ABSTRACTMetastatic Ewing sarcoma (MES) has a poor prognosis. This multicenter observational study provides real‐world data on treatment patterns of patients with MES in France. Treatment characteristics, outcomes such as time to next treatment (TTNT) and overall survival (OS), and prognostic factors of patients aged ≥ 12 years treated for a MES in 11 French reference network centers were retrieved from our national database. From 2008 to 2018, 156 patients with MES were included: 82 were metastatic at diagnosis (upfront metastatic cohort), 74 had developed secondary metastases after treatment of a localized disease (metastatic relapse cohort). 94% of patients received systemic treatment, with a median of three lines (1–11), 61% had at least one loco‐regional procedure and 42% of patients participated in a clinical trial in the metastatic setting. Median OS from metastatic diagnosis was 20.3 months [95% CI 14.0; 27.7] in the metastatic relapse cohort and 31.4 months [95% CI 26.5; 42.5] in the upfront metastatic cohort (p = 0.02). Median TTNT in first metastatic line was 16.8 months [95% CI 12.9; 21.3] in the upfront metastatic cohort and 7.4 months [95% CI 5.0; 11.1] in the metastatic relapse cohort, 5.8 months [95% CI 3.6; 7.3] in 2nd line and 3.8 months [95% CI 2.8; 5.7] in 3rd line, without significant difference between cohorts and treatment regimens. Patients with upfront MES have a longer OS than patients with relapsing disease, mainly due to first metastatic line dose‐dense polychemotherapy and loco‐regional procedures in selected patients. Main regimens used at relapse are associated with the same range of benefit and survival, while TKIs such as regorafenib or cabozantinib have modest activity. Inclusion in clinical trials should be prioritized.

Though 18F-FDG-PET CT scan is established as a standard of care imaging technique for staging and restaging in Ewing Sarcoma (ES), its utility in the prognostic assessment of residual masses post definitive radiotherapy to the primary is still unknown. This study audits the above. Children ≤ 15 years of age with ES treated from January 2012 to December 2023 who received definitive radiotherapy to the primary and had an 18F-FDG-PET CT 3 months post completion of RT (PET-3) were analyzed retrospectively. Disease status of the soft tissue component based on PET-3 was classified as having either no residual, anatomic residual with no FDG-avidity, or FDG-avid residual. One hundred and forty-nine patients formed the study cohort. Metastatic disease was present in 46.3% (n = 69). PET3 showed FDG-avid residual in 17.4% (n = 26), anatomic residual in 30.2% (n = 45), and no residual in 52.3% (n = 78) of patients. Five-year EFS of those with no residual, anatomic residual, and FDG-avid residual in the whole cohort were 52.8% (95% CI: 40.4%-65.2%), 45.9% (95% CI: 28.4%-63.4%), 42.6% (95% CI: 20.7%-64.6%) (p = 0.038). Among patients with FDG-avid residual disease, 5-year EFS was 22.9% (95% CI: 5.0%-45.2%) for metastatic cases compared to 75.0% (95% CI: 44.0%-100%) for localized disease (p = 0.003). A PET3 SUVmax > 3.9 was prognostic for EFS (HR-9.59, 95% CI: 3.94-23.3, p ≤ 0.001) in the metastatic cohort. In patients with metastatic Ewing Sarcoma treated with definitive radiotherapy of the primary, the persistence of FDG-avid residual on post-treatment PET/CT signifies a markedly poor prognosis. In particular, a PET3 SUVmax exceeding 3.9 delineates an ultra-high-risk cohort.

Metastatic Ewing sarcoma (EWS) has a poor prognosis. While metastasis-directed therapy benefits oligometastatic disease, the role of comprehensive all-site radiotherapy in widespread disease remains underexplored. This study aimed to preliminarily assess its short-term efficacy and safety. This retrospective analysis included 21 consecutive metastatic EWS patients treated with helical tomotherapy (Aug 2024-Jun 2025). All known metastases and unresected primary tumors received radiotherapy (45-55Gy in 20 fractions for most sites; 12-45Gy for lung/pleural metastases). Concurrent systemic therapies included chemotherapy, tyrosine kinase inhibitors (TKIs), immune checkpoint inhibitors (ICIs), or combinations. Primary endpoints were local objective response rate (ORR, RECIST 1.1) at 2months post-radiotherapy and acute toxicity (CTCAE 5.0). Systemic therapy subgroup analyses were exploratory. Median follow-up was 6months (range 2-11). Among 77 target lesions, ORR was 61.0% (CR 48.1%), and disease control rate (DCR) was 97.4%. Soft tissue lesions had a significantly higher CR rate than bone metastases (78.3% vs 3.2%, p < 0.001), potentially influenced by RECIST 1.1 limitations and treatment heterogeneity. Median progression-free survival (PFS) was 6.0months (95% CI: 2.30-9.70), and median overall survival (OS) was 8.0months (95% CI: 6.69-9.31). Combination systemic therapy was associated with improved PFS (HR 3.94, 95%CI 1.32-11.78; p = 0.014), with chemotherapy-based regimens yielding the best PFS (8.5months). TKI-containing regimens were linked to shorter PFS (4.0 vs 8.5months, p = 0.048) due to selection bias. Acute toxicity was manageable, with grade 3 thrombocytopenia (19.0%) and pneumonitis (4.7%) resolving with treatment. All-site radiotherapy achieves promising short-term local control with manageable toxicity in metastatic EWS, demonstrating feasibility and a lesion site-specific response warranting further investigation. Systemic therapy, particularly chemotherapy, correlates with improved PFS. Given high systemic progression rates, long-term outcomes may benefit from integration with more effective systemic therapies, supporting future prospective trials.

Survival rates for metastatic Ewing sarcoma (EwS) have remained persistently low over recent decades, highlighting the need for more effective chemotherapeutic options. Potential targets may be found within the Neuropeptide Y (NPY) signalling pathway that has been implicated in EwS cell survival. However, confounding factors include hypoxia that modulates NPY signalling, dipeptidyl peptidase-4 (DPP4/CD26) that cleaves NPY and interactions via NPY signalling from infiltrating immune cells. We investigated these interactions in A673 and SK-ES-1 EwS cell lines and THP-1 monocytes to identify therapeutic targets suitable for drug repurposing. Both EwS cell lines secreted NPY into conditioned media and extracellular vesicles. Recombinant NPY enhanced viability of both A673 and SK-ES-1 cells; however, the NPY1R antagonist BMS-193885 reduced viability in A673 cells only. Recombinant DPP4 widely promoted EwS viability and, under hypoxic conditions, it increased cell metabolism. The DPP4 inhibitor linagliptin, which is used clinically, consistently suppressed EwS viability with elevated sensitivity under hypoxia, where there was increased cell death of SK-ES-1 cells. Conversely, in THP-1 monocytes, NPY suppressed metabolism, BMS-193885 increased live-cell staining and DPP4 induced cell death. These findings suggest that NPY and DPP4 enhance EwS survival through autocrine/paracrine signalling while reducing monocyte viability. Thus, targeting the NPY/DPP4 signalling axis may provide therapeutic benefit by directly suppressing EwS growth and enhancing efficacy of immunotherapy.

160P Cabozantinib in late-line therapy for metastatic Ewing sarcoma: A single-center experience

Metastatic Ewing's sarcoma in serous effusions: a clinicopathological analysis of three cases

Ewing sarcoma (EWS) is the second most common primary malignant bone and soft tissue tumor. Relapsed EWS is difficult to treat, with poor long-term survival. Isolated lung relapse occurs in about a third of relapsed patients, and there is no standard of care. We aim to review the available literature with the goal of providing guidance for the management of isolated pulmonary relapsed EWS using the 2 major types of pulmonary-directed radiation: whole lung irradiation (WLI) and stereotactic body radiation therapy (SBRT). PubMed and Web of Science were reviewed for studies that evaluated patients with pulmonary relapsed or metastatic EWS who received radiation. Twelve articles met criteria for inclusion; 8 reviewed the use of WLI and 4 reviewed SBRT. Patients who received WLI had improved progression-free survival compared with those who received SBRT, though most had already achieved disease control. There were low rates of toxicity reported with both modalities. WLI appears to be beneficial in patients who have a complete resection before undergoing radiation, while SBRT is indicated to areas of active disease. We suggest future studies test a combination of SBRT in areas of active disease with low-dose WLI to decrease the risk for future metastases.

Metastatic Ewing Sarcoma remains a critical therapeutic challenge with 5-year survival below 30%. The EWSR1-FLI1 fusion oncogene is undruggable by conventional approaches, requiring integrated bioengineering solutions. We present CRISPR-TTP, a modular architecture combining high-fidelity CRISPR-Cas9 genome engineering (>94% efficiency), FUS-programmable temporally controlled delivery via HOF-nanoparticles (1-2mm spatial resolution), dendritic cell autovaccination, and PD-1 blockade. A multimodal AI system orchestrates real-time personalization and optimization. In silico modeling predicts ∼96.3% tumor growth inhibition and a ∼65% improvement in median survival. CD8+ T-cell infiltration increases ∼3.2-fold. AI-optimized sgRNA prediction accuracy reaches 89.3%. This CC0-licensed architecture defines a new standard for integrated, spatiotemporally programmable precision oncology and is suitable for compassionate-use-ready translational deployment.

Background: Ewing sarcoma (ES) is a highly aggressive malignant tumor that predominantly affects children and young adults. Despite advances in multimodal therapy, relapse and refractory disease remain the leading causes of treatment failure. High-dose chemotherapy followed by autologous stem cell transplantation (HDCT-ASCT) has been proposed as a consolidation strategy for high-risk or relapsed ES; however, its clinical value remains controversial. Methods: We retrospectively analyzed 46 consecutive patients with locally advanced or metastatic ES who underwent HDCT-ASCT after at least one prior systemic therapy line. Clinical, pathological, and transplant-related variables were evaluated for associations with overall survival (OS), post-transplant OS (OS-2), and progression-free survival (PFS). Survival was estimated using the Kaplan–Meier method, and prognostic factors were assessed by Cox proportional hazards modeling. Results: The median age at diagnosis was 23 years (range: 14–55). Median OS from diagnosis was 42 months, while post-transplant OS-2 and PFS were 8 and 5 months, respectively. Younger patients (≤23 years) had significantly longer OS (50 vs. 34 months; p = 0.027). Liver metastasis predicted inferior OS (HR = 5.411; p = 0.006), whereas lung metastasis was associated with shorter OS-2 (HR = 2.672; p = 0.025) and PFS (HR = 6.037; p = 0.016). Treatment-related mortality was low (2.1%), though hematologic toxicity was universal. Overall, HDCT-ASCT provided transient disease control, with modest benefit confined to younger, chemosensitive, and medically fit patients. Conclusions: In this real-world cohort, HDCT-ASCT was feasible and safe but offered limited survival advantage in heavily pretreated Ewing sarcoma. Prognosis was primarily influenced by age and metastatic distribution, particularly hepatic and pulmonary involvement. These findings support a risk-adapted, biology-driven approach reserving HDCT-ASCT for selected patients and highlight the need for post-transplant maintenance strategies integrating targeted or immunotherapeutic modalities.

Ewing sarcoma (ES) is a rare but extremely aggressive bone and soft-tissue tumor. Clinical outcomes for patients with metastatic or recurrent ES remain poor, particularly for patients who are resistant to chemotherapy. This underscores an urgent need for alternative treatment strategies for these patients. A deep and comprehensive understanding of the cell-cell communications in ES may help identify new therapeutic approaches. We first applied single-cell RNA sequencing (scRNA-seq) data analysis to map the cell-cell communication network within the ES tumor microenvironment (TME). Then, based on the cell-cell communication map, we inferred that multi-kinase anti-angiogenic inhibitors might effectively treat ES. Therefore, we investigated the anti-tumor efficacy of a novel multi-kinase inhibitor, KC1036, which primarily targets VEGFR2, MET, and AXL in ES cancer cell lines. The efficacy of KC1036 in ES was further validated in cell line-derived xenograft (CDX) models and a treatment-naïve patient-derived xenograft (PDX) model. We plotted a comprehensive cell-cell communication map of ES, where ES was characterized by highly immunosuppressive TME, strong autocrine signal NPY-NPY1R in tumor cells, wide activation of receptor kinase signaling pathways in cancer-associated fibroblasts (CAFs) (e.g., AXL, MET, FGFR, PDGFR, and KIT), and robust activation of tumor angiogenesis pathways (e.g., VEGFA/B-VEGFR1/2). Multi-kinase inhibitor KC1036 effectively inhibited ES tumor growth in both CDX and PDX models with superior efficacy compared to pazopanib, cabozantinib, and doxorubicin (DOX). The novel anti-angiogenic inhibitor, KC1036, is effective in treating ES in the preclinical models.

Surface expression of the disialoganglioside subtype GD2 has been observed on Ewing sarcoma (ES) cells, making it a suitable target for immunotherapy with the anti-GD2 antibody dinutuximab beta (DB). Here we report our experience of using DB in a cohort of 13 patients with GD2-positive, metastatic ES, in both the frontline (n=9) and relapsed/refractory (n=4) settings, when added to standard chemotherapeutic regimens. Outcomes were compared with 24 patients, primarily with localized ES, who were also treated at our center with standard therapy alone (without DB). Patients treated with DB had a median overall survival (OS) of 1877 days in the frontline setting and 810 days in the relapsed/refractory setting. Median time to progression was 1811 days and 782 days, respectively. In contrast, those treated with standard therapy alone in our center demonstrated a median OS of 1547 days and 210 days in the frontline and relapsed/refractory setting, respectively, with median times of progression of 1261 days and 113 days. DB treatment was well tolerated, with no new or unexpected adverse events reported. Anti-GD2 immunotherapy with DB represents a promising therapeutic option to improve outcomes in patients with metastatic ES, in both the frontline and relapsed/refractory settings.

Ewing Sarcoma (ES) is a rare, malignant bone neoplasm that is primarily diagnosed in childhood and adolescence. The aggressive nature of this neoplasm requires the use of surgery, radiation and a rigorous chemotherapy regimen. Metastatic ES carries a poor prognosis, which necessitates the development of new therapeutic agents. Mithramycin was tested in targeted therapy due to its specific inhibitory effects on the EWS-FLI1 fusion protein which is present in >85% of ES tumors. We tested the combination of Mithramycin with chemotherapeutic agents vincristine (VCR) and Etoposide (Eto) for inducing higher cytotoxicity against ES cells, CHLA10 and TC205. Cardiomyocyte cell line, H9C2 was used to test the effect on non-malignant cells. Cell viability was measured using the CellTiter-Glo kit, and the combination index was evaluated to determine the type of combination response (antagonistic, additive, or synergistic). Apoptotic cells were measured post-treatment with vehicle (DMSO, control), monotherapy (mithramycin or etoposide), or combination therapy (mithramycin + etoposide) using BD LSRII flow cytometer and analyzed utilizing FlowJo software V8.0. The apoptotic protein marker c-PARP in both treatment and control groups was analyzed using Western blot analysis. The results showed higher cytotoxicity for combination treatment when compared to individual agents, and the combination index confirmed the response as synergistic. H9C2 cells did not demonstrate significant decreases in cell viability when treated with combination therapy, highlighting the specificity of the treatment toward its target tissue. Flow cytometry confirmed the underlying mechanism as upregulation of apoptosis which is further supported by an increase in effector caspases 3/7 and elevated expression of c-PARP. These in vitro assays using ES cells provide preliminary evidence for the benefit of chemotherapy and mithramycin combination.

Ewing sarcoma is a rare malignant tumor that primarily affects children and young adults, commonly metastasizing to the lungs, bone, and bone marrow. CNS involvement is extremely rare, especially with the primary presentation of intracranial hemorrhage. Current protocols do not include routine neuroimaging, potentially delaying the detection of brain metastasis. Additionally, Ewing sarcoma patients are at risk of developing chemotherapy-induced thrombocytopenia that can significantly increase the possibility of intracranial hemorrhage. A 19-year-old male with metastatic Ewing sarcoma of the pelvis presented with sudden-onset severe headache, seizures, and unresponsiveness. Imaging revealed a large left frontoparietal intraparenchymal hemorrhage with midline shift requiring emergency surgical decompression. Histopathology confirmed metastatic Ewing sarcoma. Despite proper management, the patient's medical condition deteriorated, leading to subsequent death. Early and interval brain imaging should be considered in patients diagnosed with Ewing sarcoma, especially those undergoing chemotherapy with thrombocytopenic effects. Earlier detection of CNS involvement may facilitate timely intervention and improve outcomes. Future research should explore optimized surveillance strategies and the connection between metastatic intracranial Ewing sarcoma and the unusual presentation of hemorrhage. https://thejns.org/doi/10.3171/CASE25246.

Vascular endothelial growth factor (VEGF) is an angiogenic marker and is implicated in the carcinogenesis and prognostication of cancers. However, its prognostic potential in Ewing sarcoma (ES) merits exploration. Histopathologically confirmed consecutive ES cases registered at our institute from 2014 to 2018 were analyzed. Immunohistochemical staining for VEGF was performed on tumor tissues, and they were further classified based on VEGF intensity. There were 105 patients (53 non-metastatic and 52 metastatic). VEGF immunostaining in non-metastatic and metastatic cohorts was negative in 20 (37.7%) and 21 (40.4%), mildly positive in 13 (24.5%) and 9 (17.3%) cases, moderately positive in 14 (26.4%) and 16 (30.8%), and was intensely positive in 6 (11.3%) and 7 (13.5%) patients, respectively. VEGF immunoexpression of up to 25% was seen in 14 (13.3%) and 10 (9.5%) patients within the non-metastatic and metastatic cohorts, respectively. For the non-metastatic cohort, the median EFS (months) was 78.6 (95% CI: 61.34-NA), whereas the median OS was not reached. The median EFS and OS (months) were 10.8 (95% CI: 6.41-17.1) and 13.5 (95% CI: 8.08-21.2), respectively, for the metastatic cohort. Metastatic ES patients having either VEGF immunostaining in >25% of tumor cells or moderate/strong immunostaining were found to have inferior OS (P = 0.008, HR = 10.565). In addition, metastatic patients who were treatment naïve had inferior OS, whereas patients who underwent definitive surgery and completed treatment had superior OS (P = 0.003, P = 0.015, and P = 0.009, respectively). VEGF was found to be an independent prognostic marker in metastatic ESs. This may translate to therapeutic relevance but needs validation in the subsequent, larger prospective studies.

Background: Ewing sarcoma is a rare, aggressive malignancy of bone and soft tissue, primarily affecting adolescents and young adults. Even with access to standard chemotherapy and radiotherapy, patients with metastatic or recurrent Ewing sarcoma, particularly in lower-resource settings, continue to experience limited survival outcomes. Case Presentation: We report the first known use of Next Generation Sequencing (NGS) testing to guide individualised treatment for Ewing sarcoma in Iran. A 17-year-old male presented with a paraspinal tumour and rapid neurological decline. Following surgical resection and histopathologic confirmation of Ewing sarcoma, he received standard chemoradiotherapy. Genomic profiling, including Tumour Mutational Burden (TMB) and circulating tumour cell immunocytochemistry, was conducted to identify precision therapeutic options. The patient’s TMB was low (2 mutations/Mb), pMMR and MSI-low, ruling out immunotherapy candidacy. However, VEGFR1/2 expression supported the inclusion of Sorafenib. A customised regimen combining oncology and repurposed non-oncology agents was initiated. After seven treatment cycles, imaging revealed resolution of lung metastases and a reduction in spinal involvement, accompanied by significant functional recovery. Conclusion: This case illustrates the feasibility and clinical significance of integrating molecular diagnostics into the treatment of rare cancers in low-resource settings. Even when immunotherapy is excluded, precision medicine can offer novel pathways to disease control and improved outcomes

10008 Background: Regorafenib monotherapy has shown interesting but limited activity against relapsed Ewing sarcoma. We present the first results of the phase Ib study to identify the maximum tolerated dose (MTD) of regorafenib in combination with standard multimodal treatment in patients with newly diagnosed multi-metastatic Ewing sarcoma (NCT05830084). Methods: International multi-center phase Ib study of the combination of regorafenib with interval-compressed chemotherapy (VDC/IE) in patients aged 2-50 years with newly diagnosed metastatic (excluding lung/pleura only) Ewing sarcoma. VDC/IE chemotherapy was administered at the standard doses. Regorafenib was given orally for 21 days of a 28-day cycle (from day 5) at a starting dose level of 66 mg/m 2 /day (capped at 120 mg, DL0, 80% of the pediatric recommended phase 2 dose (RP2D)) and escalated to 82 mg/m 2 /day (100% RPD2, capped at 160 mg, DL1) or de-escalated to 50 mg/m 2 /day (60% RPD2, DL-1). The study implemented the Bayesian Optimal Interval (BOIN) design (Yuan et al, Clin Cancer Res 2016). Primary tumour local treatment was surgery and/or radiotherapy. Adjuvant therapy consisted of VC/IE cycles or high dose chemotherapy consolidation with Busulfan/Melphalan (BuMel) and autologous stem cell rescue (ASCR). Regorafenib was given concomitant to adjuvant VC/IE cycles and primary tumor radiotherapy to the extremities but permanently discontinued in those receiving BuMel/ASCR or primary tumour radiotherapy to sites other than extremities. Results: Thirteen patients (DL0: n=2, DL1: n= 11) with a median age of 15.2 years (range, 8.1-23.5), were enrolled between June 2023 and December 2024 in 7 centres and 3 countries. All were evaluable for toxicity. One dose-limiting toxicity (DLT) occurred in one patient at DL1 (pressure ulcer grade 2, requiring regorafenib dose interruption and reduction in a 17-year old patient). After the DLT period, one patient had regorafenib dose interruption/reduction. One patient presented with a grade 3 veno-occlusive disease after Bu-Mel/ASCR. Detailed toxicity data after the DLT period and pharmacokinetic data will be presented. At data cut-off of 21/01/2025, two patients had experienced disease progression before primary tumour local treatment, eight patients had finished all treatment cycles (three received Bu-Mel/ASCR consolidation) and three patients were on therapy. Conclusions: Regorafenib combined with VDC/IE chemotherapy is well tolerated with a MTD of 82 mg/m 2 /day (capped 160 mg). The efficacy of the addition of regorafenib to standard multimodal treatment in newly diagnosed patients with metastatic Ewing sarcoma will be tested in the Inter-Ewing-1 trial developed by the Euro Ewing Consortium (planned initiation in Q3 2025). Recruitment to the Rego-Inter-Ewing-1 continues at DL1 (maximum 24 patients), until Inter-Ewing-1 initiates. Clinical trial information: NCT05830084 .

Organotropism results from the functional versatility of metastatic cancer cells to survive and proliferate in diverse microenvironments. This adaptivity can originate in clonal variation of the spreading tumor and is often empowered by epigenetic and molecular reprogramming of cell regulatory circuits. Related to organotropic colonization of metastatic sites are environmentally-sensitive, differential responses of cancer cells to therapeutic attack. Accordingly, understanding the organotropic profile of a cancer and probing the underlying driver mechanisms are of high clinical importance. However, determining systematically the organotropism of one cancer versus the organotropism of another cancer, potentially with the granularity of comparing the same cancer type between patients or tracking the evolution of a cancer in a single patient for the purpose of personalized treatment, has remained very challenging. It requires a host organism that allows observation of the spreading pattern over relatively short experimental times. Moreover, organotropic patterns often tend to be statistically weak and superimposed by experimental variation. Thus, an assay for organotropism must give access to statistical powers that can separate ‘meaningful heterogeneity’, i.e., heterogeneity that determines organotropism, from ‘meaningless heterogeneity’, i.e., heterogeneity that causes experimental noise. Here we describe an experimental workflow that leverages the physiological properties of zebrafish larvae for an imaging-based assessment of organotropic patterns over a time-frame of 3 days. The workflow incorporates computer vision pipelines to automatically integrate the stochastic spreading behavior of a particular cancer xenograft in tens to hundreds of larvae allowing subtle trends in the colonization of particular organs to emerge above random cell depositions throughout the host organism. We validate our approach with positive control experiments comparing the spreading patterns of a metastatic sarcoma against non-transformed fibroblasts and the spreading patterns of two melanoma cell lines with previously established differences in metastatic propensity. We then show that integration of the spreading pattern of xenografts in 40 – 50 larvae is necessary and sufficient to generate a Fish Metastatic Atlas page that is representative of the organotropism of a particular oncogenotype and experimental condition. Finally, we apply the power of this assay to determine the function of the EWSR1::FLI1 fusion oncogene and its transcriptional target SOX6 as plasticity factors that enhance the adaptive capacity of metastatic Ewing sarcoma.

Abstract Ewing's sarcoma is an uncommon and highly metastatic form of sarcoma affecting children and young adults, occurring in approximately 1 in 1.5 million individuals, with a predominance in males. We present an exceptionally unique case highlighting Ewing's sarcoma metastasis to the peritoneum and omentum, identified through an 18F-fluorodeoxyglucose positron emission tomography/computed tomography (18F-FDG PET/CT) scan and its unforeseen treatment response. In contrast to previously published literature concerning primary peritoneal primitive neuroectodermal tumors, this exceedingly notable instance illuminates the occurrence of metastatic Ewing's sarcoma within the peritoneum, as opposed to originating as a primary tumor within the peritoneum, revealing diagnostic intricacies. This case accentuates several pivotal learning points that enhance our understanding of Ewing's sarcoma. Also, the noteworthy high sensitivity and specificity of 18F-FDG PET/CT allow for early detection of recurrence and treatment response, substantially improving prognosis in Ewing's sarcoma patients, aiding in timely therapeutic interventions and optimizing patient outcomes.

Abstract Objective: To date, the response rate of immunotherapy in Ewing sarcoma (EwS) has been poor. The tumor immune microenvironment (TME) has a vital role in cancer survival and progression with implications in drug resistance and immune escape. However, there is a lack of understanding of the TME of EwS. Although recent single-cell RNA sequencing of EwS and immune cells have revealed a heterogenous transcriptional landscape with distinct subsets of immune cell populations in EwS, little is known about the spatial organization and spatial microenvironmental niches of the TME of EwS. Methods: We performed Spatial Transcriptomics (ST) on 16 primary pre-treatment biopsied EwS tumors (10 with localized EwS (L-EwS); 6 with metastatic EwS (M-EwS)). Results: Integrated analysis revealed inter- and intra-tumoral heterogeneity. We inferred the stromal regions of tumors transcriptionally and noted that tumors from L-EwS had significantly more stromal signature than M-EwS (p-value &amp;lt;2.2e-16). Gene set enrichment analysis using Hallmark gene sets revealed that L-EwS tumors were enriched in epithelial mesenchymal transition (EMT) and inflammatory signatures (adjusted p-value &amp;lt;0.05) and M-EwS tumors were enriched in proliferative signatures (adjusted p-value &amp;lt;0.05). We identified a set of 46 unique genes enriched in L-EwS that overlapped with genes in EMT hallmark pathways and were predominantly extracellular matrix related (ECM-r) genes such as COL3A1, ITGB1, MMP2, ACTA2, TNC, TGFB1. These ECM-r genes spatially overlapped with the stromal regions of individual tumors. Furthermore, gene composite analysis demonstrated spatial colocalization of macrophages and cancer-associated fibroblasts (CAFs) within the ECM-r regions of the tumors. Spatially informed cell type deconvolution revealed EwS tumor regions that correlated well with histology. Moreover, majority of macrophages/monocytes in L-EwS tumors colocalized with CAFs. Deconvolution analysis also uncovered previously unreported B cell infiltrates and CD8 T cell infiltrative tumors. Spatial cellular microenvironment analysis revealed immune microenvironmental signaling throughout all tumors and unique ECM microenvironmental signaling predominantly in L-EwS compared to M-EwS (p-value &amp;lt;0.05) (TGF-B, cell adhesion and ECM remodeling signals). Conclusion: L-EwS tumors are enriched in ECM and ECM-related microenvironmental signals compared to M-EwS. Spatial cellular microenvironmental signals reveal that components of the ECM may serve as anchors for tumors to eventually metastasize, nutrients for EwS to proliferate, and most importantly as a communication network with host immune cells. Further studies will be performed on a novel immunocompetent zebrafish genetic EwS model to identify conserved mechanisms of host-tumor interactions and further dissect the role of ECM in EwS. Citation Format: Christopher Kuo, Krinio Giannikou, Nuoya Wang, Mikako Warren, Andrew Goodspeed, Masanori Hayashi, Micha S. Raredon, James F. Amatruda. Spatial transcriptomics of Ewing sarcoma: evolution of tumor associated extracellular matrix from localized to metastasis-associated primary Ewing sarcomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6440.

Abstract Patients with metastatic Ewing Sarcoma have a five-year survival rate of 15-30%. For 30 years, there are limited approved therapies, including chemotherapy, radiotherapy and surgery, which are associated with significant morbidities and secondary cancers, supporting the need for new impactful therapies. Analysis of the RNA splicing factor RNA Binding Motif Protein 39 (RBM39) in the Cancer Target Discovery and Development (CTD2) Network showed that Ewing sarcoma cell lines are especially sensitive to RBM39 loss. Here we demonstrate effects on mediators of DNA damage repair in association with significant anti-cancer effects of an orally administered novel RBM39 degrader, ST-00937. Methods and Results: A next generation RBM39 degrader ST-00937 was designed through structure-based drug design and prioritized based on in vitro anti-cancer potency, metabolic stability and oral pharmacokinetics in mice. Human Ewing sarcoma A673 cells were utilized to explore the effects of ST-00937 in this cancer type. Tumor cell viability was evaluated by CellTiter-Glo® Luminescent Cell Viability Assay (CTG) and anti-tumor efficacy was evaluated in nude mice bearing tumors established with A673 cells. Protein levels in cell lysates and tumor homogenate were measured by Western Blot. Target gene RNA splicing was evaluated by RT-PCR.ST-00937 degraded RBM39 by 6 hours of treatment and reduced in vitro A673 Ewing sarcoma cell viability with an IC50 of 170 nM. These in vitro effects translated to complete A673 tumor regressions in mice by the 11th day of twice daily ST-00937 oral dosing at 30mg/kg, without an effect on body weight. By the third day of treatment, RBM39 was undetectable in A673 tumors. Mechanically both the EWS-FLI1 driver gene fusion in A673 cells and RBM39 degradation are reported to reduce DNA damage repair pathways. Here we report RBM39 degradation in A673 cells significantly increases phosphorylated ʏH2Ax, a marker of DNA double strand breaks. This DNA damage is thought to contribute to the induction of cell apoptosis detected as increased cleaved caspase-3. Increased DNA double strand breaks is furthermore associated with negative effects on 3 proteins involved in homologous recombination repair of DNA double strand breaks; mis-splicing of BRCA1 (Breast cancer type 1) and ATM (ATM Serine/Threonine Kinase), and reduced protein levels of RAD51D (recombination repairing regulator Recombinase homolog D). Conclusion: The RBM39 molecular glue degrader ST-00937 therefore demonstrates total tumor regression in a model of Ewing sarcoma. Mechanistic studies indicate that the robust activity of ST-00937 involves deficient homologous recombination repair. Continued research in our lab will add further insights into the mechanism of action of RBM39 degraders in this cancer of high unmet need. Citation Format: Fei Liu, Yunkai Zhang, Baiyun Wang, James Finn, James Tonra, Lan Huang, Dong Liu, Haihong Jin, Xing Liu, Dan Lu. Targeting Ewing sarcoma with a novel RBM39 degrader: DNA damage repair pathway effects [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 7035.