BACKGROUND: Children ≤5 years of age with Ewing\'s sarcoma (ES) possibly have a distinct disease biology, data on which are scarce. We evaluated clinical features, outcomes, and prognostic factors of ES among children with age ≤5 years.
METHODS: Children with ES registered between 2003 and 2019 were included. Baseline clinical and treatment details were retrieved from medical records. Prognostic factors were identified using multivariable Cox regression. Clinical features and outcomes of children ≤5 years were compared with those greater than 5 years by chi-square and log-rank tests. Propensity score-matched (PSM) analysis was done to evaluate the impact of age on survival in the metastatic and localized subgroups.
RESULTS: Out of the 859 patients, 86 (10%) were ≤5 years of age (median age 4 years, 60 males [69.8%]). The most common location was the extremities (37.2%), followed by thorax (27.9%) and head and neck (H&N) (22.1%); baseline metastases were seen in 25 patients (29.8%). The median event-free-survival (EFS) and overall survival (OS) were 25.6 and 68.7 months, respectively. Metastatic disease predicted inferior OS (hazard ratio [HR] = 2.54, p = .018) and EFS (HR = 2.47, p = .007], symptom duration ≤3 months predicted an inferior OS (HR = 2.17, p = .048). Compared to age greater than 5 years, younger children had more H&N and less pelvic primaries (p < .001) and lesser baseline metastases (p = .037). PSM analysis did not reveal any significant impact of age on OS in the metastatic (HR = 1.59, p = .29) or localized cohort (HR = 1.77, p = .09).
CONCLUSIONS: Children with ES ≤5 years of age have a distinct favorable clinical presentation. However, age is not an independent prognostic factor for survival outcomes when adjusted for confounders.

UNASSIGNED: The present study evaluates the clinical outcomes of children, adolescents and adults with Ewing sarcoma and identifies the prognostic factors.
UNASSIGNED: Included in the study were 222 pediatric and adult patients diagnosed with Ewing sarcoma (EwS) who were followed up between 1992 and 2019, and whose data were analyzed retrospectively.
UNASSIGNED: The median age of 131 male and 91 female patients included in the study was 13 (1-64). The median follow-up duration of the survivors was 79 months (range, 11-182 months). The 3-year EFS rate of the 222 patients was 34 % (Confidence Interval (CI) (0.158-0.242 %) and the OS rate was 54 % (CI, 0.289-0.590 %). For the non-metastatic patients, the 3-year EFS rate was 47 % and the OS was 68 %, while for the metastatic patients the 3-year EFS rate was 13 % and the OS was 30 %. Of the patient sample, 81 (36, 5 %) survived, of whom 72 were continuously free of disease while the disease persisted in nine, and three developed a secondary neoplasm (2 of whom subsequently died while one survived disease-free). Of the 129 patients who relapsed with metastases and/or local recurrence, eight survived and are disease-free, nine are alive with uncontrolled disease; five were lost to follow-up and 107 died.
UNASSIGNED: The findings of the present study suggest metastatic disease at presentation and positive margins after surgery to be of prognostic significance in EwS. Disruptions in aggressive local treatments may reduce the chances of cure in EwS.

Sarcomas comprise between 10-15% of all pediatric malignancies. Osteosarcoma and Ewing sarcoma are the two most common pediatric bone tumors diagnosed in children and young adults. These tumors are commonly treated with surgery and/or radiation therapy and combination chemotherapy. However, there is a strong need for the development and utilization of targeted therapeutic methods to improve patient outcomes. Towards accomplishing this goal, pre-clinical models for these unique malignancies are of particular importance to design and test experimental therapeutic strategies prior to being introduced to patients due to their origination site and propensity to metastasize. Pre-clinical models offer several advantages for the study of pediatric sarcomas with unique benefits and shortcomings dependent on the type of model. This review addresses the types of pre-clinical models available for the study of pediatric solid tumors, with special attention to the bone sarcomas osteosarcoma and Ewing sarcoma.

Ewing sarcoma/primitive neuroectodermal tumor (ES/PNET) is a type of malignancy that usually appears as a tumor in the bone. However, in a few patients with ES/PNET, it can occur outside of the bone. Although extraosseous ES/PNET can appear in various parts of the body, involvement of small bowel is rare. If it does, it can present with vague abdominal pain and gastrointestinal bleeding. This case report presents a 28-year-old gentleman with extraosseous ES/PNET in the duodenum who experienced gastrointestinal bleeding as a presenting symptom.

With the increasing use of next generation sequencing in soft tissue pathology, particularly in neoplasms not fitting any World Health Organization (WHO) category, the spectrum of EWSR1 fusion-associated soft tissue neoplasms has been expanding significantly. Although recurrent EWSR1::ATF1 fusions were initially limited to a triad of mesenchymal neoplasms including clear cell sarcoma of soft tissue, angiomatoid fibrous histiocytoma and malignant gastrointestinal neuroectodermal tumor (MGNET), this family has been expanding. We herein describe 4 unclassified extra-abdominal soft tissue (n = 3) and bone (n = 1) neoplasms displaying epithelioid and round cell morphology and carrying an EWSR1::ATF1 fusion. Affected were 3 males and 1 female aged 20-56 years. All primary tumors were extra-abdominal and deep-seated (chest wall, mediastinum, deltoid, and parapharyngeal soft tissue). Their size ranged 4.4-7.5 cm (median, 6.2). One patient presented with constitutional symptoms. Surgery with (2) or without (1) neo/adjuvant therapy was the treatment. At last follow-up (8-21 months), 2 patients developed progressive disease (1 recurrence; 1 distant metastasis). The immunophenotype of these tumors is potentially misleading with variable expression of EMA (2 of 3), pankeratin (2 of 4), synaptophysin (2 of 3), MUC4 (1 of 3), and ALK (1 of 3). All tumors were negative for S100 and SOX10. These observations point to the existence of heretofore under-recognized group of epithelioid and round cell neoplasms of soft tissue and bone, driven by EWSR1::ATF1 fusions, but distinct from established EWSR1::ATF1-associated soft tissue entities. Their overall morphology and immunophenotype recapitulate that of the emerging EWSR1/FUS::CREB fusion associated intra-abdominal epithelioid/round cell neoplasms. Our cases point to a potentially aggressive clinical behavior. Recognizing this tumor type is mandatory to delineate any inherent biological and/or therapeutic distinctness from other, better-known sarcomas in the differential diagnosis including sclerosing epithelioid fibrosarcoma.

Ewing sarcoma is a cancer of bone and soft tissue in children and young adults primarily driven by the EWS-FLI1 fusion oncoprotein, which has been undruggable. Here, we report that Ewing sarcoma depends on secreted sphingomyelin phosphodiesterase 1 (SMPD1), a ceramide-generating enzyme, and ceramide. We find that G-protein-coupled receptor 64 (GPR64)/adhesion G-protein-coupled receptor G2 (ADGRG2) responds to ceramide and mediates critical growth signaling in Ewing sarcoma. We show that ceramide induces the cleavage of the C-terminal intracellular domain of GPR64, which translocates to the nucleus and restrains the protein levels of RIF1 in a manner dependent on SPOP, a substrate adaptor of the Cullin3-RING E3 ubiquitin ligase. We demonstrate that both SMPD1 and GPR64 are transcriptional targets of EWS-FLI1, indicating that SMPD1 and GPR64 are EWS-FLI1-induced cytokine-receptor dependencies. These results reveal the SMPD1-ceramide-GPR64 pathway, which drives Ewing sarcoma growth and is amenable to therapeutic intervention.

ISLET-1 (ISL1) is a LIM-homeodomain transcription factor. Selective ISL1 expression is shown in neuroendocrine, non-neuroendocrine, and some soft tissue tumors including desmoplastic small round cell tumor (DSRCT). We assessed the specificity of ISL1 (clone EP283, 1:500, Cell Marque) in 288 soft tissue tumors, which included 17 DSRCTs and other histologic mimics. Positive staining threshold for ISL1 was set to >10 % of neoplastic cell nuclei at moderate intensity. ISL1 IHC was positive in 15/16 (94 %) DSRCTs with 75 % showing diffuse (>50 %) expression. ISL1 was positive in 1/10 (10 %) Ewing sarcomas (EWS), 7/13 (54 %) alveolar rhabdomyosarcoma (RMS), 14/22 (63 %) embryonal RMS, 7/14 (50 %) synovial sarcomas, 15/16 (93 %) neuroblastoma, 1/5 (20 %) Wilms tumor, 2/4 (50 %) olfactory neuroblastoma, and all 9 Merkel cell carcinomas. Other tumors, including all CIC::DUX4 sarcomas, were negative except 3/27 leiomyosarcomas, and 1 each of angiosarcoma, myxoid liposarcomas, inflammatory myofibroblastic tumor, malignant peripheral nerve sheath tumor, tenosynovial giant cell tumor, dedifferentiated LPS, and 1 ectomesenchymoma. In summary, among the soft tissue tumors tested, ISL1 is a highly sensitive but moderately specific marker for DSRCT and may be useful to distinguish from round cell mimics including EWS and CIC::DUX4 sarcomas. The oncogenic role of ISL1 in these tumors warrants further investigation.

BACKGROUND: Adolescents and young adults (AYAs) with Ewing sarcoma have a worse prognosis than children. Population-based survival evaluations stratifying findings by important clinical factors are, however, limited. This Dutch population study comprehensively compared survival of children and AYAs with Ewing sarcoma over three decades considering diagnostic period, tissue of origin, tumor site, and disease stage.
METHODS: Data on all children (0-17 years, N = 463) and AYAs (18-39 years, N = 379) diagnosed with Ewing sarcoma in the Netherlands between 1990-2018 were collected from the Netherlands Cancer Registry with follow-up until February 2023. Five-year relative survival was calculated using the cohort method. Multivariable analyses were conducted through Poisson regression.
RESULTS: Children with Ewing sarcoma had a significantly higher 5-year relative survival than AYAs (65 % vs. 44 %). An increasing trend in survival was noted reaching 70 % in children and 53 % in AYAs in 2010-2018. Results were similar for Ewing bone sarcoma and extraosseous Ewing sarcoma. AYAs had a poorer prognosis than children for most tumor sites and regardless of disease stage. Survival probabilities were 60 % vs. 78 % for localized disease and 20 % vs. 33 % for metastatic disease. Multivariable-regression analysis, adjusted for follow-up time, diagnostic period, sex, disease stage, and tumor site, confirmed increased excess mortality among AYAs compared with children (excess HR: 1.7, 95 % CI: 1.3-2.1).
CONCLUSIONS: Despite survival improvements since the 1990s, AYAs with Ewing sarcoma in the Netherlands continue to fare considerably worse than children. This survival disparity was present irrespective of tissue of origin, tumor site, and disease stage.

Ewing sarcoma (EwS) is a cancer that arises in the bones and soft tissues, typically driven by the Ewing\'s sarcoma breakpoint region 1-Friend leukemia virus integration 1 (EWS-FLI) oncogene. Implementation of genetically modified animal models of EwS has proved difficult largely owing to EWS-FLI\'s high toxicity. The EWS-FLI1FS frameshift variant that circumvents toxicity but is still able to perform key oncogenic functions provided the first study model in Drosophila. However, the quest for Drosophila lines expressing full-length, unmodified EWS-FLI remained open. Here, we show that EWS-FLI1FS\'s lower toxicity is owed to reduced protein levels caused by its frameshifted C-terminal peptide, and report new strategies through which we have generated Drosophila lines that express full-length, unmodified EWS-FLI. Using these lines, we have found that the upregulation of transcription from GGAA-microsatellites (GGAAμSats) presents a positive linear correlation within a wide range of EWS-FLI protein concentrations. In contrast, rather counterintuitively, GGAAμSats-independent transcriptomic dysregulation presents relatively minor differences across the same range, suggesting that GGAAμSat-dependent and -independent transcriptional upregulation present different kinetics of response with regards to changing EWS-FLI protein concentration. Our results underpin the functional relevance of varying EWS-FLI expression levels and provide experimental tools to investigate, in Drosophila, the effect of the EWS-FLI \'high\' and \'low\' states that have been reported and are suspected to be important for EwS in humans.

BACKGROUND: Macrophages play important roles in phagocytosing tumor cells. However, tumors escape macrophage phagocytosis in part through the expression of anti-phagocytic signals, most commonly CD47. In Ewing sarcoma (ES), we found that tumor cells utilize dual mechanisms to evade macrophage clearance by simultaneously over-expressing CD47 and down-regulating cell surface calreticulin (csCRT), the pro-phagocytic signal. Here, we investigate the combination of a CD47 blockade (magrolimab, MAG) to inhibit the anti-phagocytic signal and a chemotherapy regimen (doxorubicin, DOX) to enhance the pro-phagocytic signal to induce macrophage phagocytosis of ES cells in vitro and inhibit tumor growth and metastasis in vivo.
METHODS: Macrophages were derived from human peripheral blood monocytes by granulocyte-macrophage colony-stimulating factor (GM-CSF) and macrophage colony-stimulating factor (M-CSF). Flow cytometry- and microscopy-based in-vitro phagocytosis assays were performed to evaluate macrophage phagocytosis of ES cells. Annexin-V assay was performed to evaluate apoptosis. CD47 was knocked out by CRISPR/Cas9 approach. ES cell-based and patient-derived-xenograft (PDX)-based mouse models were utilized to assess the effects of MAG and/or DOX on ES tumor development and animal survival. RNA-Seq combined with CIBERSORTx analysis was utilized to identify changes in tumor cell transcriptome and tumor infiltrating immune cell profiling in MAG and/or DOX treated xenograft tumors.
RESULTS: We found that MAG significantly increased macrophage phagocytosis of ES cells in vitro (p < 0.01) and had significant effect on reducing tumor burden (p < 0.01) and increasing survival in NSG mouse model (p < 0.001). The csCRT level on ES cells was significantly enhanced by DOX in a dose- and time-dependent manner (p < 0.01). Importantly, DOX combined with MAG significantly enhanced macrophage phagocytosis of ES cells in vitro (p < 0.01) and significantly decreased tumor burden (p < 0.01) and lung metastasis (p < 0.0001) and extended animal survival in vivo in two different mouse models of ES (p < 0.0001). Furthermore, we identified CD38, CD209, CD163 and CD206 as potential markers for ES-phagocytic macrophages. Moreover, we found increased M2 macrophage infiltration and decreased expression of Cd209 in the tumor microenvironment of MAG and DOX combinatorial therapy treated tumors.
CONCLUSIONS: By turning \"two keys\" simultaneously to reactivate macrophage phagocytic activity, our data demonstrated an effective and highly translatable alternative therapeutic approach utilizing innate (tumor associated macrophages) immunotherapy against high-risk metastatic ES.