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Background & Objectives: Relapse of Ewing sarcoma (ES) can occur months or years after initial remission, and salvage therapy for relapsed disease is usually ineffective. There is great need to develop biomarkers that can predict which patients are at risk for relapse, so that therapy and post-therapy evaluation can be adjusted accordingly. In the current study we sought to identify prognostic gene expression signatures in patients diagnosed with ES. Methods: Specimens were obtained from the Children's Oncology Group (COG) Biorepository. All samples were prospectively acquired from patients treated on clinical trials INT-0154 and AEWS0031. Criteria for inclusion included confirmation of localized ES, registration on a clinical trial and availability of frozen tumor. Total RNA was processed for whole genome expression profiling using Affymetrix GeneChip Human Exon 1.0 ST arrays. Affymetrix Power Tools (APT) were used to generate normalized gene-level signal intensity estimates from raw CEL file data. Differentially expressed genes with false discovery rate (FDR) of < 0.2 and absolute fold-change > 1.3 were considered to be significantly associated with outcome (survivor vs. non-survivor, relapse vs. no-relapse). Differentially expressed genes were used to create prognostic gene signatures. An independent group of ES samples from the Euro-Ewing tumor Biorepository in Muenster, Germany was similarly analyzed as a validation cohort. Results: Frozen tumor tissue was available from 254 COG patients. Following RNA and sample QC, Affymetrix CEL files were successfully generated from 56 unique patients with localized disease for whom outcome data were available. Analysis of processed gene expression data led to exclusion of 10 cases from further analysis due to issues of batch effect and outlier status. The demographic, event-free (EFS), and overall (OS) characteristics of the remaining 46 cases were shown to be representative of the INT-0154 and AEWS0031 studies as a whole. Unsupervised clustering of transcript-level data failed to demonstrate segregation of the 46 tumors into groups based on relapse or survival status. Supervised analysis of survivors vs. non-survivors identified a small number of differentially expressed genes and several statistically significant gene signatures, but none of these candidate classifiers could be internally validated. Interestingly, gene set enrichment analysis (GSEA) reproducibly revealed that integrin and chemokine receptor pathways were significantly over-represented as discriminators of EFS and OS (FDR Conclusions: Robust prognostic gene signatures that pass internal and external cross-validation were not identified in this study. These findings demonstrate the profound degree of molecular heterogeneity in ES and suggest that the heterogeneous nature of these tumors is likely to preclude identification of prognostic signatures that will be clinically useful for all cases. Whether prognostic gene signatures will be useful for differentiating among subsets of ES cases is unknown and will require larger cohort analyses. Nevertheless, these studies support recent laboratory-based discoveries implicating cell adhesion and chemokine receptor signaling in ES aggression. Further investigation of these pathways in the context of tumor cell:tumor stroma interactions is warranted. Citation Format: Samuel L. Volchenboum, Jorge Andrade, Donald A. Barkauskas, Mark Krailo, Richard Sposto, Andreas Ranft, Jenny Potratz, Uta Dirksen, Timothy J. Triche, Elizabeth Lawlor. Do prognostic gene signatures exist in Ewing sarcoma? A report from the Children's Oncology Group. [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 A72.

10529 Background: Since 1980 patients with Ewing sarcoma have been treated according to consecutive protocols (CESS) of the German Society of Pediatric Oncology and Hematology (GPOH)(supported by D...

10031 Background: The randomized Euro-EWING99-R1 trial assessed noninferiority of cyclophosphamide- vs ifosfamide-based consolidation regimens (VAC vs VAI) in SR localized ES patients. Overall, efficacy of VAC was deemed acceptable compared to VAI, with hazard ratio of event HR = 1.12 [0.89 - 1.41]. Based on its gender-stratified randomization, influence of gender on efficacy and acute toxicity were additionally explored. Methods: Impact of gender on EFS, acute toxicity by course, switches between treatment arms, and cumulative dose of alkylating agents was evaluated in multivariable models, including terms to test for heterogeneity of treatment effect by gender. Analysis was performed on the intention to treat population. Results: 856 patients (509 males, 347 females) were recruited between 2000 and 2010: 425 VAI and 431 VAC. EFS did not significantly differ between genders (p=0.33), but a marginal interaction was seen between treatment and gender (p=0.083): VAC was associated with poorer EFS in males than VAI, HR(VAC/VAI) = 1.34 [0.96 - 1.86], whereas, in females, VAC was slightly better than VAI, HR = 0.83 [0.54 - 1.28]. Similarly, males had a worse EFS than females with VAC, HR(M/F) = 1.42 [0.97 – 2.08], whereas results by gender were very similar with VAI, HR = 0.91 [0.62 – 1.33]. Severe hematological toxicity was more frequent with VAC than VAI whereas tubular renal impairment was more frequent with VAI. Severe toxicity was more frequent in females than in males, whatever the toxicity type, with no significant interaction between treatment and gender effect. 30 patients switched from VAI to VAC (21 F, 9 M) mostly due to renal toxicity, and 3 from VAC to VAI (1 F, 2 M). A reduction of alkylating agent cumulative dose >20% was more frequent in females (15% vs 9%, p=0.01), with no major difference between VAI and VAC (13% vs 10%, p=0.21). Conclusions: The marginal interaction between gender and type of alkylating agent on EFS has to be validated on external data. Differences of acute toxicity rate and compliance are not sufficient explanation. Effects of gender-dependant polymorphism/activity of metabolic enzymes (e.g. known for CYP2B6) of ifosfamide vs cyclophosphamide should be explored. Clinical trial information: NCT00020566.

9517 Background: Anthracyclines and alkylating agents are the main effective drugs known for ES. Ifosfamide and cyclophosphamide are widely used, have different toxicity profiles and unknown relati...

e20516 Background: Ewing Sarcoma rarely occurs in the head and neck region, representing less than 5% of all Ewing sarcoma cases. Consequently, data of the clinical background and outcome are quite...

10546 Background: High-dose chemotherapy (HDC) is widely used in consolidation treatment of Ewing sarcoma patients with primary disseminated or recurrent disease. Standard busulfan-based regimens are not compatible with radiotherapy to central axis sites. Treosulfan is a newly-developed bifunctional alkylating agent that has been shown to be safe in current trials of the German Society for Pediatric Hematology and Oncology (GPOH) even in heavily pre-treated patients including axial radiation. Methods: Event-free survival (EFS) and overall survival (OS) for 21 patients with primary disseminated disease (PDD) and survival after relapse for 11 relapse patients recruited from the EURO-E.W.I.N.G.99 trial 1998–2007 were analyzed. PDD-patients had received VIDE induction prior to treosulfan-based HDC (treosulfan: 36g/m2; melphalan: 140mg/m2) followed by autologous stem cell transplantation. Median follow up was 1.72 yrs. Relapse patients had received appropriate second line chemotherapy regimens prior to treosulfan-based HDC. Median follow up after relapse was 2.26 yrs. Results: Seventeen of 21 (81%) PDD-patients had a central axis site of the primary tumor, 8 patients (38%) presented with a pelvic tumor. Metastatic sites were: bone (76%), bone marrow (22%), CNS (10%), liver (15%), lymphnode (29%), other (16%), and additionally lung metastases in 57%. 1y-EFS was 0.70 (SE=.10), 2y-EFS was 0.27 (SE=0.10), and 3y-EFS was 0.16 (SE=.09). 1y-OS was 0.84 (SE=.08), 2y-OS was 0.46 (SE=.13), and 3y-OS was 0.32 (SE=.12). In the cohort of 11 relapse patients two patients (18%) had a local relapse, 3 (27%) a combined relapse, and 6 (55%) a systemic relapse. Six of 11 (55%) relapse patients had a late relapse >2 yrs from diagnosis. Survival after relapse at 3yrs was 0.70 (SE=.15). Conclusions: Treosulfan-based HDC is an alternative approach in high-risk Ewing tumor patients especially after relapse to consolidate a second remission. Further investigation and comparison to other standard HDC regimens is needed. No significant financial relationships to disclose.

Besides its role in hemato- and lymphopoiesis, bone marrow (BM) has emerged as a secondary lymphoid organ with important roles in both T cell priming and memory responses. Due to these properties, non-malignant T cells persisting within the BM of patients with acute leukemias may be involved in the immune response to leukemia and the control of minimal residual disease. Here, we investigated the phenotypic signature of residual T cells present at diagnosis in 25 pediatric patients (age 2–16 years) with B cell precursor ALL. Patients with high risk disease including Philadelphia chromosome-positive or MLL-rearranged leukemias were excluded from this analysis. Mononuclear cells were isolated from freshly aspirated BM by density gradient centrifugation and analyzed by six-color-flow cytometry using monoclonal antibodies directed towards various T-cell associated surface and intracellular markers, including CD3/CD56/TCRαβ/TCRγδ/CD86/HLA-DR (Panel 1); CD3/CD4/CD8/CCR7/CD45RA/ CD45RO (Panel 2); CD4/FoxP3/CD25/CD45RA/CD45RO/CCR7 (Panel 3). For each sample, ≥15,000 CD3+ cells (Panel 1, 2) or ≥8,000 CD4+ cells (Panel 3) were analyzed with FACS Canto and Diva Software. The Student’s t test was used to determine statistical significances between individual subgroups, and correlations were performed using the Pearson test. Consistent with published data on BM T cell subsets in healthy donors, the CD4+/CD8+ T cell ratio was 1.32±0.41. The predominant subset among CD8+ T cells (55.2±17.6%) had a naïve T cell phenotype (CD45RA+CCR7+), while 20.7±11.5% were effector memory T cells (TEM; CD45RA-CCR7-), and 7.1±6.2% were central memory T cells (TCM; CD45RA-CCR7+). No differences were found between TEL/AML1 positive or negative leukemias, or between patients stratified into standard (SR) vs. medium risk (MR) groups according to the criteria of the ALL-BFM 2000 study group. T cells bearing γδ T cell receptors have been attributed important roles in the primary immune defense against microbes and in immune control of cancer. We found that 6.9±3.0% (1.8 to 11.8%) of BM T cells were γδTCR+ (Vγ9Vδ2). A statistically significant (p

10039 Background: High dose (HD) chemotherapy using a busulfan-containing regimen has been proven advantageous for some high risk Ewing tumor (ET) patients. HD busulfan is not compatible with radiotherapy to central axis sites. Treosulfan is a bifunctional alkylating agent, structurally related to busulfan, that has shown to be safe even in heavily pretreated patients and patients at risk to treatment related toxicity. Methods: In vitro: In order to compare the cytotoxicity of busulfan and treosulfan, growth inhibition was tested by a modified (4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium-bromide (MTT) assay. In vivo: 11 patients with primary disseminated ET and contraindication for busulfan HD treatment and 11 patients with relapsed ET were referred to HD treosulfan (36 g/m2) and melphalan (140 mg/m2) followed by autologous stem cell rescue. Results: In vitro: Busulfan and treosulfan were cytotoxic in a time and dose dependent manner. Treosulfan was 15-fold more cytotoxic compared to busulfan. In vivo: The median age in patients with disseminated ET was 14.3 years, in relapsed ET 22.7 years. Sex distribution was equal. Response status prior to HD treosulfan was: stable disease in 4, partial response in 6, complete remission in 8, progressive disease in 1, not documented in 3 patients. Median follow up time in patients with disseminated ET was 0.91 years. All patients are alive. Four patients have relapsed within 2 years after HD treatment. Median follow up time in relapsed ET was 1.35 years. Seven patients are in remission, one patient is under treatment and two patients have died after relapse. HD was well tolerated with mucositis °3 in seven, °4 in three, stomatitis °3 in four, °4 in three , neutropenic infection °3 in three and reversible myelitis in one patient. Conclusions: Treosulfan HD treatment is a feasible therapeutic option without overt acute toxicity for patients with advanced ET. Efficacy and late effects remain to be evaluated. No significant financial relationships to disclose.

10012 Background: Ewing tumor treatment has resulted in long term survival rates of approx. 70% in localized, and 30% in metastatic disease. Survival following relapse has remained poor with survival rates well below 30%. Methods: All histological proven Ewing tumor patients from three consecutive GPOH trials since 1981 (CESS81 & CESS86 & EICESS92; N=1,549) were included to conduct a meta-analyses of time to and type of recurrence. The follow up has been recently updated in 2006 resulting in a maximum observation period of 25 years. The cumulative incidences of the types of first relapse excluding progress under therapy were computed with competing risk analysis. Multivariate stepwise regression analyses according to the Cox proportional hazard model researched the influences of time to and type of relapse on survival after relapse. Results: More than 70% of first recurrences occurred within 2 years, and 85% within 3 years from initial diagnosis. Most common relapses were systemic with a 3-year cumulative incidence of approx. 0.10 each for lung metastases, bone metastases and multisystem metastases including other sites, followed by local and combined relapses both with a 3-year cumulative incidence of approx. 0.05. Multivariate analyses identified both early (2.5 (p No significant financial relationships to disclose.

11501 Background: Ewing 2008R3 (EudraCT2008-003658-13) was conducted in 12 countries. It evaluated the effect of treosulfan and melphalan high dose chemotherapy followed by re-infusion of autologous hematopoietic stem cells (HDTreoMel) on event-free (EFS, primary endpoint) and overall survival (OS) in high-risk Ewing Sarcoma (EwS). Methods: Phase III, open label, prospective, multi-center, randomized controlled clinical trial. Eligible patients (pts) had disseminated EwS with metastases to bone and/or other sites, excluding pts with only pleuropulmonary metastases. Pts received 6 cycles of VIDE induction and 8 cycles of VAC consolidation therapy. Patients were randomized to receive additional HDTreoMel chemotherapy or no further treatment (control), They were further stratified by number of bone metastases (1, 2-5, > 5). One-sided adaptive inverse-normal 4-stage design, changed after the 1st interim analysis via Müller-Schäfer method. Initial sample size 185 pts, type I error rate 2.5%, power 80%. Results: 109 pts were randomized between 2009 and 2018: 55 were randomized to HDTreoMel. With a median follow-up of 3.3 years, the primary endpoint EFS was not significantly different between HDTreoMel and control in the adaptive design (HR 0.85, 95% CI 0.55-1.32, intention-to-treat). 3-year (3y) EFS was 20.9 % (95% CI 11.5-37.9%) in HDTreoMel and 19.2 % (95% CI 10.8-34.4%) in control pts. Results were similar in the per protocol collective. Subgroup analyses showed that independent of treatment, male patients had a worse outcome than female patients: 3y EFS 13.3% (95% CI 5.7-31.1%) vs 25.2% (95% CI 15.5-40.8%); p = 0.07. Patients aged < 14 had a better outcome when treated in the HDTreoMel group: 3y EFS 39.3% (95% CI 20.4-75.8%) vs 9% (95% CI 2.4-34%); p = 0.016; HR 0.40 (0.19-0.87). These effects were similar in the per protocol collective. Severe toxicities of hematology, gut, general condition and infection were more pronounced in the HDTreoMel group (p < 0.05). Conclusions: In patients with very high risk EwS, additional HDTreoMel was of no benefit for the entire cohort of patients. HDTreoMel may be of benefit for children age < 14. This observation is supported by comparable results from a non-randomized trial EE99 R3 (Ladenstein et al. JCO, 2010). Clinical trial information: NCT00987636 .