PURPOSE: Recent studies revealed divergent gene expression patterns in Ewing sarcoma (EwS) with canonical EWSR1-ETS gene fusions and undifferentiated round cell sarcomas (URCS) with EWSR1 rearrangements fused to the non-ETS gene NFATc2. Thus, the question arises whether the latter tumors really belong to EwS.
METHODS: We collected five cases matching the group of URCS with EWSR1-NFATc2 fusion and performed DNA methylation and copy number profiling. Results were compared to methylation data of 30 EwS with various EWSR1-ETS fusions and one EwS with FUS-ERG fusion, 16 URCS with CIC rearrangement and 10 URCS with BCOR alteration and a total of 81 EWSR1-associated soft tissue sarcomas including 7 angiomatoid fibrous histiocytomas, 7 clear cell sarcomas of the soft tissue, 28 desmoplastic small round cell tumors, 10 extraskeletal myxoid chondrosarcomas and 29 myxoid liposarcomas.
RESULTS: Unsupervised hierarchical clustering and t-distributed stochastic neighbor embedding analysis of DNA methylation data revealed a homogeneous methylation cluster for URCS with EWSR1-NFATc2 fusion, which clearly segregated from EwS and the other subtypes. Copy number profiles of EWSR1-NFATc2 cases showed recurrent losses on chromosome 9q and segmental gains on 20q13 and 22q12 involving the EWSR1 and NFATc2 loci, respectively.
CONCLUSION: In summary, URCS with EWSR1-NFATc2 fusion share a distinct DNA methylation signature and carry characteristic copy number alterations, which emphasizes that these sarcomas should be considered separately from EwS.

One of the still open questions in Ewing sarcoma, a rare bone tumor with weak therapeutic options, is to identify the tumor-driving cell (sub) population and to understand the specifics in the biological network of these cells. This basic scientific insight might foster the development of more specific therapeutic target patterns. The experimental approach is based on a side population (SP) of Ewing cells, based on the model cell line CADO-ES1. The SP is established by flow cytometry and defined by the idea that tumor stem-like cells can be identified by the time-course in clearing a given artificial dye. The SP was characterized by a higher colony forming activity, by a higher differentiation potential, by higher resistance to cytotoxic drugs, and by morphology. Several SP and non-SP cell fractions and bone marrow-derived mesenchymal stem cell reference were analyzed by short read sequencing of the full transcriptome. The double-differential analysis leads to an altered expression structure of SP cells centered around the AP-1 and APC/c complex. The SP cells share only a limited proportion of the full mesenchymal stem cell stemness set of genes. This is in line with the expectation that tumor stem-like cells share only a limited subset of stemness features which are relevant for tumor survival.

As the second most common malignant bone tumor in children and adolescents, Ewing sarcoma is initiated and exacerbated by a chimeric oncoprotein, most commonly, EWS-FLI1. In this study, we apply epigenomic analysis to characterize the transcription dysregulation in this cancer, focusing on the investigation of super-enhancer and its associated transcriptional regulatory mechanisms. We demonstrate that super-enhancer-associated transcripts are significantly enriched in EWS-FLI1 target genes, contribute to the aberrant transcriptional network of the disease, and mediate the exceptional sensitivity of Ewing sarcoma to transcriptional inhibition. Through integrative analysis, we identify MEIS1 as a super-enhancer-driven oncogene, which co-operates with EWS-FLI1 in transcriptional regulation, and plays a key pro-survival role in Ewing sarcoma. Moreover, APCDD1, another super-enhancer-associated gene, acting as a downstream target of both MEIS1 and EWS-FLI1, is also characterized as a novel tumor-promoting factor in this malignancy. These data delineate super-enhancer-mediated transcriptional deregulation in Ewing sarcoma, and uncover numerous candidate oncogenes which can be exploited for further understanding of the molecular pathogenesis for this disease.

BACKGROUND: Overall survival of Ewing sarcoma (EWS) remains poor and less than 30% of patients with metastatic or recurrent disease survive despite current treatments. Thus, there is a constant search for new biomarkers for diagnosis, prognosis and prediction of therapy. Numerous studies have reported the abnormal expression of miR-708-5p in tumors of different origins. However, its role in EWS remains unclear.
PROCEDURE: qRT-PCR was performed in nineteen consecutive EWS samples and twelve non-tumor bone samples from age-matched controls. Functional assays were performed in SK-ES-1 cells transfected with miR-708 lentiviral-based vectors and results analyzed in terms of clonogenicity, migration, invasion and western blot.
RESULTS: We show that miR-708-5p is downregulated in EWS tissues though no associations with any prognostic features such as HUVOS grade, event or survival were found in our cohort. Nonetheless, expression levels of this micro-RNA were inversely associated with the presence of the EWS/FLI1 translocation. When miR-708-5p was transfected into the SK-ES-1 cell line, it did not affect migration or clonogenicity, but promoted a significant increase on the invasive potential of cells endorsed with high expression of MMP2.
CONCLUSIONS: Taken together, our results suggest that despite downregulated in EWS samples, this miRNA might represent a secondary genetic alteration derived from the pleiotropic cellular effects of the abnormal EWS/FLI1 transcription factor that does not affect tumor growth but instead, is related with the promotion of tumor invasion, not being suitable for future therapeutic intervention.

Ewing sarcoma/primitive neuroectodermal tumor (Ewing/PNET sarcomas or EPS) are a group of round cell tumors. Malignant round cell tumors form a large and diverse group that includes rhabdomyosarcoma, synovial sarcoma, non-Hodgkin's lymphoma, neuroblastoma, hepatoblastoma, Wilm's tumor, desmoplastic small round cell tumor, and other morphologically similar entities. Differential diagnosis of Ewing sarcoma/primitive neuroectodermal tumor (Ewing/PNET sarcomas or EPS) is difficult. In addition to morphology and immunohistochemistry (IHC), differential diagnosis of these tumors is based on molecular analysis of the

Ewing sarcoma (ES) is a common primary malignancy in children and adolescents. Progression of treatment methods hasn't contributed a lot to the imrovement of prognosis. To identify potential prognostic biomarkers, a meta‑analysis pipeline of multi‑gene expression datasets for ES from the Gene Expression Omnibus (GEO) was performed. Three datasets were screened and differential expression genes (DEGs) in ES samples compared with normal tissues were identified through limma package and subjected to network analysis. As a result, 1,470 DEGs were obtained which were mainly involved in biological processes associated with immune response and transcription regulation. Network analysis obtained 22 core genes with high network degree and fold change. Kaplan‑Meier analysis based on ES datasets from The Cancer Genome Atlas identified five genes, including glycogen phosphorylase, muscle‑associated, myocyte‑specific enhancer factor 2C, tripartite motif containing 63, budding uninhibited by benzimidazoses1 and Ras GTPase‑activating protein 1, whose altered expression profiles are significantly associated with survival. Changes of their expression values were further confirmed through RT‑qPCR in ES cell and normal cell lines. Those genes may be considered as potential prognostic biomarkers of ES and should be helpful for its early diagnosis and treatment.

Sarcomas are cancers of the bone and soft tissue often defined by gene fusions. Ewing sarcoma involves fusions between

Ewing's sarcoma is the second most frequent bone and soft tissue sarcoma, which is commonly driven by the Ewing's sarcoma breakpoint region 1‑friend leukemia integration 1 transcription factor (EWS‑FLI1) fusion gene. Since microRNAs (miRs) can act as either oncogenes or tumor suppressor genes in human cancer, and miR‑34b has been reported to act as a tumor suppressor, the role of miR‑34b in Ewing's sarcoma was investigated in the present study. The results demonstrated that miR‑34b expression levels were higher in tumor samples compared within normal tissue samples. Notably, miR‑34b expression levels were significantly higher in EWS‑FLI1‑positive samples compared within EWS‑FLI1‑negative samples. The effects of miR‑34b expression on cell proliferation, migration and invasion were also examined. miR‑34b expression was inhibited using small interfering (si)RNA targeting the fusion gene. Transfection of a miR‑34b precursor sequence into siRNA‑treated tumor cells resulted in a significant increase in cell growth, migration and invasion compared within the control group. In addition, the adhesive ability was increased in the Ewing's sarcoma cell line RD‑ES, but not A673, following miR‑34b upregulation. Conversely, downregulation of miR‑34b expression led to a significant decrease in cell growth, migration and invasion. Notch has previously been reported to serve either oncogenic or tumor suppressive roles in human cancer. The results indicated that Notch1 and its target genes, Hes family BHLH transcription factor 1 and Hes‑related family BHLH transcription factor with YRPW motif 1, were suppressed by miR‑34b directly In conclusion, EWS‑FLI1 may modulate miR‑34b expression directly or indirectly, and miR‑34b potentially has an oncogenic role in Ewing's sarcoma by downregulating Notch1.

Ewing sarcoma (EWS) is a pediatric cancer characterized by the EWSR1-FLI1 fusion. We performed a genome-wide association study of 733 EWS cases and 1346 unaffected individuals of European ancestry. Our study replicates previously reported susceptibility loci at 1p36.22, 10q21.3 and 15q15.1, and identifies new loci at 6p25.1, 20p11.22 and 20p11.23. Effect estimates exhibit odds ratios in excess of 1.7, which is high for cancer GWAS, and striking in light of the rarity of EWS cases in familial cancer syndromes. Expression quantitative trait locus (eQTL) analyses identify candidate genes at 6p25.1 (RREB1) and 20p11.23 (KIZ). The 20p11.22 locus is near NKX2-2, a highly overexpressed gene in EWS. Interestingly, most loci reside near GGAA repeat sequences and may disrupt binding of the EWSR1-FLI1 fusion protein. The high locus to case discovery ratio from 733 EWS cases suggests a genetic architecture in which moderate risk SNPs constitute a significant fraction of risk.

Dysregulation of repetitive elements has been implicated in many cancers and other human diseases; however, the role of repetitive elements remains largely unexplored. In this issue of

Ewing sarcoma is a pediatric cancer driven by EWS-ETS transcription factor fusion oncoproteins in an otherwise stable genomic background. The majority of tumors express wild-type

Various types of repetitive sequences are dysregulated in cancer. In Ewing sarcoma, the oncogenic fusion protein EWS-FLI1 induces chromatin features typical of active enhancers at GGAA microsatellite repeats, but the function of these sites has not been directly demonstrated. Here, by combining nascent transcription profiling with epigenome editing, we found that a subset of GGAA microsatellite repeats is transcriptionally active in Ewing sarcoma and that silencing individual repeats abolishes local nascent transcription and leads to markedly reduced expression of putative target genes. Epigenome silencing of these repeat sites does not affect gene expression in unrelated cells, can prevent the induction of gene expression by EWS-FLI1, and, in the case of a GGAA repeat that controls

Ewing sarcoma is an aggressive paediatric cancer of the bone and soft tissue. It results from a chromosomal translocation, predominantly t(11;22)(q24:q12), that fuses the N-terminal transactivation domain of the constitutively expressed EWSR1 protein with the C-terminal DNA binding domain of the rarely expressed FLI1 protein. Ewing sarcoma is highly sensitive to genotoxic agents such as etoposide, but the underlying molecular basis of this sensitivity is unclear. Here we show that Ewing sarcoma cells display alterations in regulation of damage-induced transcription, accumulation of R-loops and increased replication stress. In addition, homologous recombination is impaired in Ewing sarcoma owing to an enriched interaction between BRCA1 and the elongating transcription machinery. Finally, we uncover a role for EWSR1 in the transcriptional response to damage, suppressing R-loops and promoting homologous recombination. Our findings improve the current understanding of EWSR1 function, elucidate the mechanistic basis of the sensitivity of Ewing sarcoma to chemotherapy (including PARP1 inhibitors) and highlight a class of BRCA-deficient-like tumours.

The limited delivery of chemotherapy agents to cancer cells and the nonspecific action of these agents are significant challenges in oncology. We have previously developed a customizable drug delivery and activation system in which a nucleic acid functionalized gold nanoparticle (Au-NP) delivers a drug that is selectively activated within a cancer cell by the presence of an mRNA unique to the cancer cell. The amount of drug released from sequestration to the Au-NP is determined by both the presence and the abundance of the cancer cell specific mRNA in a cell. We have now developed this technology for the potent, but difficult to deliver, topoisomerase I inhibitor SN-38. Herein, we demonstrate both the efficient delivery and selective release of SN-38 from gold nanoparticles in Ewing sarcoma cells with resulting efficacy in vitro and in vivo. These results provide further preclinical validation for this novel cancer therapy and may be extendable to other cancers that exhibit sensitivity to topoisomerase I inhibitors.

We present a case of a malignant Ewing-like neoplasm of the parotid gland in a 20-year-old woman with an EWSR1-KLF15 gene fusion that presented with pulmonary metastasis. Despite the fact that the tumor was essentially immunohistochemically negative for keratins, p63, and p40, we interpret this neoplasm as an unusual form of a high-grade myoepithelial carcinoma based on its focal plasmacytoid cytology, chondromyxoid matrix, SOX10, S100 protein, and calponin expression, and the knowledge that the EWSR1-KLF15 gene fusion has, to date, only been identified in 2 tumors, both myoepithelial carcinomas of the kidney. We also present a cytogenetic analysis of this unusual tumor. This "Ewing-like myoepithelial carcinoma" initially did not respond to 2 cycles of ifosfamide and etoposide alternated with a cycle of cytoxan, adriamycin, and vincristine, a standard regimen for Ewing sarcoma. Subsequent oral pazopanib therapy did result in a reduction of the patient's pulmonary and nodal disease.

Many cancer types are driven by oncogenic transcription factors that have been difficult to drug. Transcriptional inhibitors, however, may offer inroads into targeting these cancers. Through chemical genomics screening, we identified that Ewing sarcoma is a disease with preferential sensitivity to THZ1, a covalent small-molecule CDK7/12/13 inhibitor. The selective CDK12/13 inhibitor, THZ531, impairs DNA damage repair in an EWS/FLI-dependent manner, supporting a synthetic lethal relationship between response to THZ1/THZ531 and EWS/FLI expression. The combination of these molecules with PARP inhibitors showed striking synergy in cell viability and DNA damage assays in vitro and in multiple models of Ewing sarcoma, including a PDX, in vivo without hematopoietic toxicity.

Ewing sarcoma (EWS) is a kind of aggressive tumor of bone and soft tissues, which most occurring in children and adolescents. MicroRNAs (miRNAs) perform essential function in the progression and development of EWS, while the putative role of miR-638 in EWS remains uncertain. Accordingly, we detected the expression of miR-638 and explored its putative biological effects on the malignant phenotype of EWS cells. As expected, miR-638 was significantly down-regulated in EWS cells. Moreover, overexpression of miR-638 suppressed cell growth, induced cell apoptosis, and inhibited tubule formation of EWS cells

The literature is reviewed regarding of a rare molecularly defined group of sarcomas with rearrangement of both CIC and BCOR genes, which were originally placed into the EWSR1wt Ewing-like category. Personal experience with three cases demonstrating difficulties of this issue is added. Both groups of lesions differ not only by age and topography, but also vary in both the prognostic and the predictive parameters. CIC-rearranged tumors are very aggressive and almost never occur in the skeleton; in contrary, the BCOR-rearranged ones are predominantly bone tumors in young males behaving even better than classical Ewing sarcoma. From the morphologic point of view, it turned out to be a salient finding that these types of neoplasm might leave canonical morphotype of small blue round cell sarcoma. Instead of it, they are not uncommonly characterized as a relatively uniform spindle cell proliferation with prevailing myxoid transformation deserving much broader differential diagnosis. Our three cases reports display difficulties in reaching the correct diagnosis even by implementing sophisticated molecular techniques in routine practice. Notwithstanding of exhaustive molecular assays used, one may still encounter a lesion where original descriptive term Ewing-lie sarcoma remains uncorrected.

Ewing sarcoma is a bone malignancy of children and young adults, frequently harboring the EWS/FLI chromosomal translocation. The resulting fusion protein is an aberrant transcription factor that uses highly repetitive GGAA-containing elements (microsatellites) to activate and repress thousands of target genes mediating oncogenesis. However, the mechanisms of EWS/FLI interaction with microsatellites and regulation of target gene expression is not clearly understood. Here, we profile genome-wide protein binding and gene expression. Using a combination of unbiased genome-wide computational and experimental analysis, we define GGAA-microsatellites in a Ewing sarcoma context. We identify two distinct classes of GGAA-microsatellites and demonstrate that EWS/FLI responsiveness is dependent on microsatellite length. At close range "promoter-like" microsatellites, EWS/FLI binding and subsequent target gene activation is highly dependent on number of GGAA-motifs. "Enhancer-like" microsatellites demonstrate length-dependent EWS/FLI binding, but minimal correlation for activated and none for repressed targets. Our data suggest EWS/FLI binds to "promoter-like" and "enhancer-like" microsatellites to mediate activation and repression of target genes through different regulatory mechanisms. Such characterization contributes valuable insight to EWS/FLI transcription factor biology and clarifies the role of GGAA-microsatellites on a global genomic scale. This may provide unique perspective on the role of non-coding DNA in cancer susceptibility and therapeutic development.

Ewing sarcomas (ES) are biologically aggressive tumors of bone and soft tissues for which no cure is currently available. Most ES patients do not respond to chemotherapeutic treatments or acquire resistance. Since the PI3K/AKT/mTOR axis is often deregulated in ES, its inhibition offers therapeutic perspective for these aggressive tumors. Herein, by using splicing sensitive arrays, we have uncovered an extensive splicing program activated upon inhibition of the PI3K/AKT/mTOR signaling pathway by BEZ235. Bioinformatics analyses identified hnRNPM as a key factor in this response. HnRNPM motifs were significantly enriched in introns flanking the regulated exons and proximity of binding represented a key determinant for hnRNPM-dependent splicing regulation. Knockdown of hnRNPM expression abolished a subset of BEZ235-induced splicing changes that contained hnRNPM binding sites, enhanced BEZ235 cytotoxicity and limited the clonogenicity of ES cells. Importantly, hnRNPM up-regulation correlates with poor outcome in sarcoma patients. These findings uncover an hnRNPM-dependent alternative splicing program set in motion by inhibition of the mTOR/AKT/PI3K pathway in ES cells that limits therapeutic efficacy of pharmacologic inhibitors, suggesting that combined inhibition of the PI3K/AKT/mTOR pathway and hnRNPM activity may represent a novel approach for ES treatment.

Ewing sarcoma (ES) is the most common malignant bone tumor in children and young adults. It is characterized by chromosomal translocations fusing the EWS gene with an ETS oncogene, most frequently FLI1. In the present study, the authors aimed to investigate the function of EWS-FLI1 in autophagy in ES cells, and identified that EWS-FLI1 positively regulates autophagy in ES cells. ATG4B expression was observed markedly upregulated by EWS-FLI1 overexpression, and silencing of ATG4B dramatically inhibits autophagy in ES cells. Furthermore, apoptosis was inhibited in ATG4B overexpressed ES cells, and ATG4B-potentiated autophagy is required for ES cells survival. Taken together, the authors demonstrated the role of EWS-FLI1 and ATG4B in autophagy in ES cells, and suggested EWS-FLI1 and ATG4B as potential therapeutic targets for ES.

Ewing sarcoma is a highly aggressive bone or soft-tissue tumor mostly occurring in children and adolescents. Conventional multi-modal therapies are associated with considerable acute and chronic toxicity. Thus, more effective and in particular less toxic therapeutic strategies are urgently required. Despite the fact that Ewing sarcoma is characterized by specific EWSR1-ETS gene fusions, the resulting fusion oncoproteins are not suitable for targeted therapy due to their low immunogenicity and the ubiquitous expression of their constituents. However, functional genomics revealed several EWSR1-ETS target genes, which are only minimally expressed in normal tissues, and which could serve as surrogate-targets for (immuno-)therapeutic approaches. Moreover, functional genomic analyses yielded first mechanistic explanations for the relatively high incidence of Ewing sarcoma in Europeans, and first studies are exploring the value of circulating free DNA and/or exosomal mRNA of EWSR1-ETS fusion oncogenes as minimal-residual-disease markers in Ewing sarcoma. This review summarizes key contributions to these aspects and gives a perspective on their medical relevance.

Ewing sarcoma usually expresses the EWS/FLI fusion transcription factor oncoprotein. EWS/FLI regulates myriad genes required for Ewing sarcoma development. EWS/FLI binds GGAA-microsatellite sequences in vivo and in vitro. These sequences provide EWS/FLI-mediated activation to reporter constructs, suggesting that they function as EWS/FLI-response elements. We now demonstrate the critical role of an EWS/FLI-bound GGAA-microsatellite in regulation of the

Alterations in transcriptional regulators can orchestrate oncogenic gene expression programs in cancer. Here, we show that the BRG1/BRM-associated factor (BAF) chromatin remodeling complex, which is mutated in over 20% of human tumors, interacts with EWSR1, a member of a family of proteins with prion-like domains (PrLD) that are frequent partners in oncogenic fusions with transcription factors. In Ewing sarcoma, we find that the BAF complex is recruited by the EWS-FLI1 fusion protein to tumor-specific enhancers and contributes to target gene activation. This process is a neomorphic property of EWS-FLI1 compared to wild-type FLI1 and depends on tyrosine residues that are necessary for phase transitions of the EWSR1 prion-like domain. Furthermore, fusion of short fragments of EWSR1 to FLI1 is sufficient to recapitulate BAF complex retargeting and EWS-FLI1 activities. Our studies thus demonstrate that the physical properties of prion-like domains can retarget critical chromatin regulatory complexes to establish and maintain oncogenic gene expression programs.

Ewing sarcoma is a developmental tumor characterized by balanced chromosomal translocations and formation of new fusion genes. Despite the large amount of knowledge regarding the molecular aspects obtained in the last few years, many questions still remain. This article focuses on research on the molecular pathology and possible developments in targeted therapies in this malignancy and discusses some related bottlenecks, as well as the possible role of pathologists, the availability of samples, the lack of appropriate animal models, and the resources needed to carry out preclinical and clinical research.

The EWS-FLI1 chimeric protein uniquely expressed in Ewing's sarcoma has an obligate role in its aetiology. In our previous report we showed that ectopic expression of the DNA sequences form the junction region (a.a 251-280) can inhibit Ewing's sarcoma cell growth. In the present report, we introduced a peptide (TAT/NLS/EWS-PEP) comprising of thirty amino acids spanning the junction in conjunction with HIV-1-trans-activating (TAT) and nuclear localization signal sequence (NLS). Peptide uptake and localization studies revealed presence of peptide in ~99% of transduced cells and in the nucleus. Peptide transfection induced cytotoxicity relative to untreated and TAT-NLS peptide treated Ewing's sarcoma cells. The peptide inhibited clonogenicity, cell cycle, bromo-deoxy uridine (BrdU) uptake and invasion capacity of treated cells. The treatment also affected epithelial to mesenchymal transition (EMT) markers and EWS-FLI1 target gene expression levels. Co-immunoprecipitation experiments involving ectopically expressed full-length EWS-FLI1 protein and the peptide revealed an interaction. Additionally, we found that peptide interaction also occurs with the protein-GGAA microsatellite sequences complex known to contain EWS-FLI1. Further, in the pull-down assay, the peptide was found to interact with proteins known to potentially interact with EWS-FLI1. Based on these results we conclude that peptide could be applied in targeting EWS-FLI1 protein.

Ewing sarcoma (EWS) is a paediatric bone cancer with high metastatic potential. Cellular plasticity resulting from dynamic cytoskeletal reorganization, typically regulated via the Rho pathway, is a prerequisite for metastasis initiation. Here, we interrogated the role of the Ewing sarcoma driver oncogene EWS-FLI1 in cytoskeletal reprogramming. We report that EWS-FLI1 strongly represses the activity of the Rho-F-actin signal pathway transcriptional effector MRTFB, affecting the expression of a large number of EWS-FLI1-anticorrelated genes including structural and regulatory cytoskeletal genes. Consistent with this finding, chromatin immunoprecipitation sequencing (ChIP-seq) revealed strong overlaps in myocardin-related transcription factor B (MRTFB) and EWS-FLI1 chromatin occupation, especially for EWS-FLI1-anticorrelated genes. Binding of the transcriptional co-activator Yes-associated protein (YAP)-1, enrichment of TEAD-binding motifs in these shared genomic binding regions and overlapping transcriptional footprints of MRTFB and TEAD factors led us to propose synergy between MRTFB and the YAP/TEAD complex in the regulation of EWS-FLI1-anticorrelated genes. We propose that EWS-FLI1 suppresses the Rho-actin pathway by perturbation of a MRTFB/YAP-1/TEAD transcriptional module, which directly affects the actin-autoregulatory feedback loop. As spontaneous fluctuations in EWS-FLI1 levels of Ewing sarcoma cells in vitro and in vivo, associated with a switch between a proliferative, non-migratory EWS-FLI1-high and a non-proliferative highly migratory EWS-FLI1-low state, were recently described, our data provide a mechanistic basis for the underlying EWS-FLI1-dependent reversible cytoskeletal reprogramming of Ewing sarcoma cells.

BACKGROUND: Sarcomas are rare and heterogeneous cancers. We assessed the contribution of DICER1 mutations to sarcoma development.
METHODS: The coding region of DICER1 was sequenced in 67 sarcomas using a custom Fluidigm Access Array. The RNase III domains were Sanger sequenced in six additional sarcomas to identify hotspot DICER1 variants.
RESULTS: The median age of sarcoma diagnosis was 45.7 years (range: 3 months to 87.4 years). A recurrent embryonal rhabdomyosarcoma (ERMS) of the broad ligament, first diagnosed at age 23 years, harboured biallelic pathogenic somatic DICER1 variants (1 truncating and 1 RNase IIIb missense). We identified nine other DICER1 variants. One somatic variant (p.L1070V) identified in a pleomorphic sarcoma and one germline variant (c.2257-7A>G) may be pathogenic, but the others are considered to be benign.
CONCLUSIONS: We show that deleterious DICER1 mutations underlie the genetic basis of only a small fraction of sarcomas, in particular ERMS of the urogenital tract.

Efficient methodologies for recreating cancer-associated chromosome translocations are in high demand as tools for investigating how such events initiate cancer. The CRISPR/Cas9 system has been used to reconstruct the genetics of these complex rearrangements at native loci while maintaining the architecture and regulatory elements. However, the CRISPR system remains inefficient in human stem cells. Here, we compared three strategies aimed at enhancing the efficiency of the CRISPR-mediated t(11;22) translocation in human stem cells, including mesenchymal and induced pluripotent stem cells: (1) using end-joining DNA processing factors involved in repair mechanisms, or (2) ssODNs to guide the ligation of the double-strand break ends generated by CRISPR/Cas9; and (3) all-in-one plasmid or ribonucleoprotein complex-based approaches. We report that the generation of targeted t(11;22) is significantly increased by using a combination of ribonucleoprotein complexes and ssODNs. The CRISPR/Cas9-mediated generation of targeted t(11;22) in human stem cells opens up new avenues in modeling Ewing sarcoma.

Ewing's sarcoma is a highly aggressive malignant tumour most commonly affecting long bones in children and adolescents. It is part of the Ewing's sarcoma family of tumours (ESFTs) that also include peripheral primitive neuroectodermal tumour and Askin's tumours. ESFTs share common cytogenetic aberrations, antigenic profiles and proto-oncogene expression with an overall similar clinical course. In 99% of ESFTs, genetic translocation with molecular fusion involves the EWSR1 gene on 22q12. Approximately 30% of ESFTs are extraosseous, most commonly occurring in the soft tissues of extremities, pelvis, retroperitoneum and chest wall. Primary presentation in solid organs is very rare but has been described in multiple sites including the pancreas. Accurate diagnosis of a Ewing's sarcoma in a solid organ is critical in facilitating correct treatment. We report the case of a 17-year-old girl with cytogenetically confirmed primary pancreatic Ewing's sarcoma and provide a brief review of the published literature.

Ewing sarcoma (ES) is the second most common primary malignant tumor of bone in children and young adolescents. Most ES contain a pathognomonic translocation t(11;22)(q24;q12) that is likely a pivotal event in the tumorigenesis of these neoplasms. Many ES also contain nonrandom, numerical chromosomal aberrations, the most common of which are trisomies 8 and 12. In this study we evaluated the hypothesis that these trisomies might occur during neoplastic progression and might be associated with differences in biologic behavior. We tested this hypothesis using a combined cytogenetic and dual color fluorescence in situ hybridization approach to determine chromosome 8 and 12 copy number in 52 ES. Relative gains, primarily trisomies, of chromosomes 8 and 12 were found in 24 (46%) and 17 (33%) cases, respectively. Trisomy 8 and trisomy 12 were independent events acquired in a flexible order during ES genetic progression. Our preliminary findings also suggest a higher frequency of trisomies 8 and 12 in relapses than in primary tumors. Prospective studies will be required to determine whether either trisomy is prognostic in newly-diagnosed ES.

Comparative genomic hybridization (CGH) was used to detect copy number changes of DNA sequences in the Ewing family of tumours (ET). We analysed 20 samples from 17 patients. Fifteen tumours (75%) showed copy number changes. Gains of DNA sequences were much more frequent than losses, the majority of the gains affecting whole chromosomes or whole chromosome arms. Recurrent findings included copy number increases for chromosomes 8 (seven out of 20 samples; 35%), 1q (five samples; 25%) and 12 (five samples; 25%). The minimal common regions of these gains were the whole chromosomes 8 and 12, and 1q21-22. High-level amplifications affected 8q13-24, 1q and 1q21-22, each once. Southern blot analysis of the specimen with high-level amplification at 1q21-22 showed an amplification of FLG and SPRR3, both mapped to this region. All cases with a gain of chromosome 12 simultaneously showed a gain of chromosome 8. Comparison of CGH findings with cytogenetic analysis of the same tumours and previous cytogenetic reports of ET showed, in general, concordant results. In conclusion, our findings confirm that secondary changes, which may have prognostic significance in ET, are trisomy 8, trisomy 12 and a gain of DNA sequences in 1q.

Chromosomal data from 82 informative, unrelated Ewing's sarcoma (ES) specimens (including 20 personal specimens) were reviewed for secondary changes additional to the t(11;22)(q24;q12). Additional numerical and/or structural changes were found in 75 specimens. Trisomy 8 was observed consistently in half of the 43 cases selected for analysis of numerical changes. A nonrandom der(16) was observed as a result of an unbalanced t(1;16) in 18% of the 82 analyzed for structural changes. Consistent involvement of chromosome #16 in rearrangements with chromosome #1 may be an additional chromosome change specifically associated with ES.

Comparative genomic hybridization (CGH) was used to detect copy number changes of DNA sequences in the Ewing family of tumours (ET). We analysed 20 samples from 17 patients. Fifteen tumours (75%) showed copy number changes. Gains of DNA sequences were much more frequent than losses, the majority of the gains affecting whole chromosomes or whole chromosome arms. Recurrent findings included copy number increases for chromosomes 8 (seven out of 20 samples; 35%), 1q (five samples; 25%) and 12 (five samples; 25%). The minimal common regions of these gains were the whole chromosomes 8 and 12, and 1q21-22. High-level amplifications affected 8q13-24, 1q and 1q21-22, each once. Southern blot analysis of the specimen with high-level amplification at 1q21-22 showed an amplification of FLG and SPRR3, both mapped to this region. All cases with a gain of chromosome 12 simultaneously showed a gain of chromosome 8. Comparison of CGH findings with cytogenetic analysis of the same tumours and previous cytogenetic reports of ET showed, in general, concordant results. In conclusion, our findings confirm that secondary changes, which may have prognostic significance in ET, are trisomy 8, trisomy 12 and a gain of DNA sequences in 1q.

Ewing sarcoma (ES) is the second most common primary malignant tumor of bone in children and young adolescents. Most ES contain a pathognomonic translocation t(11;22)(q24;q12) that is likely a pivotal event in the tumorigenesis of these neoplasms. Many ES also contain nonrandom, numerical chromosomal aberrations, the most common of which are trisomies 8 and 12. In this study we evaluated the hypothesis that these trisomies might occur during neoplastic progression and might be associated with differences in biologic behavior. We tested this hypothesis using a combined cytogenetic and dual color fluorescence in situ hybridization approach to determine chromosome 8 and 12 copy number in 52 ES. Relative gains, primarily trisomies, of chromosomes 8 and 12 were found in 24 (46%) and 17 (33%) cases, respectively. Trisomy 8 and trisomy 12 were independent events acquired in a flexible order during ES genetic progression. Our preliminary findings also suggest a higher frequency of trisomies 8 and 12 in relapses than in primary tumors. Prospective studies will be required to determine whether either trisomy is prognostic in newly-diagnosed ES.

Chromosomal data from 82 informative, unrelated Ewing's sarcoma (ES) specimens (including 20 personal specimens) were reviewed for secondary changes additional to the t(11;22)(q24;q12). Additional numerical and/or structural changes were found in 75 specimens. Trisomy 8 was observed consistently in half of the 43 cases selected for analysis of numerical changes. A nonrandom der(16) was observed as a result of an unbalanced t(1;16) in 18% of the 82 analyzed for structural changes. Consistent involvement of chromosome #16 in rearrangements with chromosome #1 may be an additional chromosome change specifically associated with ES.

The der(16)t(1;16) has been detected cytogenetically in a number of malignancies including Ewing tumors (ETs). To enable fast and reliable analysis of der(16) chromosomes, we established an interphase cytogenetic approach. By using two DNA probes hybridizing to the heterochromatic portions on the long arms of chromosomes 1 and 16, this technique allows the detection of this chromosomal aberration in nonproliferating cells. Formation of the der(16) leads to partial excess of 1q material and partial loss of the long arm of chromosome 16. Double-target fluorescence in situ hybridization (FISH) experiments were performed on cytospin slides of 13 ETs, near-triploid tumor cells and normal cells to assess whether the FISH technique used permits the discrimination of nuclei harboring this aberration from nuclei without a der(16) chromosome. In five ETs, we found evidence for the presence of one or two der(16)t(1;16) chromosomes both by FISH and by conventional cytogenetics. Tumor cells displayed two signals for intact chromosomes 1, one or two additional fused signals for the der(16) chromosomes, and one signal for the intact chromosome 16. In one case without fused signals, the presence of a der(16) was demonstrated by hybridizing a painting probe for chromosome 16 simultaneously with the paracentromeric probe for chromosome 1. Our results suggest that double-target FISH on interphase nuclei offers an ideal tool for analyzing tumors prospectively and retrospectively to assess the biological role and the possible prognostic impact of the der(16) in ETs and in other solid tumors.

The cytogenetic hallmark of the Ewing family of tumors is t(11,22)(q24;q12) in its simple, complex or variant forms and/or its molecular equivalent EWS/FLI, EWS/ERG rearrangement. Additional secondary consistent chromosomal aberrations include the der(16)t(1;16) and frequently, other chromosome 1q abnormalities leading to 1q overdosage. We studied whether these secondary cytogenetic changes are correlated to clinical features and phenotypic expression which may have a prognostic impact. Successful cytogenetic evaluation was performed in eight patients with a Ewing family tumor. In four of these, in addition to the primary aberration, chromosome 1q overdosage (including two with der (16)t(1;16)) was noted. Out of these four patients, two had metastatic disease at the time of evaluation, while in the other four, disease was localized. Morphologically, the tumors with the additional 1q aberration, revealed the pPNET subtype more frequently than the typical Ewing. They also expressed a higher degree of neural differentiation by neural marker immunocytochemistry, in comparison to tumors without the 1q aberration. Determination of the prognostic significance of this finding requires a longer follow-up with a larger group of patients.

Somatic mutations and polymorphisms in the noncoding displacement (D)-loop of mitochondrial DNA (mtDNA) are present in a variety of human cancers. To investigate whether Ewing's sarcoma (EWS) harbors genetic alterations within the D-loop region and their potential association with EWS carcinogenesis, we analyzed and compared the complete mtDNA D-loop sequences from 17 pairs of tumor tissues and corresponding peripheral blood samples using the direct DNA sequencing method. Our results revealed that 12 of the 17 EWS tumor specimens (70.6%) carried 19 somatic mutations in the D-loop of mtDNA, including 11 single-base substitutions, 3 insertions and 5 deletions. Among the tested 17 patients, we screened a total of 40 germline polymorphisms including one novel sequence variant in the D-loop fragment. Most of these identified mutations and germline variations were clustered within two hypervariable segments (HVS1 and HVS2) as well as the homopolymeric C stretch between nucleotide position 303 and 309. In addition, there was no significant correlation between mtDNA D-loop mutations and various clinicopathological factors of EWS. In conclusion, our study reports for the first time that mtDNA D-loop mutations occur at a high frequency in EWS. These data provide evidence of mtDNA alterations' possible involvement in the initiation and/or progression of this rare malignancy.

Somatic mutations in mitochondrial DNA (mtDNA) have been long proposed to drive initiation and progression of human malignancies. Our previous study revealed a high prevalence of somatic mutations in the D-loop region of mtDNA in Ewing sarcoma (EWS). However, it is unclear whether somatic mutations also occur in the coding regions of mtDNA in EWS. To test this possibility, in the present study, we sequenced the whole mitochondrial genome from 20 cases of EWS specimens and their corresponding peripheral blood samples. We identified a total of 6 somatic mutations in the mtDNA coding regions in our EWS series, and 5 of them were missense or frame-shift mutations that have the potential to directly influence proper mitochondrial function. In combination with our earlier observations on the D-loop fragment, 70% (14/20) of EWS tissues appeared to harbor somatic mtDNA mutations. Among the identified 25 somatic mutations, 19 (76%) were located in the D-loop control region, 1 (4%) was in the sequence of the tRNA(Val) gene, 1 (4%) was in the mitochondrial ATP synthase subunit 6 gene, and 4 (16%) occurred in genes encoding components of the mitochondrial respiratory complexes. In addition, patients carrying somatic mtDNA mutations did not show significant association with their clinicopathologic characteristics. Together, these findings suggest that somatic mtDNA mutations occur in both protein coding and noncoding regions of mtDNA, which may play critical roles in the pathogenesis of EWS and should be further explored for its possible use as a novel marker for monitoring EWS occurrence and advancement.

Ewing's sarcoma, peripheral primitive neuroectodermal tumors, and Askin tumors are referred to as Ewing tumors (ETs), and are characterized by high MIC2 expression and a t(11;22)(q24;q12) or other rearrangements involving 22q12. In addition to these constant aberrations, facultative numerical and structural aberrations have been reported: gains of chromosomes 8 and 12, the unbalanced translocation t(1;16), and deletions at the short arm of chromosome 1. To evaluate the frequency and to study the biological impact of these facultative aberrations, we analyzed tumor specimens from 58 ET patients by classical cytogenetics and/or in situ hybridization techniques and compared these data with clinical parameters. Gains of chromosomes 8 and 12 were detected in 55% (32/58) and 24% (14/58) of the cases, respectively. Loss of chromosome 16 or der (16)t(1;16) chromosomes were found in 20% (10/51); deletions at 1p36 were observed in 18% (9/51) of the cases evaluated. The presence of these aberrations did not correlate with age and sex of the patients, with the location of the primary tumor or with the extent of disease at diagnosis by chi-square analysis and Fisher's exact test. Patients with tumors harboring gains of chromosome 8 showed a slightly better clinical outcome (n = 14/30, P = 0.17), whereas gains of chromosome 12 did not influence the clinical outcome (n = 7/30, P = 0.63). However, Kaplan and Meier analysis revealed that deletions at the short arm of chromosome 1 were associated with an unfavorable outcome in patients with localized disease (n = 6/22; P = 0.004).

BACKGROUND: Ewing sarcoma is associated with a nonrandom pattern of primary and secondary chromosomal aberrations. Whereas the finding of rearrangements of chromosome 22, usually in the form of a balanced translocation t(11;22)(q24;q12), is important diagnostically, nothing is known about the potential prognostic impact of the secondary chromosomal aberrations.
PROCEDURE: During a 1 3-year-period, short-term cultured tumor samples from 21 children and young adults with Ewing sarcoma were cytogenetically analyzed successfully.
RESULTS: Clonal chromosome aberrations were detected in 18 patients, 17 of whom had the characteristic t(11;22)(q24;q12) or variants thereof. The most frequent secondary change was +8, followed by +12, +2, +5, +9, +15, and gain of material from the long and short arms of chromosome 1. The only recurrent secondary change that was restricted to tumors from the ten patients that were dead at latest follow-up was gain of 1q material. Furthermore, all three patients with tumors with chromosome numbers over 50 had died, and the only patient with a tumor karyotype lacking chromosome 22 rearrangement was alive without evidence of disease.
CONCLUSIONS: These data and previously published results indicate that the karyotypic pattern not only may be of diagnostic significance but also may be important prognostically.

The cytogenetic hallmark of the Ewing family of tumors is t(11,22)(q24;q12) in its simple, complex or variant forms and/or its molecular equivalent EWS/FLI, EWS/ERG rearrangement. Additional secondary consistent chromosomal aberrations include the der(16)t(1;16) and frequently, other chromosome 1q abnormalities leading to 1q overdosage. We studied whether these secondary cytogenetic changes are correlated to clinical features and phenotypic expression which may have a prognostic impact. Successful cytogenetic evaluation was performed in eight patients with a Ewing family tumor. In four of these, in addition to the primary aberration, chromosome 1q overdosage (including two with der (16)t(1;16)) was noted. Out of these four patients, two had metastatic disease at the time of evaluation, while in the other four, disease was localized. Morphologically, the tumors with the additional 1q aberration, revealed the pPNET subtype more frequently than the typical Ewing. They also expressed a higher degree of neural differentiation by neural marker immunocytochemistry, in comparison to tumors without the 1q aberration. Determination of the prognostic significance of this finding requires a longer follow-up with a larger group of patients.

Comparative genomic hybridization (CGH) was used to detect copy number changes of DNA sequences in the Ewing family of tumours (ET). We analysed 20 samples from 17 patients. Fifteen tumours (75%) showed copy number changes. Gains of DNA sequences were much more frequent than losses, the majority of the gains affecting whole chromosomes or whole chromosome arms. Recurrent findings included copy number increases for chromosomes 8 (seven out of 20 samples; 35%), 1q (five samples; 25%) and 12 (five samples; 25%). The minimal common regions of these gains were the whole chromosomes 8 and 12, and 1q21-22. High-level amplifications affected 8q13-24, 1q and 1q21-22, each once. Southern blot analysis of the specimen with high-level amplification at 1q21-22 showed an amplification of FLG and SPRR3, both mapped to this region. All cases with a gain of chromosome 12 simultaneously showed a gain of chromosome 8. Comparison of CGH findings with cytogenetic analysis of the same tumours and previous cytogenetic reports of ET showed, in general, concordant results. In conclusion, our findings confirm that secondary changes, which may have prognostic significance in ET, are trisomy 8, trisomy 12 and a gain of DNA sequences in 1q.