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Context.— Molecular diagnostics play an increasing role in the diagnosis of Ewing sarcoma. The type of molecular testing used in clinical practice has been poorly described. Objective.— To describe patterns of translocation testing for newly diagnosed Ewing sarcoma. Design.— Children's Oncology Group (COG) trial AEWS1221 was a phase III randomized trial enrolling patients with newly diagnosed metastatic Ewing sarcoma from 2014 to 2019. Patients were required to have a histologic diagnosis of Ewing sarcoma, but translocation testing was not required. Sites provided types and results of any molecular diagnostics performed. Results.— Data from 305 enrolled patients were available. The most common type of molecular testing was fluorescence in situ hybridization (FISH) performed on the primary tumor (236 of 305 patients; 77.4%), with positive testing for an EWSR1 or FUS translocation in 211 (89.4%). Reverse transcription–polymerase chain reaction (RT-PCR) on the primary tumor was performed in 61 of 305 patients (20%), with positive results in 48 of 61 patients (78.7%). Next-generation sequencing was reported in 7 patients for the primary tumor and in 3 patients for metastatic sites. For all types of testing on either primary or metastatic tumor, 16 of 305 patients (5.2%) had no reported translocation testing. When evaluating all results from all testing, 44 of 305 patients (14.4%) lacked documentation of an abnormality consistent with a molecular diagnosis of Ewing sarcoma. Conclusions.— COG sites enrolling in a Ewing sarcoma trial have high rates of testing by FISH or PCR. A small proportion of patients have no translocation testing on either primary or metastatic sites. Next-generation sequencing techniques are not yet commonly used in this context.

Ewing sarcoma is a bone tumor most commonly diagnosed in adolescents and young adults. Survival for patients with recurrent or metastatic Ewing sarcoma is dismal and there is a dire need to better understand the mechanisms of cell metastasis specific to this disease. Our recent work demonstrated that microenvironmental stress leads to increased Ewing sarcoma cell invasion through Src activation. Additionally, we have shown that the matricellular protein tenascin C (TNC) promotes metastasis in Ewing sarcoma. A major role of both TNC and Src is mediation of cell–cell and cell-matrix interactions resulting in changes in cell motility, invasion, and adhesion. However, it remains largely unknown, if and how, TNC and Src are linked in these processes. We hypothesized that TNC is a positive regulator of invadopodia formation in Ewing sarcoma through its ability to activate Src. We demonstrate here that both tumor cell endogenous and exogenous TNC can enhance Src activation and invadopodia formation in Ewing sarcoma. We found that microenvironmental stress upregulates TNC expression and this is dampened with application of the Src inhibitor dasatinib, suggesting that TNC expression and Src activation cooperate to promote the invasive phenotype. This work reports the impact of stress-induced TNC expression on enhancing cell invadopodia formation, provides evidence for a feed forward loop between TNC and Src to promote cell metastatic behavior, and highlights a pathway by which microenvironment-driven TNC expression could be therapeutically targeted in Ewing sarcoma.

Background Ewing sarcomas are solid tumours of the bone and soft tissue, that usually affect children, adolescents, and young adults. The incidence is about three cases per million a year, with a peak incidence at 12 years of age. Metastatic disease is detected in about 20 % to 30% of people, and is typically found in the lungs, bone, bone marrow, or a combination of these. Presence of metastatic disease at diagnosis (primary metastatic disease) is the most important adverse prognostic factor, and is associated with a five-year survival lower than 30%. High-dose chemotherapy (HDC) followed by autologous haematopoietic cell transplantation (AHCT) is used in various solid tumours with unfavourable prognoses in children, adolescents, and young adults. It has also been used as rescue after multifocal radiation of metastases. The hypothesis is that HDC regimens may overcome the resistance to standard multidrug chemotherapy and improve survival rates. Objectives To assess the effects of high-dose chemotherapy with autologous haematopoietic cell transplantation compared with conventional chemotherapy in improving event-free survival, overall survival, quality-adjusted survival, and progression-free survival in children, adolescents, and young adults with primary metastatic Ewing sarcoma, and to determine the toxicity of the treatment. Search methods We searched CENTRAL, MEDLINE, Embase, conference proceedings from major international cancer-related conferences, and ongoing trial registers until January 2020. We also searched reference lists of included articles and review articles. Selection criteria We included randomised controlled trials (RCTs) or (historical) controlled clinical trials (CCTs) comparing the effectiveness of HDC and AHCT with conventional chemotherapy for children, adolescents, and young adults (younger than 30 years at the date of diagnostic biopsy) with primary metastatic Ewing sarcoma. Data collection and analysis We used standard methodological procedures expected by Cochrane. Main results We identified one RCT, which investigated the effects of HDC with AHCT versus conventional chemotherapy with whole lung irradiation (WLI) in people with Ewing sarcoma metastasised to the lungs only at diagnosis. Only a selection of the participants were eligible for our review (N = 267: HDC with AHCT group N = 134; control group N = 133). There may be no difference in event-free survival between the two treatment groups (hazard ratio (HR) 0.83, 95% confidence interval (CI) 0.59 to 1.17; low-certainty evidence). We downgraded one level each because of study limitations and imprecision. Overall survival and toxicity were not reported separately for the participants eligible for this review, while quality-adjusted survival and progression-free survival were not reported at all. We did not identify any studies that addressed children, adolescents, and young adults with Ewing sarcoma with metastases to other locations. Authors' conclusions In people with Ewing sarcoma with primary metastases to locations other than the lungs, there is currently no evidence from RCTs or CCTs to determine the efficacy of HDC with AHCT compared to conventional chemotherapy. Based on low-certainty evidence from one study (267 participants), there may be no difference in event-free survival between children, adolescents, and young adults with primary pulmonary metastatic Ewing sarcoma who receive HDC with AHCT and those who receive conventional chemotherapy with WLI. Further high-quality research is needed. Results are anticipated for the EuroEwing 2008R3 study, in which the effects of HDC with treosulfan and melphalan followed by AHCT on survival, in people with Ewing sarcoma with metastatic disease to bone, other sites, or both were explored. Achieving high-quality studies in a selection of people with rare sarcoma requires long-term, multi-centre, international participant inclusion.

Purpose: To analyze toxicity and outcome predictors in Ewing sarcoma patients with lung metastases treated with busulfan and melphalan (BU-MEL) followed by whole-lung irradiation (WLI). Methods: This retrospective study included 68 lung metastatic Ewing Sarcoma patients who underwent WLI after BU-MEL with autologous stem cell transplantation, as part of two prospective and consecutive treatment protocols. WLI 12 Gy for &lt, 14 years old and 15 Gy for ≥14 years old patients were applied at least eight weeks after BU-MEL. Toxicity, overall survival (OS), event-free survival (EFS) and pulmonary relapse-free survival (PRFS) were estimated and analyzed. Results: After WLI, grade 1–2 and grade 3 clinical toxicity was reported in 16.2% and 5.9% patients, respectively. The five-year OS, EFS and PRFS with 95% confidence interval (CI) were 69.8% (57.1–79.3), 61.2% (48.4–71.7) and 70.5% (56.3–80.8), respectively. Patients with good histological necrosis of the primary tumor after neoadjuvant chemotherapy showed a significant decreased risk of pulmonary relapse or death compared to patients with poor histological necrosis. Conclusions: WLI at recommended doses and time interval after BU-MEL is feasible and might contribute to the disease control in Ewing sarcoma with lung metastases and responsive disease. Further studies are needed to explore the treatment stratification based on the histological response of the primary tumor.

INTRODUCTION: Intramedullary metastasis of Ewing sarcoma is extremely rare. Here, we report an adult case of cervical intramedullary recurrent Ewing sarcoma after a 10-year disease-free survival after the initial surgery for a thoracic lesion. CASE PRESENTATION: A 39-year-old man with a history of surgery and chemoradiotherapy for thoracic Ewing sarcoma ten years ago presented with neck pain and incomplete motor paralysis in the right upper extremity, which had suddenly appeared three months before. Cervical magnetic resonance imaging revealed a tear-drop-shaped intramedullary lesion at the C3 level accompanied by diffuse edematous change. Because of the rapid progression of his myelopathy, he underwent surgery for this intramedullary lesion. Intraoperatively, the tumor exhibited an orangish exophytic appearance. The unclearness of the tumor boundary compelled us to perform a partial resection. The histopathology showed the tumor comprised small round atypical cells with immunoreactivity for Nkx2.2 and CD99, diagnosing a metastatic Ewing sarcoma. Postoperatively, although his myelopathy improved transiently and adjuvant chemotherapy radiation was undergone, he died of cranial dissemination of the tumor two months and a half later. DISCUSSION: To our knowledge, 31 cases of primary and only 4 cases of recurrent intramedullary spinal Ewing sarcoma have been reported to date; however, this is the first case of recurrent intramedullary Ewing sarcoma with a 10-year disease-free survival. Sadly, the prognosis of the current case was extremely poor. There is no clear treatment guideline for recurrent intramedullary Ewing sarcoma because of its rarity, and further collection of similar cases would be required.

Background: Few models have been used to estimate the survival rate of patients metastatic Ewing sarcoma of bone are scarce. We aimed to develop nomograms for predicting 3-, 5-year survival for these patients.Methods: We extracted 686 cases of metastatic Ewing's sarcoma diagnosed between 1973 and 2016 from the Surveillance, Epidemiological and End Results (SEER) database. Univariate and multivariate Cox analysis were used to determine independent prognostic factors. The nomograms are based on the results of multivariate Cox analysis. We also evaluate the performance of these prediction models through the analysis of time-dependent receiver operating characteristic curve, concordance index, calibration curve and decision curve.Results: Age, surgery, tumor size, treatment method and chemotherapy were considered to be important predictors of overall survival of bone metastatic Ewing's sarcoma. Based on these factors, the nomogram models were established and verified internally. These models have good identification and calibration characteristics. A risk classification system based on nomogram has also been constructed to promote risk stratification of metastatic Ewing's sarcoma and to optimize clinical management.Conclusions: We developed the first nomograms and corresponding risk classification system to predict the survival of patients with bone metastatic Ewing's sarcoma. These easy-to-use tools can help oncologists and surgeons make accurate survival assessments.

[Background] The prognosis of patients with metastatic Ewing sarcoma family of tumors (ESFT) remains poor. [Procedure] We retrospectively analyzed 57 patients diagnosed with metastatic ESFT between 2000 and 2018 to identify prognostic and therapeutic factors affecting the clinical outcome. [Results] The 3-year overall survival (OS) rate of the entire cohort was 46.8% (95% confidence interval [CI], 33.0-59.4%). Treatment-related death was not observed. Multivariate analysis identified stem cell transplantation (SCT), response to first-line chemotherapy, and bone metastasis as independent risk factors for OS. Objective response rate to first-line chemotherapy was 65.1% in the 43 evaluable patients. There was no significant difference in the response to different types of first-line chemotherapy. Among patients with lung metastasis alone, the 3-year OS rate was higher in 13 patients who received local treatment than in four who did not, although the difference was not significant. [Conclusions] One possible reason for the high OS rates was the absence of treatment-related mortality even in patients receiving SCT, which could be attributed to advances in the management of post-SCT complications. Novel first-line chemotherapy strategies need to be established to improve the disease status prior to SCT in a higher proportion of patients.

Objetivo. Presentar los resultados perinatales de dos pacientes con diagnóstico de sarcoma de Ewing y rabdomiosarcoma alveolar. Metodología. Se revisaron dos casos de pacientes con diagnóstico de sarcoma atendidas en el Instituto Nacional de Perinatología de la Ciudad de México. Resultados. Caso 1 femenino de 22 años, con embarazo de 23,0 semanas y tumoración en región glútea izquierda de 20 cm, dolor y dificultad para deambular. La radiografía de tórax informó múltiples nódulos pulmonares y la resonancia magnética, tumor en región glútea con compromiso extenso. El diagnóstico fue sarcoma de Ewing metastático en etapa IV. El manejo consistió en tratamiento sintomático con resolución del embarazo a las 28 semanas. Caso 2 femenino de 22 años con embarazo de 12,0 semanas y diagnóstico de síndrome medular metastásico. Se realizó descompresión T9-11. Se evidenció ausencia de frecuencia cardiaca fetal, por lo que se realizó manejo médico de aborto diferido.

PURPOSE: The R2Pulm trial was conducted to evaluate the effect of busulfan-melphalan high-dose chemotherapy with autologous stem-cell rescue (BuMel) without whole-lung irradiation (WLI) on event-free survival (main end point) and overall survival, compared with standard chemotherapy with WLI in Ewing sarcoma (ES) presenting with pulmonary and/or pleural metastases. METHODS: From 2000 to 2015, we enrolled patients younger than 50 years of age with newly diagnosed ES and with only pulmonary or pleural metastases. Patients received chemotherapy with six courses of vincristine, ifosfamide, doxorubicin, and etoposide (VIDE) and one course of vincristine, dactinomycin, and ifosfamide (VAI) before either BuMel or seven courses of VAI and WLI (VAI plus WLI) by randomized assignment. The analysis was conducted as intention to treat. The estimates of the hazard ratio (HR), 95% CI, and P value were corrected for the three previous interim analyses by the inverse normal method. RESULTS: Of 543 potentially eligible patients, 287 were randomly assigned to VAI plus WLI (n = 143) or BuMel (n = 144). Selected patients requiring radiotherapy to an axial primary site were excluded from randomization to avoid excess organ toxicity from interaction between radiotherapy and busulfan. Median follow-up was 8.1 years. We did not observe any significant difference in survival outcomes between treatment groups. Event-free survival was 50.6% versus 56.6% at 3 years and 43.1% versus 52.9% at 8 years, for VAI plus WLI and BuMel patients, respectively, resulting in an HR of 0.79 (95% CI, 0.56 to 1.10; P = .16). For overall survival, the HR was 1.00 (95% CI, 0.70 to 1.44; P = .99). Four patients died as a result of BuMel-related toxicity, and none died after VAI plus WLI. Significantly more patients in the BuMel arm experienced severe acute toxicities than in the VAI plus WLI arm. CONCLUSION: In ES with pulmonary or pleural metastases, there is no clear benefit from BuMel compared with conventional VAI plus WLI.

Highlights • The incidence of ES of the bone is very low, reaching a peak in adolescence. • We assessed the patterns of DM and the prognostic factors in this patient subset. • Lung and bone are the most frequently distant metastatic sites. • Bone metastasis is an independent risk factor for inferior survival., Background Ewing sarcoma (ES) of bone is accounting for the second most common type of primary bone cancer in children and adolescents. However, the patterns of distant metastasis (DM) and the effect of the sites of DM on survival outcomes were not investigated. Aims This study aimed to investigate the patterns of DM and the prognostic factors related to outcomes in primary metastatic ES of the bone. Methods Patients who were diagnosed with primary metastatic ES between 2010 and 2018 were identified from the Surveillance, Epidemiology, and End Results database. Kaplan–Meier analysis, log-rank tests, and Cox proportional-hazards regression models were used for statistical analyses. Results We identified 277 patients in this study and 95.3% of them (n = 264) receiving chemotherapy. A total of 371 sites of DM were observed. Lung was the most common distant metastatic site (n = 182, 49.1%), followed by bone (n = 139, 37.5%), distant lymph node (n = 26, 7.0%), liver (n = 14, 3.8%), and brain (n = 10, 2.7%). Three-year cause-specific survival (CSS) was 56.1% in the entire cohort. Older age (hazard ratio [HR] 2.210, P

A 45-year-old male with recurrent metastatic Ewing sarcoma presented to Memorial Sloan Kettering Cancer Center for potential enrollment in a clinical trial. Initial laboratory results were notable for increased alkaline phosphatase levels [(ALP2, 495 U/L; (reference interval: 33–97 U/L); Abbott Architect C8000] and γ-glutamyl transferase levels [GGT, 393 U/L (reference interval: 0–73 U/L); Abbott Architect C8000] but were otherwise unremarkable, including normal levels of aspartate aminotransferase (AST) and alanine transaminase (ALT). Elevations in ALP and GGT levels suggested liver disease, although the patient had no previous history of liver abnormalities. Given the patient's history of metastatic bone disease, it was suspected that the observed elevation in ALP was due to increased bone ALP isoenzyme. However, it was important to document the origin of the increased ALP as the patient was to be enrolled in an investigational trial that would define drug dosage and side effects. Elevations in ALP over the course of the trial that were attributable to liver-specific isoenzymes would be reported as a dose-limiting toxicity and could delay treatment or potentially exclude the patient from the clinical trial. Conversely, elevations in ALP originating from bone and secondary to disease would not be reported as toxicity. The laboratory was consulted regarding testing options to identify the specific isoenzyme(s) responsible …

A 49-year-old male with Ewing sarcoma and bone, pleural, lung and mediastinal lymph node metastasis was treated with cabozantinib after four lines of previous systemic treatments. He responded objectively and subjectively well for 8 months. In this heavily pretreated patient, the daily starting dose of 60 mg had to be reduced to 30 mg because of adverse events. We conclude that treatment with cabozantinib administered in further-line was active in this particular patient with metastatic Ewing sarcoma. The underlying mechanism of action remains unclear. Because of a stable disease on a long-term treatment with pazopanib targeting an anti-angiogenic pathway common to both drugs previously administered in this patient, it is hypothesized that the action of cabozantinib could be ascribed to its action on the non-common receptors AXL and c-Met. The potential of cabozantinib should be further investigated more upfront in this disease either alone or in combination with other systemic treatments.

Ewing sarcoma (ES) is an undifferentiated small round blue cell tumor most commonly originating in the bone of adolescents 10-20 years of age, although 30% are diagnosed in adults. The most important prognostic factor is the presence of metastatic disease. Results of the EURO-EWING 99 trial of ES patients showed that local treatment of not only the primary, but also of the sites of metastatic disease should be considered to improve event-free survival. The use of stereotactic body radiotherapy (SBRT) has been extensively reported for tumors of lung, liver, pancreas, and spine. The use of SBRT in these sites is well-accepted. Here, we report a detailed case of SBRT to multisite metastatic ES. We demonstrate the feasibility, safety, and efficacy of aggressive local control with multisite SBRT for the treatment of metastatic ES.

Outcomes for metastatic Ewing sarcoma and osteosarcoma are dismal and have not changed for decades. Oxidative stress attenuates melanoma metastasis, and melanoma cells must reduce oxidative stress to metastasize. We explored this in sarcomas by screening for oxidative stress sensitizers, which identified the class I HDAC inhibitor MS‐275 as enhancing vulnerability to reactive oxygen species (ROS) in sarcoma cells. Mechanistically, MS‐275 inhibits YB‐1 deacetylation, decreasing its binding to 5′‐UTRs of NFE2L2 encoding the antioxidant factor NRF2, thereby reducing NFE2L2 translation and synthesis of NRF2 to increase cellular ROS. By global acetylomics, MS‐275 promotes rapid acetylation of the YB‐1 RNA‐binding protein at lysine‐81, blocking binding and translational activation of NFE2L2, as well as known YB‐1 mRNA targets, HIF1A, and the stress granule nucleator, G3BP1. MS‐275 dramatically reduces sarcoma metastasis in vivo, but an MS‐275‐resistant YB‐1K81‐to‐alanine mutant restores metastatic capacity and NRF2, HIF1α, and G3BP1 synthesis in MS‐275‐treated mice. These studies describe a novel function for MS‐275 through enhanced YB‐1 acetylation, thus inhibiting YB‐1 translational control of key cytoprotective factors and its pro‐metastatic activity., Class 1 HDAC inhibitors including MS‐275 inhibit the RNA binding protein YB‐1 by enhancing its acetylation. This reduces its affinity to mRNA targets under cellular stress, and decreases tumor cell fitness and metastatic capacity.

The outcomes for relapsed and metastatic Ewing sarcoma (EWS) is extremely poor. Therefore, it is important to identify the tumor‐specific targets in these intractable diseases. High focal adhesion kinase (FAK) transcript expression levels in EWS cell lines are known. TAE226 is a dual inhibitor of FAK and insulin‐like growth factor‐I receptor (IGF‐IR), while PF‐562,271 is a dual inhibitor of FAK and proline‐rich tyrosine kinase 2. We compared the cytotoxicity of TAE226 and PF‐562,271 toward three EWS cell lines. TAE226 strongly inhibited proliferation of three cell lines when compared with PF‐562,271. Furthermore, we investigated the efficacy of TAE226 as well as its mechanism of action against EWS. A stable EWS cell line with FAK and IGF‐IR knocked down was established, and microarray analysis revealed dysregulated expression in various pathways. TAE226 treatment of EWS cell lines induced cell cycle arrest, apoptosis, AKT dephosphorylation, and inhibition of invasion. We demonstrated that TAE226 drastically inhibits the local growth of primary tumors and metastasis in EWS using mouse models. Furthermore, the combination of TAE226 and conventional chemotherapy proved to exert synergistic effects. TAE226 may be a candidate single agent or combined therapy drug to be developed for patients who have relapse and metastatic EWS tumors in future., Systemic TAE226 treatment potently reduced the size of local tumors and inhibited micrometastasis in vivo through cell cycle inhibition, induction of apoptosis, and inhibition of AKT signaling. Furthermore, combined therapy with TAE226 and conventional anticancer drugs for EWS has synergistic anticancer effects. Overall, the results of the present study suggest that TAE226 is a candidate single agent or combined therapy drug to be developed for patients who have relapse and metastatic EWS tumors in future.

Metastatic Ewing sarcoma has a very poor prognosis and therefore new investigations into the biologic drivers of metastatic progression are key to finding new therapeutic approaches. The tumor microenvironment is highly dynamic, leading to exposure of different regions of a growing solid tumor to changes in oxygen and nutrient availability. Tumor cells must adapt to such stress in order to survive and propagate. In the current study, we investigate how Ewing sarcoma cells respond to the stress of growth factor deprivation and hypoxia. Our findings reveal that serum deprivation leads to a reversible change in Ewing cell cytoskeletal phenotypes. Using an array of migration and invasion techniques, including gelatin matrix degradation invadopodia assays, we show that exposure of Ewing sarcoma cells to serum deprivation and hypoxia triggers enhanced migration, invadopodia formation, matrix degradation and invasion. Further, these functional changes are accompanied by and dependent on activation of Src kinase. Activation of Src, and the associated invasive cell phenotype, were blocked by exposing hypoxia and serum-deprived cells to the Src inhibitor dasatinib. These results indicate that Ewing sarcoma cells demonstrate significant plasticity in response to rapidly changing micro-environmental stresses that can result from rapid tumor growth and from necrosis-causing therapies. In response to these stresses, Ewing cells transition to a more migratory and invasive state and our data show that Src is an important mediator of this stress response. Our data support exploration of clinically available Src inhibitors as adjuvant agents for metastasis prevention in Ewing sarcoma.

Ewing sarcoma (ES) is a poorly differentiated bone and soft tissue tumor of high metastatic potential. ES mainly affects children, adolescents, and young adults (AYAs) at a frequency of ~1.5 cases per million globally. Ewing neoplasms strikingly converge on a single recurrent initiating event, which is a chromosomal translocation that generates a fusion transcript between the EWSR1 gene and a gene of the ETS family of transcription factors, most commonly FLi1 (85%). After 20 years since the discovery of the EWS-FLi1 fusion, ES remains a clinical challenge with unacceptably low survival rates, primarily due to metastasis. The bulk of research conducted to date (>95%) being focused on the primary tumor has resulted in a critical knowledge gap. To address this issue and unravel the dysregulated pathways in ES tumor evolution and metastatic dissemination, we harnessed the genome-wide insertional mechanism of transposons and the transduction efficiency of lentiviruses to engineer ES cell models. Human mesenchymal stem cells (hMSC), the putative cells of origin of ES, were engineered to constitutively express EWS-Fli1 accompanied by the inducible expression of a highly active transposase. Upon activation of the former, transposon-mediated mutagenesis will activate oncogenes and inactivate tumor suppressor genes, to mediate transformation of the transduced that can now engraft when implanted in recipient mice. Xenografted tumors are resected and the mice observed for development of distant metastases. Matching primary and metastatic tumors are sequenced to uncover the genes commonly affected by transposition in the two compartments. Pathway-oriented bioinformatic analysis will further reveal candidate metastatic driver genes. Matching of the targeted pathways with drugs will be used to validate the candidates by in vitro and in vivo metastasis assays. Citation Format: Wajih Jawhar, Paul Waterhouse, Rama Khokha, Takeaki Ishii, Robert Turcotte, Nada Jabado, Livia Garzia. Functional genomics of metastatic Ewing sarcoma [abstract]. In: Proceedings of the AACR Special Conference on the Advances in Pediatric Cancer Research; 2019 Sep 17-20; Montreal, QC, Canada. Philadelphia (PA): AACR; Cancer Res 2020;80(14 Suppl):Abstract nr A64.

Oncolytic virotherapy is associated with immunogenic cell death and antitumor inflammatory immune responses. Thereby, a local as well as systemic immunosuppressive microenvironment might be abrogated, resulting in efficient phagocytic and antigen-presenting properties. Consecutively, priming of naive T cells against a multitude of de-repressed tumor and viral antigens is claimed to elicit strong antitumor immune responses accompanied by tumor-infiltrating T cells, especially in combination with immune checkpoint blockade. These features might be beneficial for patients with relapsed and metastatic Ewing sarcoma, who have poor prognosis and lack innovative therapy concepts, and whose tumor biopsies are characterized by low or absent T-cell infiltration. Here, we explore the efficacy of the oncolytic YB-1-selective adenovirus XVir-N-31 in combination with the CDK4/6-inhibitor abemaciclib (LY2835219) in established Ewing sarcoma cell lines. We demonstrate enhanced viral replication, particle formation, and oncolysis when combined with abemaciclib in vitro. Priming of tumor cells with abemaciclib before viral infection leads to cell cycle arrest in G1 phase, with consecutive decrease in Rb, pRb, and E2F1 protein levels. Interestingly, in the Rb-mutated hence CDK4/6-inhibitor-resistant cell line SK-N-MC, the combination with abemaciclib does not result in enhanced viral replication oor oncolysis. However, the same effects as for CDK4/6-inhibitor sensitive cell lines can be induced by siRNA-mediated E2F1 knockdown, indicating the crucial role of E2F1 as a repressor for initiation of viral replication. In summary, our results further support the hypothesis of E2F1 being a repressor of adenoviral replication (Holm et al., manuscript in preparation) and suggest the use of CDK4/6-inhibitors to boost oncolytic adenoviral efficacy. Further in vivo confirmation is planned in order to prepare a clinical phase I study of XVir-N-31 in combination with a CDK4/6-inhibitor and immune checkpoint blockade for treatment-refractory pediatric sarcoma patients. Citation Format: Sebastian J. Schober, Uwe Thiel, Melanie Thiede, Maximilian Ehrenfeld, Roman Nawroth, Guenther HS Richter, Stefan E.G. Burdach, Per Sonne Holm. YB-1-based oncolytic virotherapy in combination with CDK4/6-inhibitors against Ewing sarcoma [abstract]. In: Proceedings of the AACR Special Conference on the Advances in Pediatric Cancer Research; 2019 Sep 17-20; Montreal, QC, Canada. Philadelphia (PA): AACR; Cancer Res 2020;80(14 Suppl):Abstract nr A48.

Ewing Sarcoma is a highly lethal undifferentiated tumor of bone. ES is a small round cell tumor with etiological and characteristic chromosomal translocations between TET/FET (TLS/FUS, EWSR1, and TAF15) and ETS (E26 transformation-specific) family genes. Generally, therapeutic approach for metastatic Ewing Sarcoma includes both local (surgery and radiotherapy) and systemic (chemotherapy) disease control with an overall cure rate of 20%. For extra-osseous tumors, the most common primary sites of disease are trunk, extremities, head and neck, retroperitoneum. Among other sites, Ewing Sarcoma/PNET may also rarely arise in colon and rectum. Even if colonic Ewing Sarcoma/PNET have been previously reported in 5 cases, none of those reports came from right side of the colon. In this article, we report the first case of right-sided Ewing Sarcoma with synchronous liver metastases completely responding to first line chemotherapy. Furthermore, we provide a systematic qualitative review of the current literature on adult colorectal Ewing Sarcoma using the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA).

Background The long-term morbidity of childhood cancer survivors is an emerging field as more patients are now expected to live through adulthood. Case We describe 2 adolescent patients with permanent premature ovarian failure and failure of endometrium to respond to estrogen after they received a combination of chemotherapy and pelvic radiation for metastatic Ewing sarcoma. Both girls were prepubertal at diagnosis of Ewing sarcoma. Puberty was induced with high-dose estrogen and progesterone; however, none of the patients had withdrawal bleeding. Summary and Conclusion It is critical to counsel these patients that menstruation might not be possible even with hormone replacement therapy.

Receptor tyrosine kinases (RTKs) have provided molecular targets for the development of novel, prognosis‐improving agents in many cancers; however, resistances to these therapies occur. On the cellular level, one resistance mechanism is attributed to functional RTK redundancies and compensatory cross‐signaling, leading to perception of RTKs as signaling and target networks. To provide a basis for better exploitation of this network in Ewing sarcoma, we generated comprehensive qPCR gene expression profiles of RTKs in Ewing sarcoma cell lines and 21 untreated primary tumors. Key findings confirm broad‐spectrum RTK expressions with potential for signaling redundancy. Profile analyses with regard to patient risk‐group further revealed several individual RTKs of interest. Among them, VEGFR3 and TIE1 showed high‐level expressions and also were suggestive of poor prognosis in localized tumors; underscoring the relevance of angiogenic signaling pathways and tumor‐stroma interactions in Ewing sarcoma. Of note, compared to localized disease, tumors derived from metastatic disease were marked by global high‐level RTK expressions. Nine individual RTKs were significantly over‐expressed, suggesting contributions to molecular mechanisms of metastasis. Of these, ROR1 is being pursued as therapeutic target in leukemias and carcinomas, but un‐characterized in sarcomas. We demonstrate expression of ROR1 and its putative ligand Wnt5a in Ewing sarcomas, and of an active ROR1 protein variant in cell lines. ROR1 silencing impaired cell migration in vitro. Therefore, ROR1 calls for further evaluation as a therapeutic target in metastatic Ewing sarcoma; and described as a pseudo‐kinase with several isoforms, underlines these additional complexities arising in our understanding of RTK signaling networks.

The prognosis of adolescent and young adult patients battling metastatic Ewing Sarcoma Family of Tumours (ESFT) remains less than 30% despite the development of systemic therapies. In the era of personalized medicine, novel molecular targets have been tested in preclinical or clinical settings in ESFT. In this review, we focus on early clinical and translational research that identified multiple molecular targets, including IGF-1R; mTOR; tyrosine kinase inhibitors; EWS-FLI1-related targets, and others. Overall, novel targeted therapies demonstrated modest efficacy; however pronounced and durable antineoplastic responses have been observed in small subsets of treated patients, for example with IGF-1R antibodies. Identifying outcome-predicting biomarkers and overcoming treatment resistance remain major challenges. Due to the rarity of ESFT, multi-institutional collaboration efforts of clinicians, basic and translational scientists are needed in order to understand biology of therapeutic response or resistance, which can lead to development of novel therapeutic methods and improved patient outcomes.

Background Outcomes for patients with metastatic Ewing sarcoma (ES) remain poor. We investigated whether the intensification of ifosfamide improved survival for patients with metastatic ES. Procedure We conducted a retrospective chart review of 30 patients with metastatic ES treated with the MSKCC “EFT regimen.” The regimen included an intensification of ifosfamide dosing from 1,800 mg/m2/day × 5 days per cycle to 2,800 mg/m2/day × 5 days per cycle. Results Twenty six of the 30 patients completed planned chemotherapy. Two patients experienced disease progression during therapy. There were no toxic deaths. One patient developed secondary leukemia. The 4-year event free survival (EFS) was 27% and the overall survival (OS) was 39%. Conclusions Intensification of ifosfamide was tolerated and did not increase toxicity in patients with metastatic ES. The intensification did not improve outcomes for these patients with metastatic disease. Pediatr Blood Cancer 2015;62:594–597. © 2015 Wiley Periodicals, Inc.

Background. Whole lung irradiation (WLI) is a standard treatment component for children with metastatic Ewing Sarcoma (ES), but data on WLI for adults are sparse.Design. An email survey was sent to expert sarcoma-dedicated oncologists worldwide:An adult with excellent performance status presents with primary ES in the leg and multiple pulmonary metastases. The patient achieves complete radiographic response after chemotherapy and resection of the primary. Would you give bilateral WLI to (1) this adult patient?, (2) this patient if 20 years old (yo)?, (3) this patient if 45 yo?, or (4) this patient if 60 yo? Results. 38 experts responded, including 24 adult, 1 adolescent young adult, and 13 pediatric oncologists. 63%, 63%, 62%, and 50% of respondents offered WLI to the adult, 20-year-old, 45-year-old, and 60-year-old, respectively. Pediatric oncologists more likely endorsed WLI across all ages including the adult (P=0.01), 20-year-old (P=0.005), 45-year-old (P=0.01), and 60-year-old (P=0.08). There were no significant differences between medical and radiation oncologists or between European/Australian and American providers.Conclusions. Almost two-thirds of experts surveyed supported WLI for adults with metastatic ES up to age 45 and half supported WLI for a 60-year-old. Continued collaboration across adult and pediatric oncology is needed to define evidence-based strategies across the age spectrum.

The developmental receptor NOTCH plays an important role in various human cancers as a consequence of oncogenic mutations. Here we describe a novel mechanism of NOTCH-induced tumor suppression involving modulation of the deacetylase SIRT1, providing a rationale for the use of SIRT1 inhibitors to treat cancers where this mechanism is inactivated because of SIRT1 overexpression. In Ewing sarcoma cells, NOTCH signaling is abrogated by the driver oncogene EWS-FLI1. Restoration of NOTCH signaling caused growth arrest due to activation of the NOTCH effector HEY1, directly suppressing SIRT1 and thereby activating p53. This mechanism of tumor suppression was validated in Ewing sarcoma cells, B-cell tumors, and human keratinocytes where NOTCH dysregulation has been implicated pathogenically. Notably, the SIRT1/2 inhibitor Tenovin-6 killed Ewing sarcoma cells in vitro and prohibited tumor growth and spread in an established xenograft model in zebrafish. Using immunohistochemistry to analyze primary tissue specimens, we found that high SIRT1 expression was associated with Ewing sarcoma metastasis and poor prognosis. Our findings suggest a mechanistic rationale for the use of SIRT1 inhibitors being developed to treat metastatic disease in patients with Ewing sarcoma. Cancer Res; 74(22); 6578–88. ©2014 AACR.

Metastatic Ewing sarcoma (EWS) is often resistant to current multimodal chemotherapeutic regimens. Oncolytic virus therapy (OV) is a novel therapeutic platform whereby viruses can selectively infect as well as replicate in and kill tumor cells, while sparing normal tissues. The purpose of this study is to investigate the efficacy of the biotherapeutic oncolytic agent, vesicular stomatitis virus (VSVΔM51), to kill EWS cells that are resistant to conventional therapy. Our hypothesis is that systemic delivery of VSVΔM51 can demonstrate tumor-specific killing of resistant EWS cells, as well as a significant decrease of tumor burden in EWS bearing mice.Methods. A biopsy sample was obtained from a patient with metastatic EWS and was used to establish a novel EWS cell line.In vitroassays evaluated the oncolytic effect of vesicular stomatitis virus (VSVΔM51) on this cell line. EWS xenograft mice model bearing either lung or subcutaneous tumors was established to evaluate the antitumor specific oncolytic effect of VSVΔM51 after local and systemic delivery.Results. The established EWS cell line shared similar molecular and genetic traits to the patient’s original tumor specimen. VSVΔM51 effectively infected and killed EWS cellsin vitro. In vivo,VSVΔM51 selectively infected and killed EWS and led to significant delay in tumor growth.Conclusion. This study has been designed to implement a translational link between the bedside and the bench, where a specific challenging clinical scenario guided this basic science research. This research demonstrated that a sarcoma, which is resistant to current conventional standard therapies, is still susceptible to an alternative therapeutic platform, such as OV. Adding OV to the armamentarium of sarcoma treatment can enhance the future therapeutic approach towards these cancer patients.

An 18-year-old male with a history of Charcot-Marie-Tooth disease (CMT) presented with metastatic Ewing sarcoma to the lungs. He had been followed by several healthcare professionals who ascribed his enlarging 23 cm gluteal mass to his CMT. The patient experienced a significant delay in diagnosis, not uncommon in sarcoma. This case explores the various system and cognitive errors that contributed to this delay.

Ewing sarcoma (ES) is the second most common type of primary bone malignancy, and retains a high propensity to metastasize; the prognosis of patients with disseminated disease is very poor, with an event-free survival rate of

Complementary and alternative medicine (CAM) therapies are commonly incorporated into the care of patients with paediatric cancer. Many modalities are safe and effective during cancer treatment and have proved beneficial for symptom relief and quality of life. However, situations where alternative therapy is provided without allopathic medical care supportive care resources can pose a safety risk to patients. This report describes the case of a 16-year-old Chinese girl with metastatic Ewing sarcoma who sought treatment with alternative treatment in Mexico. When her disease progressed with an ensuing significant loss of function, the centre personnel were unable to respond to her acute deterioration or provide necessary medical care. This resulted in her being stranded in a foreign country paralysed, isolated, and with large unanticipated financial expenditures.

Ewing sarcoma is the second most prevalent malignant primary bone tumor but constitutes only a small proportion of cardiac metastases. We present a case of asymptomatic Ewing sarcoma metastatic to the right ventricle. A 36-year-old man presented for evaluation and resection of a pedunculated right ventricular cardiac tumor. Three years before, he had been diagnosed with translocation-negative Ewing sarcoma, for which he had undergone chemotherapy and amputation of the left leg below the knee. We resected the right ventricular tumor. Analysis of the resected mass supported the diagnosis of metastatic Ewing sarcoma. Postoperative transthoracic echocardiograms showed normal biventricular size and function. One year later, the patient had no recurrence of the sarcoma. In addition to discussing this case, we review the relevant medical literature.

Ewing sarcoma is an aggressive, poorly differentiated neoplasm of solid bone that disproportionally afflicts the young. Despite intensive multi-modal therapy and valiant efforts, 70% of patients with relapsed and metastatic Ewing sarcoma will succumb to their disease. The persistent failure to improve overall survival for this subset of patients highlights the urgent need for rapid translation of novel therapeutic strategies. As Ewing sarcoma is associated with a paucity of mutations in readily targetable signal transduction pathways, targeting the key genetic aberration and master regulator of Ewing sarcoma, the EWS/ETS fusion, remains an important goal.

Background. The aims of this study were to determine the feasibility of the combination of low dose, anti-angiogenic chemotherapy with standard therapy for patients with metastatic Ewing sarcoma (ES), and to obtain preliminary outcome data. Procedures. Patients with metastatic ES were eligible. Therapy consisted of alternating cycles of ifosfamide–etoposide, and vincristine, doxorubicin, cyclophosphamide. Vinblastine and celecoxib were concomitantly administered. Surgical, radiotherapeutic, or combination local control therapy was given per institutional preference. Results. Thirty-five eligible patients were enrolled. Ninety percent received at least 75% of planned vinblastine/celecoxib doses. There was no excess of neurologic, infectious, hemorrhagic, or cardiovascular toxicities. However, 7 of 21 patients who received pulmonary irradiation prior to experiencing pulmonary toxicity did develop grade 2 or greater pulmonary toxicity, including two deaths of apparent radiation pneumonitis. Fourteen of 16 patients with pelvic disease received local irradiation. Hemorrhagic cystitis developed in six patients, five of whom had received pelvic irradiation. The overall 24-month event free survival was 35% (19–51%); 71% (26–92%) for the seven with isolated pulmonary metastases, 26% (10–45%) for all others. Conclusion. The combination of vinblastine/celecoxib metronomic therapy with standard ES treatment was feasible according to the protocol definitions. However, excess toxicity in irradiated areas was noted and limits the usefulness of this protocol. The 24-month EFS for those with isolated pulmonary metastases is better than historical controls, although the number of patient number is small, follow up short and we are lacking contemporaneous controls. Pediatr Blood Cancer 2012 Wiley Periodicals, Inc.

A 25-year-old female with a history of Ewing sarcoma presented with leukopenia (920/μL), neutropenia (180/μL), thrombocytopenia (147,000/μL) and macrocytic anemia (hemoglobin 12.2 g/dL, mean corpuscular volume (MCV) 90.3 fL). Metastatic Ewing sarcoma of the right gluteal muscle with pulmonary metastases had been diagnosed 7 years previously. Prior treatment had included chemotherapy, radiation therapy, and allogeneic hematopoietic stem cell transplantation (alloHSCT).

Background Robatumumab (19D12; MK-7454 otherwise known as SCH717454) is a fully human antibody that binds to and inhibits insulin-like growth factor receptor-1 (IGF-1R). This multiinstitutional study (P04720) determined the safety and clinical efficacy of robatumumab in three separate patient groups with resectable osteosarcoma metastases (Group 1), unresectable osteosarcoma metastases (Group 2), and Ewing sarcoma metastases (Group 3). Procedure Robatumumab infusions were administered every 2 weeks and were well tolerated with minimal toxicity. Centrally reviewed response data were available for 144 patients. Results Low disease burden was important for osteosarcoma response: three of 31 patients had complete response or partial response (PR) by Response Evaluation Criteria in Solid Tumors (RECIST) in resectable patients (Group 1) versus zero of 29 in unresectable patients (Group 2); median overall survival was 20 months in Group 1 versus 8.2 months in Group 2. In centrally reviewed patients with Ewing sarcoma with PET-CT data (N = 84/115), there were six PR, 23 stable disease, and 55 progression of disease by RECIST at 2 months. Patients with Ewing sarcoma had a median overall survival of 6.9 months. However, responding patients with Ewing sarcoma were allowed to continue on treatment after study closure. A minority of patients with metastatic Ewing sarcoma showed clinical responses and have remained healthy after receiving 25–115 doses of robatumumab with remissions of >4 years duration (N = 6). Conclusions These findings show that although the IGF-1R remains an attractive treatment target, additional research is needed to identify responders and/or means to achieve durable remissions in order to successfully exploit IGF-1R signal blockade in Ewing sarcoma (clinicaltrials.gov: NCT00617890).

Metastatic Ewing Sarcoma carries a poor prognosis, and novel therapeutics to prevent and treat metastatic disease are greatly needed. Recent evidence demonstrates that tumor-associated macrophages in Ewing Sarcoma are associated with more advanced disease. While some macrophage phenotypes (M1) exhibit anti-tumor activity, distinct phenotypes (M2) may contribute to malignant progression and metastasis. In this study, we show that M2 macrophages promote Ewing Sarcoma invasion and extravasation, pointing to a potential target of anti-metastatic therapy. CNI-1493 is a selective inhibitor of macrophage function and has shown to be safe in clinical trials as an anti-inflammatory agent. In a xenograft mouse model of metastatic Ewing Sarcoma, CNI-1493 treatment dramatically reduces metastatic tumor burden. Furthermore, metastases in treated animals have a less invasive morphology. We show in vitro that CNI-1493 decreases M2-stimulated Ewing Sarcoma tumor cell invasion and extravasation, offering a functional mechanism through which CNI-1493 attenuates metastasis. These data indicate that CNI-1493 may be a safe and effective adjuvant agent for the prevention and treatment of metastatic Ewing Sarcoma.

Developmental reprogramming techniques have been used to generate induced pluripotent stem (iPS) cells from both normal and malignant cells. The derivation of iPS cells from cancer has the potential to provide a unique scientific tool to overcome challenges associated with the establishment of cell lines from primary patient samples and a readily expandable source of cells that may be used to model the initial disease. In the current study we developmentally reprogrammed a metastatic Ewing sarcoma (EWS) cell line to a meta-stable embryonic stem (ES)-like state sharing molecular and phenotypic features with previously established ES and iPS cell lines. EWS-iPS cells exhibited a pronounced drug resistant phenotype despite persistent expression of the oncogenic EWS-FLI1 fusion transcript. This included resistance to compounds that specifically target downstream effector pathways of EWS-FLI1, such as MAPK/ERK and PI3K/AKT, which play an important role in EWS pathogenesis. EWS-iPS cells displayed tumor initiation abilities in vivo and formed tumors exhibiting characteristic Ewing histopathology. In parallel, EWS-iPS cells re-differentiated in vitro recovered sensitivity to molecularly targeted chemotherapeutic agents, which reiterated pathophysiological features of the cells from which they were derived. These data suggest that EWS-iPS cells may provide an expandable disease model that could be used to investigate processes modulating oncogenesis, metastasis, and chemotherapeutic resistance in EWS.

Background In the multicenter European Intergroup Cooperative Ewing's Sarcoma Studies, localized Ewing tumors of bone were treated by combination chemotherapy with surgery and/or radiotherapy. Patients with primary metastases (pm-pts) were treated in high risk protocols. Patients and methods One hundred seventy-seven pm-pts were registered from January 1990 to December 1995, 171 were evaluable for survival analyses. Thirty-six pm-pts received myeloablative megatherapy with stem cell rescue following conventional treatment. Bilateral whole lung irradiation (WLI) was administered in 57 pm-pts with pulmonary involvement. Event-free survival (EFS) rates were estimated by Kaplan-Meier analysis. Prognostic factors were identified by log-rank statistics, Cox procedures and logistic regression. Results Eighty-nine deaths were recorded by 1 February 1997, EFS four years after diagnosis for all 171 pm-pts was 0.27. EFS for isolated lung metastases was 0.34, for bone/bone marrow (BM) metastases, 0.28, and for combined lung plus bone/BM metastases, 0.14 (P Conclusions EFS four years after diagnosis in patients with disseminated Ewing tumors is 0.27. Whole lung irradiation and megatherapy improve outcome in subgroups of patients with disseminated Ewing tumors is 0.27. Whole lung irradiation and megatherapy improve outcome in subgroups of patients with disseminated Ewing disease.

Background: The prognosis is still poor in metastatic Ewing sarcoma (ES) patients. We aimed to assess importance of 18F-PET/CT in 2 pediatric local invasive/ metastatic Ewing sarcoma patients which have been performed antiVEGF and Sorafenib besides their neo-adjuvant and adjuvant chemotherapies (ChT) to improve their prognosis. Response to therapy has been followed by 18F-PET/CT in addition to MR imaging. Patients: First patient was a 12 year-old-girl who was suffering from right leg pain and diagnosed with Ewing sarcoma (ES). 18F-PET/CT showed a locally invasive mass with 14x5 cm dimensions located on one third of proximal tibia with a 5,1 SUVmax value was observed . After 4 cycles of conventional ChT beside antiVEGFSorafenib, SUVmax: 2,1 and tumor necrosis was 80%. Patient received local Radiotherapy (RT) and still in remission after 18 months. Second patient was a 13 year-old-girl who admitted with swelling and pain on right scapula. MR imaging revealed a mass causing deterioration on right scapula and surrounding soft tissue. Diagnosed as ES. PET/CT showed that: primary lesion SUV max 12,4 value, beside another metastatic lesion on left fifth rib with 3,4 SUVmax,confirmed by MRI and biopsi. In addition to conventional neo-adjuvant ChT regimen for ES, antiVEGF and sorafenib were added. There was no 18F-PET/CT involvement during pre-surgical evaluation. Tumor necrosis was %98 and surgical border was tumor negative. Postoperative adjuvant chemotherapy with antiVEGF and sorafenib continued untill end of the treatment protocol. Besides, local RT. Primary region of the tumor and metastatic lesion were still 18F-PET/CT imaging negative, by the enf of the therapy. Patient is still well after 15 months. Conclusion: 18F-PET/CT imaging can predict tumor response to neoadjuvant/adjuvant ChT which includes promising antiVEGF and sorafenib therapies in pediatric ES patients in the manner of the primary tumor necrosis ratio and metastatic evaluations.

Ewing sarcoma occurs in children, adolescents and young adults. High STAT3 levels have been reported in approximately 50% of patients with Ewing sarcoma, and may be important in tumorigenesis. Protein tyrosine phosphatase delta (PTPRD) is a tumor suppressor that inhibits STAT3 activation. To date, while somatic mutations in PTPRD have been reported in diverse tumors, germline mutations of PTPRD have not been investigated in Ewing sarcoma or other cancers. We identified a novel germline mutation in the PTPRD gene in three of eight patients (37.5%) with metastatic Ewing sarcoma. Although the functional impact in two of the patients is unclear, in one of them the aberration was annotated as a W775stop germline mutation, and would be expected to lead to gene truncation and, hence, loss of the STAT3 dephosphorylation function of PTPRD. Since STAT3 is phosphorylated after being recruited to the insulin growth factor receptor (IGF-1R), suppression of IGF-1R could attenuate the enhanced STAT3 activation expected in the presence of PTPRD mutations. Of interest, two of three patients with germline PTPRD mutations achieved durable complete responses following treatment with IGF-1R monoclonal antibody-based therapies. Our pilot data suggest that PTPRD germline mutations may play a role in the development of Ewing sarcoma, a disease of young people, and their presence may have implications for therapy.

Ewing sarcoma is a malignant pediatric bone and soft tissue tumor. Children with metastatic Ewing sarcoma have a cure rate of less than 30% (De loris MA et al, 2013). The major hurdle of curing Ewing sarcoma is the lack of specific and druggable targets. EWS/FLI is the master regulator of Ewing sarcoma and functions as an aberrant transcription factor to deregulate downstream targets which eventually leads to cancer phenotypes. By comparing genes deregulated by EWS/FLI in RNA-sequencing analyses across multiple model systems, we identified protein phosphatase 1 regulatory subunit 1A (PPP1R1A), a potent protein phosphatase 1 (PP1) inhibitor, as one of the core targets of EWS/FLI (Niedan S et al, 2014; Sankar S et al, 2013; Tirode F et al, 2007). Public data mining and western blotting analysis has demonstrated that PPP1R1A is highly expressed at both RNA and protein levels in Ewing sarcoma, but not the putative cell of origin, mesenchymal stem cells, or other sarcomas. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis showed that PPP1R1A is up-regulated by EWS/FLI. Further data mining with published chromatin immunoprecipitation (ChIP)-Seq, Encyclopedia of DNA Elements (ENCODE), and formaldehyde-assisted isolation of regulatory elements (FAIRE) data suggested, and subsequent luciferase reporter assay validated, that PPP1R1A is directly targeted by EWS/FLI via a GGAA microsatellite enhancer element. We next studied the role of PPP1R1A in Ewing sarcoma by functional assays in vitro and in vivo. Intriguingly, depletion of PPP1R1A caused a loss of oncogenic transformation of patient-derived Ewing sarcoma cells in soft agar, and limited xenograft tumor growth in NOD-SCID mice (p = 0.0001). Furthermore, reduction of PPP1R1A resulted in decreased cell migration in wound healing and Boyden chamber assays, and limited lung metastasis in an orthotopic mouse model. Further studies demonstrated that activation and phosphorylation at Thr35 of PPP1R1A by protein kinase A (PKA), is required for PPP1R1A to promote tumorigenesis and metastasis, as evidenced by rescuing of PPP1R1A-knockdown induced phenotypes by constitutively active (T35D) but not wild-type PPP1R1A. Consistently, we found that Ewing sarcoma but not HEK293 cells were sensitive to the treatment of PKA inhibitors (H89 and PKI 5-24). Finally, we found that further mutating of the PP1 binding site (R8A/K9A) in T35D PPP1R1A rendered this constitutively active mutant unable to rescue the knockdown phenotype, indicating that PP1 binding and inhibition by activated PPP1R1A is indispensable for PPP1R1A function. Collectively, our data suggest that PPP1R1A and related pathway plays a positive role in tumorigenesis and metastasis in Ewing sarcoma. Therefore, targeting PKA/PPP1R1A/PP1 pathway has potential therapeutic value in the treatment of both primary and metastatic Ewing sarcoma. Citation Format: Wen Luo, Changxin Xu, Janet Ayello, Filemon Dela Cruz, Jeremy Rosenblum, Stephen Lessnick, Mitchell S. Cairo. Protein phosphatase 1 regulatory subunit 1A (PPP1R1A) promotes tumor growth and metastasis via inhibition of protein phosphatase 1 in Ewing sarcoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 544.

This is an open-access article distributed under the terms of the Creative Commons Attribution Non Commercial License.-- et al., The European Network for Cancer Research in Children and Adolescents (ENCCA) provides an interaction platform for stakeholders in research and care of children with cancer. Among ENCCA objectives is the establishment of biology-based prioritization mechanisms for the selection of innovative targets, drugs, and prognostic markers for validation in clinical trials. Specifically for sarcomas, there is a burning need for novel treatment options, since current chemotherapeutic treatment protocols have met their limits. This is most obvious for metastatic Ewing sarcoma (ES), where long term survival rates are still below 20%. Despite significant progress in our understanding of ES biology, clinical translation of promising laboratory results has not yet taken place due to fragmentation of research and lack of an institutionalized discussion forum. To fill this gap, ENCCA assembled 30 European expert scientists and five North American opinion leaders in December 2011 to exchange thoughts and discuss the state of the art in ES research and latest results from the bench, and to propose biological studies and novel promising therapeutics for the upcoming European EWING2008 and EWING2012 clinical trials.

A 35-year-old female presented with pain and swelling of the distal left radius. A diagnosis of Ewing sarcoma was made and she underwent neoadjuvant chemotherapy and surgery. Macroscopic viable areas remained on the map of the surgical specimen; as such, she was classified as a poor responder and received high dose adjuvant chemotherapy. She remained disease-free for five years, until age 40. A vaginal polyp was then detected during a routine gynaecologic examination. It was removed and histopathology revealed metastatic Ewing sarcoma. To our knowledge, this is the first reported case of a vaginal metastasis of Ewing sarcoma.

BACKGROUND Although radiotherapy is an accepted component of curative treatment for Ewing sarcoma (EWS), to the authors' knowledge, there are scant data evaluating its use for palliation. The authors reviewed the Duke University Medical Center experience to evaluate treatment response and response durability. METHODS Between 1980 and 2002, 21 patients with metastatic EWS received palliative radiotherapy. Pain was the primary indication for treatment. The majority of patients were male (n = 16 patients), and the median age at diagnosis was 11.6 years (range, 2.7-28.8 yrs). Fifty-two percent of patients had metastases at initial diagnosis. For the others, the median interval from initial diagnosis to metastases was 1.7 years. RESULTS Sixty-three metastatic sites were irradiated (median dose, 30 gray [Gy]; range, 4.5-68.5 Gy), and a median of 3 sites were treated per patient (range, 1-16 sites per patient). At the time of last follow-up, 1 patient with a solitary brain metastasis has been disease free for 3.4 years after resection and cranial radiotherapy; all other patients died of their disease. Censoring this survivor, patients lived for a median of 1.0 year after metastatic diagnosis (range, from 17 days to 6.8 years), 41 days of which were spent in treatment (range, 1-93 days). Of all sites, 55% had a complete clinical response of symptoms, and 29% had a partial response. The median response duration was 4.0 months (range, 10 days to 4.8 years). Only the survivor was noted to have a treatment complication (growth hormone insufficiency). CONCLUSIONS It was possible to treat metastatic EWS effectively with palliative radiotherapy. Because these patients live a median of 1 year after diagnosis of metastases, providing symptom relief without a protracted treatment course is valuable and appropriate therapy. Cancer 2006. © 2006 American Cancer Society.

Metastasis is the major cause of disease-related death in Ewing sarcoma. Patients, who present with clinically overt disseminated disease at diagnosis and those who relapse early with distant metastases have a poor outcome despite multi-modal high-dose chemotherapy. Therefore, new treatment options are highly warranted. Ewing sarcoma pathogenesis is driven by the chimeric ETS oncogene EWS-FLI1. We here describe regulation of sirtuin SIRT1 by EWS-FLI1 and its role in metastasis. SIRT1 belongs to a family of NAD+-dependent group III deacetylases that target histone and non-histone proteins in response to metabolic stress resulting in widespread gene expression changes through epigenetic and direct transcriptional mechanisms. Among SIRT1 target proteins is the tumor suppressor p53. SIRT1 overexpression prevents p53 acetylation, which is otherwise required for the induction of its transcriptional activity. Also, SIRT1 is involved in negative regulation of several signaling pathways including NOTCH that we had previously described as being inactivated in Ewing sarcoma by the EWS-FLI1 fusion oncogene. Studying the molecular basis of EWS-FLI1 mediated functional p53 perturbation in Ewing sarcoma, we here describe a feed-back regulatory loop in which the NOTCH effector HEY1, which is epigenetically repressed by SIRT1, in turn suppresses SIRT1 expression leading to histone H4K16 and H3K56 acetylation and to p53 activation, and consequently to Ewing sarcoma cell death in vitro. By immunohistochemistry, the study of almost 400 Ewing sarcoma patient samples revealed high SIRT1 expression in about one third of primary tumors and a highly significant association of SIRT1 positivity with Ewing sarcoma metastases. The analysis of 18 paired primary tumor/metastasis samples indicated that SIRT1 was more frequently observed in lung metastases (88%) than in bone marrow metastases (55%), and was already detectable in the corresponding primary tumors in more than 60% of cases. In vitro treatment of Ewing sarcoma cell lines with a pharmacological SIRT1/2 inhibitor resulted in cell death with IC50 values between 0.8 and 3.5 μmol that depended on SIRT1 expression level and p53 status. The greatest sensitivity was obtained with cells that express high SIRT1 and are wildtype for p53. Consistent with this in vitro result, SIRT1 inhibition prohibited tumor cell migration and proliferation in a zebrafish yolk sac xenotransplantation model of a SIRT1 positive, wildtype p53 Ewing sarcoma cell line, but not of a mutant p53 cell line with low SIRT1 expression. Together, these results suggest that SIRT1 expression, which is enabled by EWS-FLI1 mediated suppression of the NOTCH signaling pathway resulting in p53 inactivation, may be therapeutically targeted to fight metastasis using pharmacological sirtuin inhibitors. Supported by EU-FP7 grant 259348 (“ASSET”) and by the Austrian Research Fund FWF, ERA-NET grant I1225-B19 (“PROVABES”). This abstract is also presented as Poster A38. Citation Format: Jozef Ban, Argyrou Fourtouna, Isidro Machado, Stephan Niedan, Ewa Snaar-Jagalska, Dave NT Aryee, Maximilian Kauer, Marco Alberghini, Adrienne Flanagan, Katia Scotlandi, Sandra J. Strauss, Elisabeth R. Lawlor, Antonio Llombart-Bosch, Heinrich Kovar. Inhibition of deacetylase SIRT1 offers a novel treatment option in metastatic Ewing sarcoma. [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr PR10.

Novel treatment strategies in Ewing sarcoma include molecularly targeted drugs and antibodies as well as cellular therapies. Preclinical in vivo models are needed that recapitulate the biology of multifocal disease and reflect the activity of novel therapies against systemic (micro)metastatic disease. Here, we used whole body magnetic resonance imaging techniques to monitor the engraftment and metastatic spread of human Ewing sarcoma xenografts in mice and to address the therapeutic efficacy of adoptive T cell transfer. Of 18 mice receiving intravenous injections of 2x106 VH-64 cells, all developed disseminated tumor growth detectable by whole-body MRI within 31 days. All mice had lung tumors, with a median of 19 tumors (range 1 to 60) per mouse. Sixteen mice had additional tumor manifestations, including bone and/or bone marrow (n=10), soft tissues (n=5), and kidney (n=13). Interobserver agreement was high, with an intraclass correlation of 0.929 for tumor numbers. Dissection and histological analysis confirmed the presence of CD99+ small blue round cell tumors in bones, lungs and kidneys in all examined specimens. Sequential whole body T2 MRI scans at weekly intervals following an initial scan 3 weeks after tumor inoculation revealed in vivo growth of tumors at all sites. To add further tissue information, we performed parallel diffusion weighted whole body imaging with background signal suppression (DWIBS). DWIBS effectively visualized metastatic Ewing sarcoma growth in bones, retroperitoneal organs, and soft tissues, whereas, as expected, susceptibility artifacts in air-filled spaces prevented effective detection of lung tumors. To assess the therapeutic efficacy of adoptive T cell transfer against disseminated Ewing sarcomas in this model, further cohorts of 9 mice each received transfusions of 1x107 14.G2a-28ζ gene-modified human GD2-specific T cells following tumor inoculation. Control mice received non-transduced T cells. The numbers of mice developing tumors and the numbers of tumors at extrapulmonary localizations sites were not noticeably different between treated and control mice. However, animals receiving GD2-targeted gene-modified T cell therapy had lower numbers of pulmonary tumors than controls (p Citation Format: Lennart Liebsch, Sareetha Kailayangiri, Laura Beck, Bianca Altvater, Raphael Koch, Marc Hotfilder, Janine Ring, Cornelius Faber, Volker Vieth, Claudia Rossig. Assessment of therapeutic responses of disseminated Ewing sarcoma xenografts to adoptive therapy with chimeric receptor gene-modified T cells in mice by whole body magnetic resonance imaging. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3978. doi:10.1158/1538-7445.AM2013-3978

Based on preclinical biology (1, 2), this translational trial aimed to assess the efficacy and tolerability of the PARP inhibitor, olaparib, in patients with advanced Ewing sarcoma progressing after prior chemotherapy. In this uncontrolled phase II pilot, adult participants with radiographically measureable disease received 400 mg of olaparib capsules, twice daily, until disease progression or drug intolerance. Tumor measurements were determined by CT or MRI, at 6 and 12 weeks after starting olaparib administration, and then every 8 weeks thereafter. Tumor response determinations were made according to RECIST 1.1, and adverse event determinations were made according to CTCAE, version 4.0. A total of 22 participants were planned to be enrolled using a conventional 2-step phase II study design. If no objective responses were observed after 12 participants had been followed for at least 3 months, further accrual would be stopped. 12 participants were enrolled, and all were evaluable. A single case each of grade 3 anemia and grade 3 thrombocytopenia was observed (see Table One). Otherwise, no significant or unexpected toxicities were observed while participants were receiving study drugs. There were 0 PR/CR, 4 SD (duration 10.9, 11.4, 11.9, and 18.4 wks), and 8 PD as best response. Of the SD, 2 had minor responses (-16.7% and -11.7% by RECIST 1.1). The median time to disease progression was 5.7 weeks. Further enrollment was therefore discontinued. This study is the first report of a prospective phase II trial to evaluate the safety and efficacy of a PARP inhibitor in patients with Ewing sarcoma. Olaparib tablets were safe and well tolerated when administered to this small cohort at the 400 mg BID dose, although the median duration of dosing was for only 5.7 weeks. However, no significant responses were seen, and the short average interval to disease progression underscores the aggressiveness of this disease. Preclinical modeling supports exquisite in vivo sensitivity of this pathway in combination with chemotherapy, and such a trial of olaparib with temozolomide will begin shortly at our Center. Toxicity Type # of Patients Grade 1 2 3 4 Anemia 1 0 0 0 0 Blood and lymphatic systems disorders: other 1 1 0 0 0 Fatigue 1 1 0 0 0 Fever 1 1 0 0 0 Pain 2 1 0 1 0 Rash maculo-papular 1 1 0 0 0 Nausea 1 1 1 0 0 Vomiting 1 1 0 0 0 Urinary tract infection 1 1 0 0 0 Platelet count decreased 2 1 1 0 Hypoglycemia 1 1 0 0 0 Metabolism and nutrition disorders: other 1 1 0 0 0 Arthritis 1 1 0 0 0 Musculoskeletal and connective tissue disorder: other 1 1 0 0 0 Dizziness 1 1 0 0 0 Nervous system disorders: other 1 1 1 0 0 Cough 3 3 0 0 0 Postnasal drip 1 1 0 0 0 Respiratory, thoracic and mediastinal disorders: other 1 1 0 0 0 Hypertension 1 1 0 0 0 Total 25 19 3 0 0 Citation Format: Edwin Choy, James Butrynski, David Harmon, Jeffrey Morgan, Suzanne George, Andrew Wagner, David D'Adamo, Greg Cote, Yael Rubinstein, Cyril Benes, Daniel Haber, Jose Baselga, George Demetri. Translation of preclinical predictive sensitivity of Ewing sarcoma to PARP inhibition: Phase II study of olaparib in adult patients with recurrent/metastatic Ewing sarcoma following failure of prior chemotherapy. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-174. doi:10.1158/1538-7445.AM2013-LB-174

Primary metastatic Ewing sarcoma involving the bone and/or bone marrow has remained largely incurable. Relapses after intensive multimodal treatment regimens are attributed to a rare subpopulation of residual cells capable of reinitiating tumor growth. Cellular immunotherapy with chimeric antigen receptor (CAR) transduced T cells is a promising strategy to eradicate minimal residual disease and prevent relapse. Based on the observation that Ewing sarcoma cells express the ganglioside antigen GD2, we have explored the capacity of T cells transduced with GD2-specific CARs to functionally interact with Ewing sarcoma cells. Surface GD2 expression was detected on 10 of 11 Ewing sarcoma cell lines by flow cytometry analysis, and on 11 of 12 primary Ewing tumor samples by immunofluorescence staining. To obtain GD2-specific effector T cells, CD3/CD28 stimulated T cells from five healthy donors were retrovirally transduced with the second generation GD2-specific CAR 14.G2aCD28ζ, resulting in CAR surface expression in 83.8±2.7% (81.1-87.8%) of the cells. Ewing sarcoma cell lines expressing various levels of GD2 were used as target cell lines for 14.G2aCD28ζ-mediated cytolysis in a 51Cr release assay. Both GD2hi VH-64 and GD2intermediate Cado-ES-1 cells were efficiently lysed by gene-modified T cells (36.6±7.7% and 25.3%±8.2 lysis at a 40:1 effector-to-target cell ratio) in the absence of background cytolysis of GD2-negative tumor cells and by non-transduced T cells. Moreover, efficient and antigen-specific granzyme B secretion was found after coincubation of various Ewing sarcoma cell lines and a primary Ewing sarcoma cell culture with CAR gene-modified T cells. 14.G2aCD28ζ T cells further eradicated Ewing sarcoma cells in a serum-free spheroid model for anchorage-independent micrometastatic tumor growth. Functional activation of CAR-expressing T cells in response to GD2 positive Ewing sarcoma cells was further demonstrated by significant secretion of IFN-γ and TNFα and significant proliferative responses to various cell lines expressing low, intermediate or high levels of GD2. Thus, GD2 is a promising target antigen for the redirected antitumor activity of T cells not only in neuroblastoma and melanoma, but also in Ewing sarcoma. To address the preclinical in vivo efficacy of 14.G2aCD28ζ T cells, we have now established an in vivo model of disseminated disease by intravenous transfer of Ewing sarcoma cells to sublethally irradiated NOD/scid mice. Magnetic resonance imaging allows to visualize metastatic tumor manifestations in xenotransplanted mice. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4754. doi:10.1158/1538-7445.AM2011-4754