Primary Ewing's sarcoma of the uterine cervix: a case report and review of the literature.

BACKGROUND: Ewing's sarcoma (ES) is an aggressive cancer of bone and soft tissue, most of which tend to occur in the bone. Extraosseous Ewing's sarcoma (EES) of the cervix is extremely rare. CASE PRESENTATION: In the present work, we reported a 39-year-old cervical EES patient with a 2.5*2.1*1.8 cm tumor mass. According to previous literatures, our case is the smallest tumor found in primary cervical ES ever. The patient initially came to our hospital due to vaginal bleeding, and then the gynecological examination found a neoplasm between the cervical canal and partially in the external cervical orifice. The diagnosis of EES was confirmed below: Hematoxylin & Eosin staining (H&E) revealed small round blue malignant cells in biopsy specimens. Immunohistochemistry (IHC) showed the positive staining for CD99, NKX2.2, and FLI1. Disruption of EWSR1 gene was found by fluorescence in situ hybridization (FISH), and the EWSR1-FLI1 gene fusion was determined by next-generation sequencing (NGS). The patient received laparoscopic wide hysterectomy, bilateral adnexectomy, pelvic lymphadenectomy, and postoperative adjuvant chemotherapy and remained disease free with regular follow-up for 1 year. CONCLUSIONS: Through a systematic review of previously reported cervical ES and this case, we highlighted the importance of FISH and NGS for the accuracy of ESS diagnosis, which could assist on the optimal treatment strategy. However, due to the rarity of the disease, there is no standard treatment schemes. Investigation on molecular pathological diagnosis and standardization of treatment regimens for cervical ES are critical to patients' prognosis.

[Detection of EWSR1 gene rearrangement by fluorescence in situ hybridization in bone and soft tissue tumors: clinical application evaluation and atypical signal analysis].

Objective: To investigate the clinical application of EWSR1 gene rearrangement by fluorescence in situ hybridization (FISH) in bone and soft tissue tumors and to analyze the cases with atypical signal pattern. Methods: The cases detected for EWSR1 gene rearrangement by FISH in Beijing Jishuitan Hospital, Capital Medical University from 2014 to 2021 were collected, and the value of detecting EWSR1 gene rearrangement for diagnosing bone and soft tissue tumors was analyzed. The cases with atypical positive signals were further analyzed by next generation sequencing (NGS). Results: FISH using EWSR1 break-apart probe kit was successfully performed in 97% (205/211) of cases, 6 cases failed. Four of the 6 failures were due to improper decalcification, 1 case due to signal overlap caused by thick slices, and 1 case due to signal amplification and disorder. EWSR1 gene rearrangements were positive in 122 cases (122/205, 59%), atypical positive signal in 8 cases (8/205, 4%), and negative in 75 cases (75/205, 37%). In cases testing positive, the percentage of positive cells ranged from 34% to 98%, with 120 cases (120/122, 98%) showing a positive cell percentage greater than 50%. Among the 205 successfully tested cases, 156 cases were histologically diagnosed as Ewing's sarcoma, of which 110 were positive (110/156, 71%), 7 were atypical positive (7/156, 4%), and 39 were negative (39/156, 25%). Nine cases were histologically diagnosed as clear cell sarcoma of soft tissue, of which 6 were positive (6/9), 1 was atypical positive (1/9), and 2 were negative (2/9). Five cases were histologically diagnosed as extraskeletal myxoid chondrosarcoma, of which 2 were positive (2/5) and 3 were negative (3/5). Three cases were histologically diagnosed as angiomatoid fibrous histiocytoma, of which 2 were positive (2/3) and 1 was negative (1/3). Two cases were histologically diagnosed as myoepithelioma of soft tissue, of which 1 was positive (1/2) and 1 was negative (1/2). One case was histologically diagnosed as olfactory neuroblastoma with a positive result. The 29 other tumor cases including osteosarcoma, synovial sarcoma, and malignant melanoma and others were all negative. Basing on histology as the standard for diagnosis and considering atypical positive cases as negative, comparing with the 29 cases of other tumors as control group, the sensitivity for diagnosing Ewing's sarcoma through the detection of EWSR1 gene rearrangement was 71%, and the specificity was 100%; the sensitivity for diagnosing clear cell sarcoma of soft tissue was 67%, and the specificity was 100%; the sensitivity for diagnosing extraskeletal myxoid chondrosarcoma was 40%, and the specificity was 100%; the sensitivity for diagnosing angiomatoid fibrous histiocytoma was 67%, and the specificity was 100%; the sensitivity for diagnosing myoepithelioma of soft tissue was 50%, and the specificity was 100%; the sensitivity for diagnosing olfactory neuroblastoma was 100%, and the specificity was 100%. Four of 8 cases with atypical positive signals analyzed by NGS showed EWSR1 rearrangement, including EWSR1::FLI1 in one case of Ewing sarcoma, EWSR1::NFATC2 in one case of EWSR1::NFATC2-rearranged sarcoma, EWSR1::ATF1 in one case of clear cell sarcoma of soft tissue and EWSR1::NR4A3 in one case of extraskeletal myxoid chondrosarcoma. Conclusions: Detection of EWSR1 rearrangement by FISH is of utmost significance in the diagnosis of bone and soft tissue tumors. Cases with atypical positive signals should be further scrutinized, correlating with their histomorphology and verifying by NGS if necessary.

Prognostic value of PRR11 and immune cell infiltration in Ewing sarcoma.

Ewing's sarcoma (ES) is the second most common bone and soft tissue malignancy in children and adolescents with a poor prognosis. The identification of genes with prognostic value may contribute to the prediction and treatment of this disease. The GSE17679, GSE68776, GSE63155, and GSE63156 datasets were downloaded from the Gene Expression Omnibus database and qualified. Prognostic value of differentially expressed genes (DEGs) between the normal and tumor groups and immune cell infiltration were explored by several algorithms. A prognostic model was established and validated. Finally, functional analyses of the DEGs were performed. Proline rich 11 (PRR11) and mast cell infiltration were noted as the key indicators for the prognosis of ES. Kaplan-Meier and scatter plots for the training and two validation sets showed that patients in the low-PRR11 expression group were associated with better outcomes than those in the high-PRR11 expression group. The concordance indices and calibration analyses of the prognostic model indicated good predictive accuracy in the training and validation sets. The area under the curve values obtained through the receiver operating characteristic analysis for 1-, 3-, 5-year prediction were ≥ 0.75 in the three cohorts, suggesting satisfactory sensitivity and specificity of the model. Decision curve analyses suggested that patients could benefit more from the model than the other strategies. Functional analyses suggested that DEGs were mainly clustered in the cell cycle pathway. PRR11 and mast cell infiltration are potential prognostic indicators in ES. PRR11 possibly affects the prognosis of patients with ES through the cell cycle pathway.

Clinical outcomes of patients with CIC-rearranged sarcoma: a single institution retrospective analysis.

PURPOSE: CIC-rearranged sarcomas represent a type of undifferentiated small round cell sarcoma (USRCS) characterized by poor survival, rapid development of chemotherapy resistance, and high rates of metastasis. We aim to contribute to the growing body of knowledge regarding diagnosis, treatment, clinical course, and outcomes for these patients. METHODS: This case series investigates the clinical courses of ten patients with CIC-rearranged sarcoma treated at the Johns Hopkins Hospital from July 2014 through January 2024. Clinical data were retrospectively extracted from electronic medical records. RESULTS: Patients ranged from 10 to 67 years of age at diagnosis, with seven patients presenting with localized disease and three with metastatic disease. Tumors originated from soft tissues of various anatomic locations. Mean overall survival (OS) was 22.1 months (10.6-52.2), and mean progression-free survival (PFS) was 16.7 months (5.3-52.2). Seven patients received intensive systemic therapy with an Ewing sarcoma-directed regimen or a soft tissue sarcoma-directed regimen. Three patients experienced prolonged disease-free survival without systemic treatment. CONCLUSION: Most patients in this case series demonstrated aggressive clinical courses consistent with those previously described in the literature, although we note a spectrum of clinical outcomes not previously reported. The diversity of clinical courses underscores the need for an improved understanding of individual tumor biology to enhance clinical decision-making and patient prognosis. Despite its limitations, this article broadens the spectrum of reported clinical outcomes, providing a valuable addition to the published literature on this rare cancer.

Oncogenic ETS fusions promote DNA damage and proinflammatory responses via pericentromeric RNAs in extracellular vesicles.

Aberrant expression of the E26 transformation-specific (ETS) transcription factors characterizes numerous human malignancies. Many of these proteins, including EWS:FLI1 and EWS:ERG fusions in Ewing sarcoma (EwS) and TMPRSS2:ERG in prostate cancer (PCa), drive oncogenic programs via binding to GGAA repeats. We report here that both EWS:FLI1 and ERG bind and transcriptionally activate GGAA-rich pericentromeric heterochromatin. The respective pathogen-like HSAT2 and HSAT3 RNAs, together with LINE, SINE, ERV, and other repeat transcripts, are expressed in EwS and PCa tumors, secreted in extracellular vesicles (EVs), and are highly elevated in plasma of patients with EwS with metastatic disease. High human satellite 2 and 3 (HSAT2,3) levels in EWS:FLI1- or ERG-expressing cells and tumors were associated with induction of G2/M checkpoint, mitotic spindle, and DNA damage programs. These programs were also activated in EwS EV-treated fibroblasts, coincident with accumulation of HSAT2,3 RNAs, proinflammatory responses, mitotic defects, and senescence. Mechanistically, HSAT2,3-enriched cancer EVs induced cGAS-TBK1 innate immune signaling and formation of cytosolic granules positive for double-strand RNAs, RNA-DNA, and cGAS. Hence, aberrantly expressed ETS proteins derepress pericentromeric heterochromatin, yielding pathogenic RNAs that transmit genotoxic stress and inflammation to local and distant sites. Monitoring HSAT2,3 plasma levels and preventing their dissemination may thus improve therapeutic strategies and blood-based diagnostics.

Insights into Molecular Diversity within the FUS/EWS/TAF15 Protein Family: Unraveling Phase Separation of the N-Terminal Low-Complexity Domain from RNA-Binding Protein EWS.

The FET protein family, comprising FUS, EWS, and TAF15, plays crucial roles in mRNA maturation, transcriptional regulation, and DNA damage response. Clinically, they are linked to Ewing family tumors and neurodegenerative diseases such as amyotrophic lateral sclerosis. The fusion protein EWS::FLI1, the causative mutation of Ewing sarcoma, arises from a genomic translocation that fuses a portion of the low-complexity domain (LCD) of EWS (EWSLCD) with the DNA binding domain of the ETS transcription factor FLI1. This fusion protein modifies transcriptional programs and disrupts native EWS functions, such as splicing. The exact role of the intrinsically disordered EWSLCD remains a topic of active investigation, but its ability to phase separate and form biomolecular condensates is believed to be central to EWS::FLI1's oncogenic properties. Here, we used paramagnetic relaxation enhancement NMR, microscopy, and all-atom molecular dynamics (MD) simulations to better understand the self-association and phase separation tendencies of the EWSLCD. Our NMR data and mutational analysis suggest that a higher density and proximity of tyrosine residues amplify the likelihood of condensate formation. MD simulations revealed that the tyrosine-rich termini exhibit compact conformations with unique contact networks and provided critical input on the relationship between contacts formed within a single molecule (intramolecular) and inside the condensed phase (intermolecular). These findings enhance our understanding of FET proteins' condensate-forming capabilities and underline differences between EWS, FUS, and TAF15.

Chromoplexy Is a Frequent Early Clonal Event in EWSR1-Rearranged Round Cell Sarcomas That Can Be Detected Using Clinically Validated Targeted Sequencing Panels.

Chromoplexy is a phenomenon defined by large-scale chromosomal chained rearrangements. A previous study observed chromoplectic events in a subset of Ewing sarcomas (ES), which was linked to an increased relapse rate. Chromoplexy analysis could potentially facilitate patient risk stratification, particularly if it could be detected with clinically applied targeted next-generation sequencing (NGS) panels. Using DELLY, a structural variant (SV) calling algorithm that is part of the MSK-IMPACT pipeline, we characterized the spectrum of SVs in EWSR1-fused round cell sarcomas, including 173 ES and 104 desmoplastic small round cell tumors (DSRCT), to detect chromoplexy and evaluate its association with clinical and genomic features. Chromoplectic events were detected in 31% of the ES cases and 19% of the DSRCT cases. EWSR1 involvement accounted for 76% to 93% of these events, being rearranged with diverse noncanonical gene partners across the genome, involving mainly translocations but also intrachromosomal deletions and inversions. A major breakpoint cluster was located on EWSR1 exons 8-13. In a subset of cases, the SVs disrupted adjacent loci, forming deletion bridges. Longitudinal sequencing and breakpoint allele fraction analysis showed that chromoplexy is an early event that remains detectable throughout disease progression and likely develops simultaneously with the driver fusion. The presence of chromoplexy was validated in an external ES patient cohort with whole exome sequencing. Chromoplexy was significantly more likely to be present in cases that were metastatic at presentation. Together, this study identifies chromoplexy as a frequent genomic alteration in diverse EWSR1-rearranged tumors that can be captured by targeted NGS panels. SIGNIFICANCE: Chromoplexy is detectable using targeted NGS in a substantial portion of EWSR1-rearranged round cell sarcomas as an early and persistent clonal event, expanding the genomic complexity of fusion-associated sarcomas.

CIC-Rearranged Sarcoma.

CIC-rearranged sarcoma is a rare type of small round cell sarcoma. The tumors often affect the deep soft tissues of patients in a wide age range. They are highly aggressive, respond poorly to chemotherapy, and have a worse outcome than Ewing sarcoma. CIC-rearranged sarcoma has characteristic and recognizable histology, including lobulated growth, focal myxoid changes, round to epithelioid cells, and minimal variation of nuclear size and shape. Nuclear ETV4 and WT1 expression are useful immunohistochemical findings. CIC fusion can be demonstrated using various methods; however, even next-generation sequencing suffers from imperfect sensitivity, especially for CIC::DUX4.

AURKA inhibition induces Ewing's sarcoma apoptosis and ferroptosis through NPM1/YAP1 axis.

Ewing's sarcoma (ES) is a rare and highly aggressive malignant tumor arising from bone and soft tissue. Suffering from intractable or recurrent diseases, the patients' therapy options are very limited. It is extremely urgent to identify novel potential therapeutic targets for ES and put them into use in clinical settings. In the present study, high-throughput screening of a small molecular pharmacy library was performed. The killing effect of the Aurora kinase A (AURKA) inhibitor TCS7010 in ES cells was identified, and AURKA was selected as the research object for further study. Disparate suppressants were adopted to study the cell death manner of TCS7010. TCS7010 and RNA silencing were used to evaluate the functions of AURKA in the apoptosis and ferroptosis of ES cells. Co-immunoprecipitation assay was used to investigate the correlation of AURKA and nucleophosmin1 (NPM1) in ES. Nude-mice transplanted tumor model was used for investigating the role of AURKA in ES in vivo. Investigations into the protein activities of AURKA were conducted using ES cell lines and xenograft models. AURKA was found to be prominently upregulated in ES. The AURKA expression level was remarkably connected to ES patients' shorter overall survival (OS) and event-free survival (EFS). Furthermore, AURKA inhibition markedly induced the apoptosis and ferroptosis of ES cells and attenuated tumorigenesis in vivo. On the part of potential mechanisms, it was found that AURKA inhibition triggered the apoptosis and ferroptosis of ES cells through the NPM1/Yes1 associated transcriptional regulator (YAP1) axis, which provides new insights into the tumorigenesis of ES. AURKA may be a prospective target for clinical intervention in ES patients.

Updates on WHO classification for small round cell tumors: Ewing sarcoma vs. everything else.

The WHO Classification of Soft Tissue and Bone Tumours currently recognizes four categories of undifferentiated small round cell sarcoma: Ewing sarcoma, round cell sarcoma with EWSR1-non-ETS fusions including NFATc2 and PATZ1, CIC-rearranged sarcoma, and sarcoma with BCOR genetic alterations. These neoplasms frequently pose significant diagnostic challenges due to rarity and overlapping morphologic and immunohistochemical findings. Further, molecular testing, with accompanying pitfalls, may be needed to establish a definitive diagnosis. This review summarizes the clinical, histologic, immunohistochemical, and molecular features of these neoplasms. In addition, differential diagnosis and areas of uncertainty and ongoing investigation are discussed.

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.

The Chromatin Remodeler CHD4 Sustains Ewing Sarcoma Cell Survival by Controlling Global Chromatin Architecture.

Ewing sarcoma is an aggressive cancer with a defective response to DNA damage leading to an enhanced sensitivity to genotoxic agents. Mechanistically, Ewing sarcoma is driven by the fusion transcription factor EWS-FLI1, which reprograms the tumor cell epigenome. The nucleosome remodeling and deacetylase (NuRD) complex is an important regulator of chromatin function, controlling both gene expression and DNA damage repair, and has been associated with EWS-FLI1 activity. Here, a NuRD-focused CRISPR/Cas9 inactivation screen identified the helicase CHD4 as essential for Ewing sarcoma cell proliferation. CHD4 silencing induced tumor cell death by apoptosis and abolished colony formation. Although CHD4 and NuRD colocalized with EWS-FLI1 at enhancers and super-enhancers, CHD4 promoted Ewing sarcoma cell survival not by modulating EWS-FLI1 activity and its oncogenic gene expression program but by regulating chromatin structure. CHD4 depletion led to a global increase in DNA accessibility and induction of spontaneous DNA damage, resulting in an increased susceptibility to DNA-damaging agents. CHD4 loss delayed tumor growth in vivo, increased overall survival, and combination with PARP inhibition by olaparib treatment further suppressed tumor growth. Collectively, these findings highlight the NuRD subunit CHD4 as a therapeutic target in Ewing sarcoma that can potentiate the antitumor activity of genotoxic agents. SIGNIFICANCE: CRISPR/Cas9 screening in Ewing sarcoma identifies a dependency on CHD4, which is crucial for the maintenance of chromatin architecture to suppress DNA damage and a promising therapeutic target for DNA damage repair-deficient malignancies.

Adamantinoma-Like Ewing Sarcoma Mimicking Merkel Cell Carcinoma in the Parotid Gland: A Diagnostic Pitfall.

Adamantinoma-like Ewing sarcoma (ALES) is a newly described rare entity, which shows EWSR1::FLI1 rearrangement characteristic of Ewing sarcoma. This can be diagnostically challenging as it manifests histologically with epithelial differentiation and has diffuse keratin expression as well as p40 and p60 positivity. We hereby report a case of ALES in a 33-year-old woman with a past medical history of breast carcinoma who presented with a right-sided parotid mass. CT scan of the neck showed a heterogenous mass within the superficial lobe, measuring 17 mm in diameter for which the patient underwent superficial parotidectomy. Histopathology of the mass revealed a malignant neoplasm formed of solid nests, cords and sheets of cells with minimal cytoplasm and monomorphic nuclei with granular chromatin and indistinct nucleoli. Brisk mitotic activity and tumor necrosis were also present. The tumor showed strong and diffuse reactivity for pankeratin (clone AE1/AE3) and keratin 20, both in a dot-like pattern, raising the suspicion of metastatic Merkel cell carcinoma; however, molecular studies showed EWSR1::FLI1 rearrangement, supporting the diagnosis of ALES. In summary, it is prudent to have knowledge about this entity to avoid its misdiagnosis as other malignancies of the head and neck region which exhibit a different clinical course, prognosis and hence treatment modalities.

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.

Engagement of CD99 Activates Distinct Programs in Ewing Sarcoma and Macrophages.

Ewing sarcoma (EWS) is the second most common pediatric bone tumor. The EWS tumor microenvironment is largely recognized as immune-cold, with macrophages being the most abundant immune cells and their presence associated with worse patient prognosis. Expression of CD99 is a hallmark of EWS cells, and its targeting induces inhibition of EWS tumor growth through a poorly understood mechanism. In this study, we analyzed CD99 expression and functions on macrophages and investigated whether the concomitant targeting of CD99 on both tumor and macrophages could explain the inhibitory effect of this approach against EWS. Targeting CD99 on EWS cells downregulated expression of the "don't eat-me" CD47 molecule but increased levels of the "eat-me" phosphatidyl serine and calreticulin molecules on the outer leaflet of the tumor cell membrane, triggering phagocytosis and digestion of EWS cells by macrophages. In addition, CD99 ligation induced reprogramming of undifferentiated M0 macrophages and M2-like macrophages toward the inflammatory M1-like phenotype. These events resulted in the inhibition of EWS tumor growth. Thus, this study reveals what we believe to be a previously unrecognized function of CD99, which engenders a virtuous circle that delivers intrinsic cell death signals to EWS cells, favors tumor cell phagocytosis by macrophages, and promotes the expression of various molecules and cytokines, which are pro-inflammatory and usually associated with tumor regression. This raises the possibility that CD99 may be involved in boosting the antitumor activity of macrophages.

Surface and Global Proteome Analyses Identify ENPP1 and Other Surface Proteins as Actionable Immunotherapeutic Targets in Ewing Sarcoma.

PURPOSE: Ewing sarcoma is the second most common bone sarcoma in children, with 1 case per 1.5 million in the United States. Although the survival rate of patients diagnosed with localized disease is approximately 70%, this decreases to approximately 30% for patients with metastatic disease and only approximately 10% for treatment-refractory disease, which have not changed for decades. Therefore, new therapeutic strategies are urgently needed for metastatic and refractory Ewing sarcoma. EXPERIMENTAL DESIGN: This study analyzed 19 unique Ewing sarcoma patient- or cell line-derived xenografts (from 14 primary and 5 metastatic specimens) using proteomics to identify surface proteins for potential immunotherapeutic targeting. Plasma membranes were enriched using density gradient ultracentrifugation and compared with a reference standard of 12 immortalized non-Ewing sarcoma cell lines prepared in a similar manner. In parallel, global proteome analysis was carried out on each model to complement the surfaceome data. All models were analyzed by Tandem Mass Tags-based mass spectrometry to quantify identified proteins. RESULTS: The surfaceome and global proteome analyses identified 1,131 and 1,030 annotated surface proteins, respectively. Among surface proteins identified, both approaches identified known Ewing sarcoma-associated proteins, including IL1RAP, CD99, STEAP1, and ADGRG2, and many new cell surface targets, including ENPP1 and CDH11. Robust staining of ENPP1 was demonstrated in Ewing sarcoma tumors compared with other childhood sarcomas and normal tissues. CONCLUSIONS: Our comprehensive proteomic characterization of the Ewing sarcoma surfaceome provides a rich resource of surface-expressed proteins in Ewing sarcoma. This dataset provides the preclinical justification for exploration of targets such as ENPP1 for potential immunotherapeutic application in Ewing sarcoma. See related commentary by Bailey, p. 934.

Suggested role for neutrophil extracellular trap formation in Ewing sarcoma immune microenvironment.

Ewing sarcoma (EWS) is a highly aggressive cancer with a survival rate of 70%-80% for patients with localized disease and under 30% for those with metastatic disease. Tumor-infiltrating neutrophils (TIN) can generate extracellular net-like DNA structures known as neutrophil extracellular traps (NETs). However, little is known about the presence and prognostic significance of tumor-infiltrating NETs in EWS. Herein, we investigated 46 patients diagnosed with EWS and treated in the Tel Aviv Medical Center between 2010 and 2021. TINs and NETs were identified in diagnostic biopsies of EWS by immunofluorescence. In addition, NETs were investigated in neutrophils isolated from peripheral blood samples of EWS patients at diagnosis and following neoadjuvant chemotherapy. The relationships between the presence of TINs and NETs, pathological and clinical features, and outcomes were analyzed. Our results demonstrate that TIN and NETs at diagnosis were higher in EWS patients with metastatic disease compared with those with local disease. High NET formation at diagnosis predicted poor response to neoadjuvant chemotherapy, relapse, and death from disease (p < 0.05). NET formation in peripheral blood samples at diagnosis was significantly elevated among patients with EWS compared with pediatric controls and decreased significantly following neoadjuvant chemotherapy. In conclusion, NET formation seems to have a role in the EWS immune microenvironment. Their presence can refine risk stratification, predict chemotherapy resistance and survival, and serve as a therapeutic target in patients with EWS.

Therapeutic Hope on the Surface of Ewing Sarcoma.

A detailed analysis of the Ewing sarcoma surfacesome has arrived. Robust expression of surface CDH11 and ENPP1 was identified. This "comprehensive catalog" of the Ewing surfacesome serves as a fresh roadmap to development of new therapeutic approaches, including immunotherapies and multi-modality therapeutic combinations, to target aggressive Ewing tumor subpopulations. See related article by Mooney et al., p. 1022.

[Ewing's sarcoma of central nervous system: a clinicopathological analysis of six cases].

Objective: To investigate the clinicopathological characteristics, pathological diagnosis of Ewing's sarcoma of the central nervous system. Methods: Six cases of Ewing's sarcoma of the central nervous system diagnosed at the First Affiliated Hospital of Nanjing Medical University, Nanjing, China from 2015 to 2022 were collected. The clinical manifestations, histological morphology, immunophenotype and molecular genetics of these cases were analyzed. The related literature was reviewed. Results: There were four males and two females, with a male to female ratio of 2∶1. The onset age was 17-40 years, with a median age of 23 years. All 6 tumors were located in the spinal cord (2 cases of cervical vertebra, 1 case of thoracic vertebra, 2 cases of lumbar vertebra, and 1 case of sacral vertebra). The patients' clinical manifestations were mostly lumbago, weakness and numbness of lower limbs/limbs. In 1 case, the tumor recurred and metastasized to the suprasellar region and the third ventricle. Microscopically, the tumor showed diffuse infiltrative growth. In some cases, the tumor was closely related to the spinal meninges. The tumor cells were arranged in sheet, lobular, thin-rope, and nest-like patterns. Homer-Wright rosette was visible. The tumor cells were small to medium in size, and most of them had scant cytoplasm. A few cells had clear cytoplasm. Some areas were rhabdoid. The tumor cell nuclei showed focal mild pleomorphism. The chromatin was uniform and delicate while the nucleoli were not obvious. Mitosis was commonly seen. The tumor was separated by fibrous connective tissue and may be accompanied by mucinous degeneration. Immunohistochemistry showed that all tumors were positive for CD99, NKX2.2, Fli1, ERG. ATRX, H3K27me3, INI1 and BRG1 were all retained. Immunohistochemical stains for EMA, GFAP and Olig2 were negative. The Ki-67 proliferation index was 30%-70%. EWSR1 break-apart FISH test was positive. Conclusions: Ewing's sarcoma is rare in the central nervous system and needs to be distinguished from a variety of neoplasms with primitive undifferentiated small cell morphology. Immunohistochemistry and molecular genetics may be required for a proper diagnosis.

[The expression of neuraminidase-1 in Ewing sarcoma and its impact on sarcoma cell proliferation and migration].

Objective: To investigate the expression of neuraminidase-1 (NEU1) in Ewing sarcoma (ES) tissue and its effect on the proliferation and migration of ES cells. Methods: To obtain datasets of ES from the National Center for Biotechnology Information's High-Throughput Gene Expression Omnibus (GEO) for the analysis of NEU1 expression in ES; to acquire ES patient dataset from the International Cancer Genome Consortium (ICGC) database and apply Kaplan-Meier survival analysis to investigate the relationship between NEU1 and the prognosis of ES patients; adopting both univariate and multivariate Cox regression analysis to determine whether NEU1 is a prognostic factor for ES; adopting the Kyoto Encyclopedia of Genes and Genomes (KEGG) annotation to analyze the potential mechanism of NEU1 in regulating the malignant biological behavior of ES; adopting the real-time fluorescence quantitative polynucleotide chain reaction (RT-qPCR) to verify the expression of NEU1 in the human bone marrow mesenchymal stem cells (hBMSC) and the ES cell line RD-ES; adopting the transfection technology to knock down the expression of NEU1 in ES cell lines and divide them into two groups: shRNA-NEU1 and shRNA-NC to explore the effects of altered NEU1 expression on ES malignant behavior; adopting the cell counting kit (CCK-8) and cell clone formation experiment to detect the proliferation ability of two groups of cells; adopting the scratch healing experiment to test the cell migration ability of the two groups. Results: We retrieved and analyzed data from the GEO database, including GSE17674 (44 ES tissues and 18 normal tissues) and GSE17679 (87 ES tissues and 18 normal tissues), and found that NEU1 expression was significantly higher in ES tissues compared to normal control tissues (P<0.001). The complete gene expression and clinical information of 56 ES patients obtained from the ICGC database revealed that the ES patients with high NEU1 expression (n=28) had a significantly lower overall survival rates at different time points compared to those with low NEU1 expression (n=28) (HR=2.830, 95%CI:1.324-6.051, P=0.005). Univariate analysis indicated that NEU1 could impact ES patient prognosis (HR=1.049, 95%CI: 1.008-1.092, P=0.019), and multivariate analysis further suggested that NEU1 could serve as a risk factor for ES prognosis (HR=1.087, 95%CI: 1.028-1.148, P=0.003). KEGG results show that MAPK signaling pathway and cell adhesion molecule signaling pathway were potential mechanisms regulating the malignant process of ES. The RT-qPCR results showed that the expression level of NEU1 in the RD-ES cell line is significantly higher than that in the control cell hBMSC (2 184.23±527.32 vs 1.00±0.08, P<0.001). The CCK-8 experiment results show that the proliferation of RD-ES cells in the NEU1 knockdown group was lower than that in the control group at 24, 48, and 72 hours (0.494±0.126 vs 0.696±0.118, 0.657±0.096 vs 1.142±0.182, 1.053±0.064 vs 1.980±0.146, all P<0.001). The results of single cell clone formation experiment show that the number of colony formation in the low expression NEU1 group was significantly lower than that in the control group (184.2±123.9 vs 362.8±78.0, P=0.021). The cell scratch healing experiment finds that the average scratch distance of the NEU1 knockdown group was significantly lower than that of the control group (19.6%±5.7% vs 56.0%±7.6%, P<0.001). Conclusion: NEU1 may be a prognostic factor in ES, and its abnormal expression in ES can affect the proliferation and migration ability of the ES cells, leading to poor prognosis in ES patients.