoneum of middle-aged adults (2 females and 1 male), and discovered incidentally by imaging or during surgery performed for unrelated reasons. None presented with symptoms or had a known history of asbestos exposure. All three cases were cellular epithelioid neoplasms with heterogeneous architectural patterns comprising mostly solid nests and sheets with variably papillary and trabecular areas against collagenous stroma. Cytologically, the cells were monomorphic, polygonal, epithelioid cells with dense eosinophilic cytoplasm and centrally located nuclei. Overt mitotic activity or tumor necrosis was absent. All cases showed strong diffuse immunoreactivity for pancytokeratin, CK7, and nuclear WT1, patchy to negative calretinin, retained BAP1 expression, and were negative for Ber-EP4 and MOC31. RNA-sequencing confirmed in-frame gene fusion transcripts involving EWSR1 exon 7/8 and YY1 exon 2/3. By unsupervised clustering analysis, the methylation profiles of EWSR1::YY1-fused mesotheliomas clustered similarly with EWSR1/FUS::ATF1-fused mesotheliomas and conventional mesotheliomas, suggesting a mesothelioma epigenetic signature. All three patients underwent surgical resection or cytoreductive surgery of the masses. On follow-up imaging, no recurrence or progression of disease was identified. Our findings suggest that EWSR1::YY1-fusion defines a small subset of peritoneal epithelioid mesothelioma in middle-aged adults without history of asbestos exposure.

Formononetin, J1 and J2 have different effects on endothelial cells via EWSAT1-TRAF6 and its downstream pathway.

Formononetin is a natural isoflavone compound found mainly in Chinese herbal medicines such as astragalus and red clover. It is considered to be a typical phytooestrogen. In our previous experiments, it was found that formononetin has a two-way regulatory effect on endothelial cells (ECs): low concentrations promote the proliferation of ECs and high concentrations have an inhibitory effect. To find a specific mechanism of action and provide a better clinical effect, we performed a structural transformation of formononetin and selected better medicinal properties for formononetin modifier J1 and J2 from a variety of modified constructs. The MTT assay measured the effects of drugs on human umbilical vein endothelial cell (HUVEC) activity. Scratch and transwell experiments validated the effects of the drugs on HUVEC migration and invasion. An in vivo assessment effect of the drugs on ovariectomized rats. Long-chain non-coding RNA for EWSAT1, which is abnormally highly expressed in HUVEC, was screened by gene chip, and the effect of the drug on its expression was detected by PCR after the drug was applied. The downstream factors and their pathways were analysed, and the changes in the protein levels after drug treatment were evaluated by Western blot. In conclusion, the mechanism of action of formononetin, J1 and J2 on ECs may be through EWSAT1-TRAF6 and its downstream pathways.

Combinatorial Drug Screening Identifies Ewing Sarcoma-specific Sensitivities.

Improvements in survival for Ewing sarcoma pediatric and adolescent patients have been modest over the past 20 years. Combinations of anticancer agents endure as an option to overcome resistance to single treatments caused by compensatory pathways. Moreover, combinations are thought to lessen any associated adverse side effects through reduced dosing, which is particularly important in childhood tumors. Using a parallel phenotypic combinatorial screening approach of cells derived from three pediatric tumor types, we identified Ewing sarcoma-specific interactions of a diverse set of targeted agents including approved drugs. We were able to retrieve highly synergistic drug combinations specific for Ewing sarcoma and identified signaling processes important for Ewing sarcoma cell proliferation determined by EWS-FLI1 We generated a molecular target profile of PKC412, a multikinase inhibitor with strong synergistic propensity in Ewing sarcoma, revealing its targets in critical Ewing sarcoma signaling routes. Using a multilevel experimental approach including quantitative phosphoproteomics, we analyzed the molecular rationale behind the disease-specific synergistic effect of simultaneous application of PKC412 and IGF1R inhibitors. The mechanism of the drug synergy between these inhibitors is different from the sum of the mechanisms of the single agents. The combination effectively inhibited pathway crosstalk and averted feedback loop repression, in EWS-FLI1-dependent manner. Mol Cancer Ther; 16(1); 88-101. ©2016 AACR.

Preclinical Justification of pbi-shRNA EWS/FLI1 Lipoplex (LPX) Treatment for Ewing's Sarcoma.

The EWS/FLI1 fusion gene is well characterized as a driver of Ewing's sarcoma. Bi-shRNA EWS/FLI1 is a functional plasmid DNA construct that transcribes both siRNA and miRNA-like effectors each of which targets the identical type 1 translocation junction region of the EWS/FLI1 transcribed mRNA sequence. Previous preclinical and clinical studies confirm the safety of this RNA interference platform technology and consistently demonstrate designated mRNA and protein target knockdown at greater than 90% efficiency. We initiated development of pbi-shRNA EWS/FLI1 lipoplex (LPX) for the treatment of type 1 Ewing's sarcoma. Clinical-grade plasmid was manufactured and both sequence and activity verified. Target protein and RNA knockdown of 85-92% was demonstrated in vitro in type 1 human Ewing's sarcoma tumor cell lines with the optimal bi-shRNA EWS/FLI1 plasmid. This functional plasmid was placed in a clinically tested, liposomal (LP) delivery vehicle followed by in vivo verification of activity. Type 1 Ewing's sarcoma xenograft modeling confirmed dose related safety and tumor response to pbi-shRNA EWS/FLI1 LPX. Toxicology studies in mini-pigs with doses comparable to the demonstrated in vivo efficacy dose resulted in transient fever, occasional limited hypertension at low- and high-dose assessment and transient liver enzyme elevation at high dose. These results provide the justification to initiate clinical testing.

Exenteration and Custom Implant Brachytherapy as a Treatment for Recurrent Primary Extraskeletal Orbital Ewing Sarcoma.

A 6-year-old boy initially presented to an outside hospital with a right orbital mass with biopsy positive for translocation involving EWS RNA-binding protein 1 gene and imaging consistent with primary extraskeletal Ewing sarcoma (ES). There was no evidence of metastatic disease. Patient underwent gross tumor resection and adjuvant chemotherapy (VAdriaC/IE) followed by postoperative 45-Gy proton beam radiation. After 19 months, a solitary in-field local recurrence occurred, which was unsuccessfully surgically resected. Thereafter, treatment commenced with irinotecan and temozolomide, and the patient presented to the center of the authors. MRI showed locally recurrent disease without evidence of metastatic disease. Right orbital exenteration was performed, and an orbital mold was fashioned to deliver brachytherapy. There were no complications. The patient had no evidence of recurrent disease at 37-month follow up. This is the first report of orbital implant brachytherapy for recurrent primary ES of the orbit, and an additional report of primary extraskeletal ES of the orbit, which is a rare primary orbital tumor.

RNA sequencing identifies fusion of the EWSR1 and YY1 genes in mesothelioma with t(14;22)(q32;q12).

Mesothelioma is a rare but very aggressive tumor derived from mesothelial cells. A number of often complex but nonrandom cytogenetic abnormalities have been found in these tumors, resulting in loss of chromosome bands 14q32 and 22q12 in more than 35% of the cases. In this study, we used RNA sequencing to search for fusion transcripts in a mesothelioma carrying a t(14;22)(q32;q12) as the sole chromosomal aberration and found an EWSR1-YY1 and its reciprocal YY1-EWSR1 fusion transcript. Screening 15 additional cases of mesothelioma from which we had RNA but no cytogenetic information, we identified one more tumor carrying an EWSR1-YY1 fusion gene but not the reciprocal YY1-EWSR1 transcript. RT-polymerase chain reaction and sequencing showed that in both cases exon 8 of EWSR1 (nucleotide 1,139, accession number NM_013986 version 3, former exon 7 in sequence with accession number X66899) was fused to exon 2 of YY1 (nucleotide 1,160, accession number NM_003403 version 3). The EWSR1 breakpoint in exon 8 in the EWSR1-YY1 chimeric transcript is similar to what is found in other fusions involving EWSR1 such as EWSR1-FLI1, EWSR1-DDIT3, and EWSR1-ATF1. The EWSR1-YY1-encoded protein is an abnormal transcription factor with the transactivation domain of EWSR1 and the DNA-binding domain of YY1. This is the first study to detect a specific fusion gene in mesothelioma (the reason how frequent the EWSR1-YY1 fusion is remains uncertain) and also the first time that direct involvement of YY1 in oncogenesis has been demonstrated.

The proto-oncoprotein SYT interacts with SYT-interacting protein/co-activator activator (SIP/CoAA), a human nuclear receptor co-activator with similarity to EWS and TLS/FUS family of proteins.

The proto-oncoprotein SYT is involved in the unique translocation t(X;18) found in synovial sarcoma SYT-SSX fusions. SYT has a conserved N-terminal domain (SNH domain) that interacts with the human paralog of Drosophila Brahma (hBRM) and Brahma-related gene 1 (BRG1) chromatin remodeling proteins and a C-terminal transactivating sequence rich in glutamine, proline, glycine, and tyrosine (QPGY domain). Here we reported the isolation of the ribonucleoprotein SYT-interacting protein/co-activator activator (SIP/CoAA), which specifically binds the QPGY domain of SYT and also the SYT-SSX2 translocation fusion. SIP/CoAA is a general nuclear co-activator and an RNA splicing modulator that contains two RNA recognition motifs and multiple hexapeptide repeats. We showed that the region consisting of the hexapeptide motif (YQ domain) is similar to the hexapeptide repeat domain found in EWS and in TLS/FUS family proteins. The YQ domain also resembles the QPGY region of SYT itself and like all these other domains acts as a transcriptional activator in reporter assays. Most interestingly, the last 84 amino acids adjacent to YQ down-modulate by 25-fold the YQ transactivation of the reporter gene, and both domains are important for SIP/CoAA binding to SYT. In addition, SYT acts together with SIP/CoAA in stimulating estrogen and glucocorticoid receptor-dependent transcriptional activation. Activation is hormone-dependent and requires functional hBRM and/or BRG1. The stimulation is strongly reduced if the N-terminal region of hBRM/BRG1 (amino acids 1-211) is deleted. This region encompasses the SNF11 binding domain (amino acids 156-211), which interacts specifically with SYT in vivo and in vitro.

Molecular cloning and subcellular localisation of the snRNP-associated protein 69KD, a structural homologue of the proto-oncoproteins TLS and EWS with RNA and DNA-binding properties.

We recently isolated and characterised a 69 kDa protein (69KD) found associated with spliceosomal small nuclear ribonucleoproteins (snRNPs). Here, we report the molecular cloning of a cDNA encoding this protein, its nucleic acid binding properties and its subcellular localisation. Sequence analysis of the 69KD cDNA revealed: (1) that 69KD shares structural similarity with the human RNA binding proteins TLS and EWS (95% and 65% identity, respectively), the products of two genes frequently targeted by tumour-specific chromosomal translocations; (2) that 69KD contains a consensus RNA binding domain (CS-RBD) and three Arg/Gly-rich RNA binding motifs, structural features typical of many RNA binding proteins, in particular of hnRNP proteins; and (3) that 69KD contains a single putative Cys2/Cys2 zinc finger domain, a characteristic of many DNA-binding proteins. Consistent with its possession of these motifs, 69KD display a general nucleic acid binding activity, with a strong preference for guanyl and uridyl-rich RNA sequences, as well as for single-stranded and double-stranded DNA. The functional significance of this affinity for nucleic acids remains unclear. However, based on the established association of 69KD with the Sm core domain of snRNPs in vivo, these motifs might help mediate 69KD binding to snRNPs or be involved in some, as yet, unknown aspect of RNA metabolism. Consistent with both possibilities, 69KD is detected within typical snRNP containing subnuclear structures referred to as speckles, and is also more widely distributed throughout the nucleoplasm, as observed for many hnRNP proteins.

Oncogenic conversion of a novel orphan nuclear receptor by chromosome translocation.

A recurrent t(9;22) (q22;q12) chromosome translocation has been described in extraskeletal myxoid chondrosarcoma (EMC). Fluorescent in situ hybridization experiments performed on one EMC tumour indicated that the chromosome 22 breakpoint occurred in the EWS gene. Northern blot analysis revealed an aberrant EWS transcript which is cloned by a modified RT-PCR procedure. This transcript consists of an in-frame fusion of the 5' end of EWS to a previously unidentified gene, which was named TEC. This fusion transcript was detected in six of eight EMC studied, and three different junction types between the two genes were found. In all junction types, the putative translation product contained the amino-terminal transactivation domain of EWS linked to the entire TEC protein. Homology analysis showed that the predicted TEC protein contains a DNA-binding domain characteristic of nuclear receptors. The highest identity scores were observed with the NURR1 family of orphan nuclear receptors. These receptors are involved in the control of cell proliferation and differentiation by modulating the response to growth factors and retinoic acid. This work provides, after the PML/RAR alpha gene fusion, the second example of the oncogenic conversion of a nuclear receptor and the first example involving the orphan subfamily. Analysis of the disturbance induced by the EWS/TEc protein in the nuclear receptor network and their target genes may lead to new approaches for EMC treatment.