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The advent of dose intensified interval compressed therapy has improved event-free survival for patients with localized Ewing sarcoma (EwS) to 78% at 5 years. However, nearly a quarter of patients with localized tumors and 60-80% of patients with metastatic tumors suffer relapse and die of disease. In addition, those who survive are often left with debilitating late effects. Clinical features aside from stage have proven inadequate to meaningfully classify patients for risk-stratified therapy. Therefore, there is a critical need to develop approaches to risk stratify patients with EwS based on molecular features. Over the past decade, new technology has enabled the study of multiple molecular biomarkers in EwS. Preliminary evidence requiring validation supports copy number changes, and loss of function mutations in tumor suppressor genes as biomarkers of outcome in EwS. Initial studies of circulating tumor DNA demonstrated that diagnostic ctDNA burden and ctDNA clearance during induction are also associated with outcome. In addition, fusion partner should be a pre-requisite for enrollment on EwS clinical trials, and the fusion type and structure require further study to determine prognostic impact. These emerging biomarkers represent a new horizon in our understanding of disease risk and will enable future efforts to develop risk-adapted treatment.

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.

Wnt/β-catenin signaling is active in small subpopulations of Ewing sarcoma cells, and these cells display a more metastatic phenotype, in part due to antagonism of EWS-FLI1-dependent transcriptional activity. Importantly, these β-catenin-activated Ewing sarcoma cells also alter secretion of extracellular matrix (ECM) proteins. We thus hypothesized that, in addition to cell-autonomous mechanisms, Wnt/β-catenin-active tumor cells might contribute to disease progression by altering the tumor microenvironment (TME). Analysis of transcriptomic data from primary patient biopsies and from β-catenin-active versus -nonactive tumor cells identified angiogenic switch genes as being highly and reproducibly upregulated in the context of β-catenin activation. In addition, in silico and in vitro analyses, along with chorioallantoic membrane assays, demonstrated that β-catenin-activated Ewing cells secreted factors that promote angiogenesis. In particular, activation of canonical Wnt signaling leads Ewing sarcoma cells to upregulate expression and secretion of proangiogenic ECM proteins, collectively termed the angiomatrix. Significantly, our data show that induction of the angiomatrix by Wnt-responsive tumor cells is indirect and is mediated by TGF-β. Mechanistically, Wnt/β-catenin signaling antagonizes EWS-FLI1-dependent repression of TGF-β receptor type 2, thereby sensitizing tumor cells to TGF-β ligands. Together, these findings suggest that Wnt/β-catenin-active tumor cells can contribute to Ewing sarcoma progression by promoting angiogenesis in the local TME.

Tumor: tumor microenvironment (TME) interactions are critical for tumor progression and the composition and structure of the local extracellular matrix (ECM) are key determinants of tumor metastasis. We recently reported that activation of Wnt/beta-catenin signaling in Ewing sarcoma cells induces widespread transcriptional changes that are associated with acquisition of a metastatic tumor phenotype. Significantly, ECM protein-encoding genes were found to be enriched among Wnt/beta-catenin induced transcripts, leading us to hypothesize that activation of canonical Wnt signaling might induce changes in the Ewing sarcoma secretome. To address this hypothesis, conditioned media from Ewing sarcoma cell lines cultured in the presence or absence of Wnt3a was collected for proteomic analysis. Label-free mass spectrometry was used to identify and quantify differentially secreted proteins. We then used in silico databases to identify only proteins annotated as secreted. Comparison of the secretomes of two Ewing sarcoma cell lines revealed numerous shared proteins, as well as a degree of heterogeneity, in both basal and Wnt-stimulated conditions. Gene set enrichment analysis of secreted proteins revealed that Wnt stimulation reproducibly resulted in increased secretion of proteins involved in ECM organization, ECM receptor interactions, and collagen formation. In particular, Wnt-stimulated Ewing sarcoma cells up-regulated secretion of structural collagens, as well as matricellular proteins, such as the metastasis-associated protein, tenascin C (TNC). Interrogation of published databases confirmed reproducible correlations between Wnt/beta-catenin activation and TNC and COL1A1 expression in patient tumors. In summary, this first study of the Ewing sarcoma secretome reveals that Wnt/beta-catenin activated tumor cells upregulate secretion of ECM proteins. Such Wnt/beta-catenin mediated changes are likely to impact on tumor: TME interactions that contribute to metastatic progression.

// Laurie K. Svoboda 1 , Natashay Bailey 1 , Raelene A. Van Noord 2 , Melanie A. Krook 1 , Ashley Harris 1 , Cassondra Cramer 1 , Brooke Jasman 1 , Rajiv M. Patel 3, 4 , Dafydd Thomas 3 , Dmitry Borkin 3 , Tomasz Cierpicki 3 , Jolanta Grembecka 3 , Elizabeth R. Lawlor 1, 3 1 Department of Pediatrics and Communicable Diseases, University of Michigan Medical School, Ann Arbor, MI 48109, USA 2 Department of Surgery, University of Michigan Medical School, Ann Arbor, MI 48109, USA 3 Department of Pathology, University of Michigan Medical School, Ann Arbor, MI 48109, USA 4 Department of Dermatology, University of Michigan Medical School, Ann Arbor, MI 48109, USA Correspondence to: Elizabeth R. Lawlor, email: elawlor@med.umich.edu Keywords: Ewing sarcoma, trithorax, MLL, menin, HOX Received: August 15, 2016 Accepted: November 14, 2016 Published: November 18, 2016 ABSTRACT Developmental transcription programs are epigenetically regulated by the competing actions of polycomb and trithorax (TrxG) protein complexes, which repress and activate genes, respectively. Ewing sarcoma is a developmental tumor that is associated with widespread de-regulation of developmental transcription programs, including HOX programs. Posterior HOXD genes are abnormally over-expressed by Ewing sarcoma and HOXD13 , in particular, contributes to the tumorigenic phenotype. In MLL1 fusion-driven leukemia, aberrant activation of HOXA genes is epigenetically mediated by the TrxG complex and HOXA gene expression and leukemogenesis are critically dependent on the protein-protein interaction between the TrxG proteins MLL1 and menin. Based on these data, we investigated whether posterior HOXD gene activation and Ewing sarcoma tumorigenicity are similarly mediated by and dependent on MLL1 and/or menin. Our findings demonstrate that Ewing sarcomas express high levels of both MLL1 and menin and that continued expression of both proteins is required for maintenance of tumorigenicity. In addition, exposure of Ewing sarcoma cells to MI-503, an inhibitor of the MLL1-menin protein-protein interaction developed for MLL1-fusion driven leukemia, leads to loss of tumorigenicity and down-regulated expression of the posterior HOXD gene cluster. Together these data demonstrate an essential role for MLL1 and menin in mediating tumor maintenance and posterior HOXD gene activation in Ewing sarcoma. A critical dependency of these tumors on the MLL1-menin interaction presents a potentially novel therapeutic target.

Background: Previous studies have shown that aberrant activation of the Wnt/β-catenin pathway is associated with many malignant neoplasms. This includes some soft-tissue sarcoma phenotypes, most notably synovial sarcoma, implicating potential targets for novel molecular therapies. Objective: We investigate the level of Wnt/β-catenin pathway activation present in leiomyosarcomas relative to synovial sarcomas, using expression of LEF1 and β-catenin as surrogates. Methods: Cancer outlier profile analysis was performed on messenger RNA expression datasets in Oncomine (70 synovial sarcomas, 178 leiomyosarcomas). Results for LEF1 and β-catenin messenger RNA expression were reported in terms of median-centered intensity. Separate immunohistochemical studies were performed on tissue microarrays created from 77 synovial sarcomas and 89 leiomyosarcomas using antibodies to LEF1 and β-catenin. Tumors with unequivocal strong nuclear staining involving ⩾5% of cells were interpreted as positive. Results: Cancer outlier profile analysis demonstrated a higher level of LEF1 messenger RNA expression in synovial sarcomas than in leiomyosarcomas ( p Conclusion: These results provide further evidence that aberrant activation of the Wnt/β-catenin pathway is present in most synovial sarcomas, but not in most leiomyosarcomas. While targeting the constituents of this pathway might be effective in the treatment of synovial sarcomas, it is not likely to be an effective strategy in the treatment of leiomyosarcomas.

Ewing sarcomas are characterized by the presence of EWS/ETS fusion genes in the absence of other recurrent genetic alterations and mechanisms of tumor heterogeneity that contribute to disease progression remain unclear. Mutations in the Wnt/β-catenin pathway are rare in Ewing sarcoma but the Wnt pathway modulator LGR5 is often highly expressed, suggesting a potential role for the axis in tumor pathogenesis. We evaluated β-catenin and LGR5 expression in Ewing sarcoma cell lines and tumors and noted marked intra- and inter-tumor heterogeneity. Tumors with evidence of active Wnt/β-catenin signaling were associated with increased incidence of tumor relapse and worse overall survival. Paradoxically, RNA sequencing revealed a marked antagonism of EWS/ETS transcriptional activity in Wnt/β-catenin–activated tumor cells. Consistent with this, Wnt/β-catenin–activated cells displayed a phenotype that was reminiscent of Ewing sarcoma cells with partial EWS/ETS loss of function. Specifically, activation of Wnt/β-catenin induced alterations to the actin cytoskeleton, acquisition of a migratory phenotype, and upregulation of EWS/ETS–repressed genes. Notably, activation of Wnt/β-catenin signaling led to marked induction of tenascin C (TNC), an established promoter of cancer metastasis, and an EWS/ETS–repressed target gene. Loss of TNC function in Ewing sarcoma cells profoundly inhibited their migratory and metastatic potential. Our studies reveal that heterogeneous activation of Wnt/β-catenin signaling in subpopulations of tumor cells contributes to phenotypic heterogeneity and disease progression in Ewing sarcoma. Significantly, this is mediated, at least in part, by inhibition of EWS/ETS fusion protein function that results in derepression of metastasis-associated gene programs. Cancer Res; 76(17); 5040–53. ©2016 AACR.

// Melanie A. Krook 1, 2 , Allegra G. Hawkins 1, 2 , Rajiv M. Patel 3 , David R. Lucas 3 , Raelene Van Noord 1, 2 , Rashmi Chugh 4 , Elizabeth R. Lawlor 1, 2, 3 1 Translational Oncology Program, University of Michigan, Ann Arbor, MI, USA 2 Department of Pediatrics and Communicable Diseases, University of Michigan, Ann Arbor, MI, USA 3 Department of Pathology, University of Michigan, Ann Arbor, MI, USA 4 Department of Internal Medicine, University of Michigan, Ann Arbor, MI, USA Correspondence to: Elizabeth R. Lawlor, email: elawlor@umich.edu Keywords: CXCR4, epigenetics, plasticity, Ewing sarcoma Received: May 02, 2016 Accepted: July 28, 2016 Published: August 12, 2016 ABSTRACT Tumor heterogeneity is a major impediment to cancer cures. Tumor cell heterogeneity can arise by irreversible genetic mutation, as well as by non-mutational mechanisms, which can be reversibly modulated by the tumor microenvironment and the epigenome. We recently reported that the chemokine receptor CXCR4 is induced in Ewing sarcoma cells in response to microenvironmental stress. In the current study, we investigated plasticity of CXCR4 expression in vivo and assessed whether CXCR4 impacts on tumor growth. Our studies showed that Ewing sarcoma cells convert between CXCR4 negative and CXCR4 positive states in vivo and that positive cells are most abundant adjacent to areas of necrosis. In addition, tumor volumes directly correlated with CXCR4 expression supporting a role for CXCR4 in growth promotion. Mechanistically, our results show that, in ambient conditions where CXCR4 expression is low, the CXCR4 promoter exists in a poised, bivalent state with simultaneous enrichment of both activating (H3K4me3) and repressive (H3K27me3) post-translational histone modifications. In contrast, when exposed to stress, CXCR4 negative cells lose the H3K27me3 mark. This loss of promoter bivalency is associated with CXCR4 upregulation. These studies demonstrate that stress-dependent plasticity of CXCR4 is, in part, mediated by epigenetic plasticity and a bivalent promoter.

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.

Background The prognostic significance of having extraskeletal (EES) versus skeletal Ewing sarcoma (ES) in the setting of modern chemotherapy protocols is unknown. The purpose of this study was to compare the clinical characteristics, biologic features, and outcomes for patients with EES and skeletal ES. Methods Patients had localized ES and were treated on two consecutive protocols using five-drug chemotherapy (INT-0154 and AEWS0031). Patients were analyzed based on having an extraskeletal (n = 213) or skeletal (n = 826) site of tumor origin. Event-free survival (EFS) was estimated using the Kaplan–Meier method, compared using the log-rank test, and modeled using Cox multivariate regression. Results Patients with extraskeletal ES (EES) were more likely to have axial tumors (72% vs. 55%; P 8 cm (9% vs. 17%; P < 0.01), and less likely to be white (81% vs. 87%; P < 0.001) compared to patients with skeletal ES. There was no difference in key genomic features (type of EWSR1 translocation, TP53 mutation, CDKN2A mutation/loss) between groups. After controlling for age, race, and primary site, EES was associated with superior EFS (hazard ratio = 0.69; 95% confidence interval: 0.50–0.95; P = 0.02). Among patients with EES, age ≥18, nonwhite race, and elevated baseline erythrocyte sedimentation rate were independently associated with inferior EFS. Conclusion Clinical characteristics, but not key tumor genomic features, differ between EES and skeletal ES. Extraskeletal origin is a favorable prognostic factor, independent of age, race, and primary site.

Background: Ewing sarcoma is the second most common pediatric bone cancer. Metastatic disease is almost always fatal, and there is currently no method to predict which patients are at risk for metastasis. Better therapies are needed to prevent and treat metastatic disease, which means the mechanisms that drive Ewing sarcoma metastasis must be better elucidated. It is becoming increasingly clear that interactions between tumor cells and the tumor microenvironment (TME) play an essential role in metastasis, but the specific mechanisms through which the TME contributes to Ewing sarcoma progression remain largely unknown. Our previous work has demonstrated that activation of canonical Wnt and TGF-β pathways in a subset of Ewing sarcoma cells induces changes in gene expression and protein secretion that are associated with enhanced metastatic engraftment, changes in the extracellular matrix (ECM), and increased angiogenesis. As bone, the primary site for Ewing sarcoma, is an excellent source of ligands for both pathways, we hypothesize that crosstalk between Wnt/TGF-β-activated tumor cells and the local bone microenvironment contributes to osteolysis and metastatic progression. Methods: Ewing sarcoma cells were treated with control or Wnt3a media +/- recombinant TGF-β1, and then the mRNA levels of target genes were measured by Q-RT-PCR. ELISA and Luminex assays were also performed to determine the levels of secreted proteins. Conditioned media collected from stimulated cells were used to treat osteoblast precursor cells, which were then assayed for effects on differentiation and function. Subcutaneous, femur, and vossicle transplant xenograft models were established in mice and are being validated. Results: We have demonstrated that in response to Wnt3a and TGF-β1, Ewing sarcoma cells upregulate expression of canonical Wnt targets (e.g., LEF1), ECM-associated genes (e.g., TNC, COL1A1, and MMP2), and the pro-osteolytic factor PTHrP. We have also shown that osteoblast precursor cells can be induced to differentiate in the presence of conditioned media from Ewing sarcoma cell lines. Immunohistochemical staining is being used to evaluate Wnt and TGF-β pathway activation, angiogenesis, osteolysis, and the ECM in our bone tumor xenograft models. Conclusions: Wnt3a/TGF-β1-stimulated Ewing sarcoma cells upregulate expression of genes associated with the ECM and osteolysis. We are in the process of demonstrating the functional consequences of those changes using in vitro conditioned media experiments and in vivo transplant models. Understanding the mechanisms by which these pathways contribute to Ewing sarcoma phenotypes is important for the development of new therapeutics for patients with metastatic disease. Citation Format: Kelsey Temprine, Sydney Treichel, Allegra Hawkins, Tahra Suhan, Wei Jiang, Parker Acevedo, Amy Koh, Kurt Hankenson, Laurie K. McCauley, Elizabeth R. Lawlor. Investigating the role of tumor:bone microenvironment crosstalk in Ewing sarcoma progression [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 B74.

Multi-agent chemotherapeutic regimes remain the cornerstone treatment for Ewing sarcoma, the second most common bone malignancy diagnosed in pediatric and young adolescent populations. We have reached a therapeutic ceiling with conventional cytotoxic agents, highlighting the need to adopt novel approaches that specifically target the drivers of Ewing sarcoma oncogenesis. As KDM1A/lysine-specific demethylase 1 (LSD1) is highly expressed in Ewing sarcoma cell lines and tumors, with elevated expression levels associated with worse overall survival (P = 0.033), this study has examined biomarkers of sensitivity and mechanisms of cytotoxicity to targeted KDM1A inhibition using SP-2509 (reversible KDM1A inhibitor). We report, that innate resistance to SP-2509 was not observed in our Ewing sarcoma cell line cohort (n = 17; IC50 range, 81 –1,593 nmol/L), in contrast resistance to the next-generation KDM1A irreversible inhibitor GSK-LSD1 was observed across multiple cell lines (IC50 > 300 μmol/L). Although TP53/STAG2/CDKN2A status and basal KDM1A mRNA and protein levels did not correlate with SP-2509 response, induction of KDM1B following SP-2509 treatment was strongly associated with SP-2509 hypersensitivity. We show that the transcriptional profile driven by SP-2509 strongly mirrors KDM1A genetic depletion. Mechanistically, RNA-seq analysis revealed that SP-2509 imparts robust apoptosis through engagement of the endoplasmic reticulum stress pathway. In addition, ETS1/HIST1H2BM were specifically induced/repressed, respectively following SP-2509 treatment only in our hypersensitive cell lines. Together, our findings provide key insights into the mechanisms of SP-2509 cytotoxicity as well as biomarkers that can be used to predict KDM1A inhibitor sensitivity in Ewing sarcoma. Mol Cancer Ther; 17(9); 1902–16. ©2018 AACR.

Relapse of Ewing sarcoma (ES) can occur months or years after initial remission and salvage therapy for relapsed disease is usually ineffective. Thus, 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. For the current study we performed whole genome expression profiling on two independent cohorts of clinically annotated ES tumors in an effort to identify and validate prognostic gene signatures. ES specimens were obtained from the Children's Oncology Group (COG) and whole genome expression profiling performed using Affymetrix Human Exon 1.0 ST arrays. Lists of differentially expressed genes between survivors and non-survivors were used to identify prognostic gene signatures. An independent cohort of tumors from the Euro-Ewing cooperative group was similarly analyzed as a validation cohort. Unsupervised clustering of gene expression data failed to segregate tumors based on outcome. Supervised analysis of survivors vs. non-survivors revealed a small number of differentially expressed genes and several statistically significant gene signatures. Gene specific enrichment analysis (GSEA) demonstrated that integrin and chemokine genes were associated with survival in tumors where stromal contamination was present. Tumors that did not harbor stromal contamination showed no association of any genes or pathways with clinical outcome. Our results reflect the challenges of performing RNA-based assays on archived bone tumor specimens. In addition, they reveal a key role for tumor stroma in determining ES prognosis. Future biologic and clinical investigations should focus on elucidating the contribution of tumor:microenvironment interactions on ES progression and response to therapy. Key words: ES, gene expression profiling, prognostic signature.

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.

BACKGROUND Ewing sarcoma (ES) is driven by fusion of the Ewing sarcoma breakpoint region 1 gene (EWSR1) with an E26 transformation-specific (ETS) transcription factor (EWS-ETS), most often the Friend leukemia integration 1 transcription factor (FLI1). Neuropeptide Y (NPY) is an EWS-FLI1 transcriptional target; it is highly expressed in ES and exerts opposing effects, ranging from ES cell death to angiogenesis and cancer stem cell propagation. The functions of NPY are regulated by dipeptidyl peptidase IV (DPPIV), a hypoxia-inducible enzyme that cleaves the peptide and activates its growth-promoting actions. The objective of this study was to determine the clinically relevant functions of NPY by identifying the associations between patients' ES phenotype and their NPY concentrations and DPP activity. METHODS NPY concentrations and DPP activity were measured in serum samples from 223 patients with localized ES and 9 patients with metastatic ES provided by the Children's Oncology Group. RESULTS Serum NPY levels were elevated in ES patients compared with the levels in a healthy control group and an osteosarcoma patient population, and the elevated levels were independent of EWS-ETS translocation type. Significantly higher NPY concentrations were detected in patients with ES who had tumors of pelvic and bone origin. A similar trend was observed in patients with metastatic ES. There was no effect of NPY on survival in patients with localized ES. DPP activity in sera from patients with ES did not differ significantly from that in healthy controls and patients with osteosarcoma. However, high DPP levels were associated with improved survival. CONCLUSIONS Systemic NPY levels are elevated in patients with ES, and these high levels are associated with unfavorable disease features. DPPIV in serum samples from patients with ES is derived from nontumor sources, and its high activity is correlated with improved survival. Cancer 2015;121:697–707. © 2014 American Cancer Society.

Background Advances in cancer therapeutics depend on reliable in vivo model systems. To develop biologically relevant xenografts, ultrasound was utilized for tissue-directed implantation of neuroblastoma (NB) cell line and patient-derived tumors in the adrenal gland, and for renal subcapsular engraftment of Ewing's sarcoma (ES). Materials and methods NB xenografts were established by direct adrenal injection of luciferase-transfected NB cell lines (IMR32, SH-SY5Y, SK-N-BE2) or NB patient-derived tumor cells (UMNBL001, UMNBL002). ES xenografts were established by renal subcapsular injection of TC32, A673, CHLA-25, or A4573 cells. Progression was monitored by in vivo imaging. Results Tumors progressed to local disease with metastasis evident by 5 weeks. Metastatic sites included cortical bone, lung, liver, and lymph nodes. Xenografted tumors retained immunochemical features of the original cancer. Conclusion Human NB adrenal xenografts, including two patient-derived orthotopic, and ES renal subcapsular xenografts were established by ultrasound without open surgery. Tissue-directed implantation is an effective technique for developing metastatic preclinical models.

Ewing sarcoma is the second most common bone cancer in pediatric patients. Although the primary cause of death in Ewing sarcoma is metastasis, the mechanism underlying tumor spread needs to be elucidated. To this end, the role of the CXCR4/SDF-1a chemokine axis as a mediator of Ewing sarcoma metastasis was investigated. CXCR4 expression status was measured in primary tumor specimens by immunohistochemical staining and in multiple cell lines by quantitative reverse transcriptase PCR and flow cytometry. Migration and invasion of CXCR4-positive Ewing sarcoma cells toward CXCL12/SDF-1a were also determined. Interestingly, while CXCR4 status was disparate among Ewing sarcoma cells, ranging from absent to high-level expression, its expression was found to be highly dynamic and responsive to changes in the microenvironment. In particular, upregulation of CXCR4 occurred in cells that were subjected to growth factor deprivation, hypoxia, and space constraints. This upregulation of CXCR4 was rapidly reversed upon removal of the offending cellular stress conditions. Functionally, CXCR4-positive cells migrated and invaded toward an SDF-1a gradient and these aggressive properties were impeded by both the CXCR4 small-molecule inhibitor AMD3100, and by knockdown of CXCR4. In addition, CXCR4-dependent migration and invasion were inhibited by small-molecule inhibitors of Cdc42 and Rac1, mechanistically implicating these Rho-GTPases as downstream mediators of the CXCR4-dependent phenotype. Implications: This study reveals the highly plastic and dynamic nature of CXCR4 expression in Ewing sarcoma and supports a model in which stress-induced upregulation of CXCR4 contributes to tumor metastasis to lung and bone marrow, which express high levels of SDF-1a. Mol Cancer Res; 12(6); 953–64. ©2014 AACR.

Ewing sarcomas predominantly arise in pelvic and stylopod bones (i.e., femur and humerus), likely as a consequence of EWS-FLI1 oncogene-induced transformation of mesenchymal stem/progenitor cells (MSCs). MSCs located in the embryonic superficial zone cells (eSZ) of limbs express anatomically distinct posterior Hox genes. Significantly, high expression of posterior HOXD genes, especially HOXD13, is a hallmark of Ewing sarcoma. These data drove our hypothesis that Hox genes in posterior skeleton MSCs contribute to Ewing sarcoma tumorigenesis. We isolated eSZ cells from stylopod and zeugopod (i.e., tibia/fibula, radius/ulna) bones, from wild-type and Hoxd13 mutant embryos, and tested the impact of EWS-FLI1 transduction on cell proliferation, gene expression, and tumorigenicity. Our data demonstrate that both stylopod and zeugopod eSZ cells tolerate EWS-FLI1 but that stylopod eSZ cells are relatively more susceptible, demonstrating changes in proliferation and gene expression consistent with initiation of malignant transformation. Significantly, loss of Hoxd13 had no impact, showing that it is dispensable for the initiation of EWS-FLI1-induced transformation in mouse MSCs. These findings show that MSCs from anatomically distinct sites are differentially susceptible to EWS-FLI1-induced transformation, supporting the premise that the dominant presentation of Ewing sarcoma in pelvic and stylopod bones is attributable to anatomically-defined differences in MSCs.

// Kristina von Heyking 1 , Laura Roth 1, * , Miriam Ertl 1, * , Oxana Schmidt 1 , Julia Calzada-Wack 2 , Frauke Neff 2 , Elizabeth R. Lawlor 3 , Stefan Burdach 1 , Gunther H.S. Richter 1 1 Laboratory for Functional Genomics and Transplantation Biology, Children’s Cancer Research Center and Department of Pediatrics, Klinikum rechts der Isar, Technische Universitat Munchen, Munich Comprehensive Cancer Center (CCCM), and German Translational Cancer Research Consortium (DKTK), Munich, Germany 2 Institute of Pathology, Helmholtz Zentrum Munchen - German Research Center for Environmental Health (GmbH), Neuherberg, Germany 3 Departments of Pediatrics and Pathology, University of Michigan, Ann Arbor, Michigan, United States of America * These authors contributed equally to this work Correspondence to: Gunther H.S. Richter, email: guenther.richter@tum.de Keywords: HOXD, metastasis, Ewing sarcoma, WNT signaling, endochondral development Received: December 02, 2015 Accepted: May 13, 2016 Published: May 30, 2016 ABSTRACT Microarray analysis revealed genes of the posterior HOXD locus normally involved in bone formation to be over-expressed in primary Ewing sarcoma (ES). The expression of posterior HOXD genes was not influenced via ES pathognomonic EWS/ETS translocations. However, knock down of the dickkopf WNT signaling pathway inhibitor 2 (DKK2) resulted in a significant suppression of HOXD10, HOXD11 and HOXD13 while over-expression of DKK2 and stimulation with factors of the WNT signaling pathway such as WNT3a, WNT5a or WNT11 increased their expression. RNA interference demonstrated that individual HOXD genes promoted chondrogenic differentiation potential, and enhanced expression of the bone-associated gene RUNX2 . Furthermore, HOXD genes increased the level of the osteoblast- and osteoclast-specific genes, osteocalcin ( BGLAP ) and platelet-derived growth factor beta polypeptide ( PDGFB ), and may further regulate endochondral bone development via induction of parathyroid hormone-like hormone ( PTHLH ). Additionally, HOXD11 and HOXD13 promoted contact independent growth of ES, while in vitro invasiveness of ES lines was enhanced by all 3 HOXD genes investigated and seemed mediated via matrix metallopeptidase 1 (MMP1). Consequently, knock down of HOXD11 or HOXD13 significantly suppressed lung metastasis in a xeno-transplant model in immune deficient mice, providing overall evidence that posterior HOXD genes promote clonogenicity and metastatic potential of ES.

Despite multimodal treatment, long term outcome for patients with Ewing sarcoma is still poor. The second >European interdisciplinary Ewing sarcoma research summit> assembled a large group of scientific experts in the field to discuss their latest unpublished findings on the way to the identification of novel therapeutic targets and strategies. Ewing sarcoma is characterized by a quiet genome with presence of an EWSR1-ETS gene rearrangement as the only and defining genetic aberration. RNAsequencing of recently described Ewing-like sarcomas with variant translocations identified them as biologically distinct diseases. Various presentations adressed mechanisms of EWS-ETS fusion protein activities with a focus on EWS-FLI1. Data were presented shedding light on the molecular underpinnings of genetic permissiveness to this disease uncovering interaction of EWS-FLI1 with recently discovered susceptibility loci. Epigenetic context as a consequence of the interaction between the oncoprotein, cell type, developmental stage, and tissue microenvironment emerged as dominant theme in the discussion of the molecular pathogenesis and inter- and intratumor heterogeneity of Ewing sarcoma, and the difficulty to generate animal models faithfully recapitulating the human disease. The problem of preclinical development of biologically targeted therapeutics was discussed and promising perspectives were offered from the study of novel in vitro models. Finally, it was concluded that in order to facilitate rapid pre-clinical and clinical development of novel therapies in Ewing sarcoma, the community needs a platform to maintain knowledge of unpublished results, systems and models used in drug testing and to continue the open dialogue initiated at the first two Ewing sarcoma summits., The conference was supported by the European Union’s Seventh Framework Programme grants 261743(ENCCA) and 259348 (ASSET).

Purpose: We report a novel analytic method, named intercohort co-analysis or Ican, which aids in the discovery of genes with predictive value for the progression or outcome of diseases from small-size cohorts. We tested this premise in Ewing's sarcoma (ES), a highly metastatic cancer of bone and soft tissues that lacks validated molecular metastasis and prognostic indicators. Experimental Design: To uncover genes significantly expressed in ES patient subsets, we first determined a nonarbitrary gene expression significance cutoff based on expression levels in validated expressing and nonexpressing tissues. We next searched for genes that were consistently significantly expressed in several ES cohort and cell line datasets. Significantly expressed genes were independently validated by quantitative reverse transcription-PCR in an additional ES cohort. Results: Analysis of ES cohorts revealed marked intercohort gene expression variability. After filtering out the intercohort variability, CXCR4 and CXCR7 were found to be consistently associated with specific ES subsets. Pairwise analyses showed CXCR4 to correlate with ES metastases, and CXCR4 and CXCR7 to patient survival, but not with several other clinicopathological variables. Conclusion: Ican is a powerful novel method to identifying genes consistently associated with particular disease states in cancers for which large cohorts are not available, currently the case of most cancers. We report for the first time that high CXCR4 expression preferentially associates with metastatic ES, and that of CXCR7 with poor patient survival. Clin Cancer Res; 16(14); 3769–78. ©2010 AACR.

Ewing sarcoma is an aggressive paediatric cancer of the bone and soft tissue. It results from a chromosomal translocation, predominantly t(11;22)(q24:q12), that fuses the N-terminal transactivation domain of the constitutively expressed EWSR1 protein with the C-terminal DNA binding domain of the rarely expressed FLI1 protein1. Ewing sarcoma is highly sensitive to genotoxic agents such as etoposide, but the underlying molecular basis of this sensitivity is unclear. Here we show that Ewing sarcoma cells display alterations in regulation of damage-induced transcription, accumulation of R-loops and increased replication stress. In addition, homologous recombination is impaired in Ewing sarcoma owing to an enriched interaction between BRCA1 and the elongating transcription machinery. Finally, we uncover a role for EWSR1 in the transcriptional response to damage, suppressing R-loops and promoting homologous recombination. Our findings improve the current understanding of EWSR1 function, elucidate the mechanistic basis of the sensitivity of Ewing sarcoma to chemotherapy (including PARP1 inhibitors) and highlight a class of BRCA-deficient-like tumours.

11528Background: Ewing Sarcoma (ES) afflicts 225 children and 180 adults in the US annually, but the vast majority of research is in ped pts. In multiple series, adults have poorer outcomes. The ai...

Purpose Ewing sarcoma family tumors (ESFTs) exhibit chromosomal translocations that lead to the creation of chimeric fusion oncogenes. Combinatorial diversity among chromosomal breakpoints produces varying fusions. The type 1 EWS-FLI1 transcript is created as a result of fusion between exons 7 of EWS and 6 of FLI1, and retrospective studies have reported that type 1 tumors are associated with an improved outcome. We have re-examined this association in a prospective cohort of patients with ESFT treated according to current Children's Oncology Group (COG) treatment protocols. Methods Frozen tumor tissue was prospectively obtained from patients diagnosed with ESFT, and reverse transcriptase polymerase chain reaction (RT-PCR) was used to determine translocation status. Analysis was confined to patients with localized tumors who were diagnosed after 1994 and treated according to COG protocols. Translocation status was correlated with disease characteristics, event-free survival (EFS), and overall survival (OS). Results RT-PCR identified chimeric fusion oncogenes in 119 of 132 ESFTs. Eighty-nine percent of identified transcripts were EWS-FLI1, and of these, 58.8% were type 1. Five-year EFS and OS rates for patients with type 1 and non–type 1 fusions diagnosed between 2001 and 2005 were equivalent (type 1: EFS, 63% ± 7%; OS, 83% ± 6%; non–type 1: EFS, 71% ± 9%; OS, 79% ± 8%). Conclusion Current intensive treatment protocols for localized ESFT have erased the clinical disadvantage that was formerly observed in patients with non–type 1 EWS-FLI1 fusions.

Tumor heterogeneity contributes to tumor relapse and metastasis and is a major impediment to cancer cures. Sources of tumor heterogeneity are diverse and include the contributions of tumor cells, nontumor cells, and the extracellular matrix (ECM). Tumor cell heterogeneity can arise by irreversible genetic mutation, as well as by nonmutational mechanisms, which can be reversibly modulated by changes in the epigenome and the tumor microenvironment. In carcinomas, such nonmutational tumor cell plasticity plays a key role in metastatic progression. In particular, in response to cues from the microenvironment, aggressive carcinoma cells activate an epithelial-to-mesenchymal transition (EMT) program that results in transition of cells from proliferative to metastasis-initiating states. Bone and soft tissue sarcomas also frequently progress to lethal metastatic disease. However, unlike their epithelial counterparts, sarcoma cells are mesenchymal by nature and the contribution of cell plasticity to sarcoma metastasis remains to be fully elucidated. We have discovered that Ewing sarcoma, a highly metastatic tumor with peak incidence in adolescents and young adults, is profoundly plastic. When Ewing sarcoma cells are exposed to hypoxia or deprived of growth factors---microenvironmental stresses that are encountered in vivo---they undergo phenotypic transitions from relatively nonmotile to highly migratory and invasive states. These transitions are associated with upregulation of CXCR4 and activation of Src and are, at least in part, epigenetically determined. In addition, we have shown that activation of the Wnt/beta-catenin pathway by exposure to canonical Wnt ligands induces similar phenotypic transitions in Wnt-responsive cells. Interestingly, these studies also showed that the impact of Wnt/beta-catenin activation on the Ewing sarcoma cell phenotype is in large part mediated by a seemingly paradoxical inhibition of the EWS/FLI1-dependent transcriptional signature. Significantly, recent studies from other groups have demonstrated heterogeneity of EWS/FLI1 expression among individual Ewing sarcoma cells in vitro and in vivo, and cells with reduced EWS/FLI1 levels have been shown to be more metastatic. Thus, these studies together implicate tumor cell heterogeneity and cell plasticity as key contributors to Ewing sarcoma invasion and metastasis. This talk will review the published data that support cell plasticity and cell state transitions as key drivers of Ewing sarcoma metastasis. In addition, new unpublished data will be presented in support of our hypothesis that Wnt/beta-catenin-activated Ewing sarcoma cells alter tumor:tumor microenvironment crosstalk to promote bone resorption and metastasis. Specifically, results of proteomics studies that define the secretome of Wnt-activated cells will be presented. The results of these studies show that Wnt/beta-catenin activated Ewing sarcoma cells secrete ECM proteins that contribute to matrix remodeling. Thus, heterogeneity and plasticity of Ewing sarcoma tumor cells in response to canonical Wnt ligands in the tumor microenvironment leads to changes in the ECM and these changes, in turn, induce changes in both the tumor cells and the tumor microenvironment that promote tumor metastasis. Citation Format: Elizabeth R. Lawlor. Cell plasticity and metastatic progression of Ewing sarcoma [abstract]. In: Proceedings of the AACR Conference on Advances in Sarcomas: From Basic Science to Clinical Translation; May 16-19, 2017; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(2_Suppl):Abstract nr IA26.

Ewing sarcoma is an aggressive paediatric cancer of the bone and soft tissue. It results from a chromosomal translocation, predominantly t(11;22)(q24:q12), that fuses the N-terminal transactivation domain of the constitutively expressed EWSR1 protein with the C-terminal DNA binding domain of the rarely expressed FLI1 protein. Ewing sarcoma is highly sensitive to genotoxic agents such as etoposide, but the underlying molecular basis of this sensitivity is unclear. Here we show that Ewing sarcoma cells display alterations in regulation of damage-induced transcription, accumulation of R-loops and increased replication stress. In addition, homologous recombination is impaired in Ewing sarcoma owing to an enriched interaction between BRCA1 and the elongating transcription machinery. Finally, we uncover a role for EWSR1 in the transcriptional response to damage, suppressing R-loops and promoting homologous recombination. Our findings improve the current understanding of EWSR1 function, elucidate the mechanistic basis of the sensitivity of Ewing sarcoma to chemotherapy (including PARP1 inhibitors) and highlight a class of BRCA-deficient-like tumours.

Ewing sarcoma is the second most common bone cancer in children and adolescents. Patients with metastatic disease have less than a 25% chance of survival making it imperative to understand the mechanisms that drive metastasis. We previously showed that activation of Wnt/β-catenin signaling in Ewing sarcoma cells alters the tumor cell transcriptome, promoting transition to a more metastatic cellular phenotype. In addition, primary tumors with high-level Wnt activation were associated with worse clinical outcomes, supporting a role for canonical Wnt signaling in metastatic progression. Significantly, extracellular matrix (ECM) protein genes were among the most affected by Wnt/β-catenin, suggesting that activation of the pathway might lead to alterations in the local tumor microenvironment (TME). Crosstalk between the TME and tumor cells regulates fundamental aspects of the metastatic cascade including invasion, migration, and angiogenesis. It is our goal to define the impact of Wnt/β-catenin signaling on the TME and elucidate how these changes contribute to metastasis. Our specific hypothesis is that activation of Wnt/β-catenin in Ewing sarcoma cells leads to changes in the tumor secretome that result in altered TME composition and tumor:TME crosstalk. To address this hypothesis, conditioned media from Ewing sarcoma cell lines cultured in the presence and absence of Wnt3a was collected for proteomic analysis. We used label-free mass spectrometry with spectral counting to quantify proteins that passed a ≤1% FDR threshold. Significance was defined as greater than 4.5 or less than 0.2 fold difference in counts between Wnt3a and control conditions. Western blot of conditioned media was used to validate identified proteins. Preliminary analysis of the TC32 cell line identified 49 proteins up regulated in Wnt3a treated media in comparison to control. Significantly, Tenascin C (TNC), was among the most significantly up regulated proteins, validating our prior data that TNC is a transcriptional target of Wnt/β-catenin and a promoter of the metastatic phenotype in vivo. Gene ontology analysis of the significantly differentially expressed proteins revealed enrichment of proteins involved in ECM-receptor signaling, ECM organization, cell adhesion, and collagen catabolism. These biologic processes all play critical roles in tumor:TME interactions and together contribute to the early steps of adhesion, migration and invasion that are responsible for initiation of metastasis. These preliminary studies confirm that Wnt/β-catenin-dependent changes in the Ewing sarcoma transcriptome are associated with changes in tumor cell secretome. We are now validating these findings in additional cell lines and updated results will be presented. These studies will elucidate how changes in tumor cells can lead to changes in the local TME and in tumor:TME crosstalk that promote metastatic progression of Ewing sarcoma. Citation Format: Allegra Hawkins, Colin Sperring, Venkatesha Basrur, Alexey Nesvizhskii, Elizabeth Lawlor. Activation of Wnt/beta-catenin alters the Ewing sarcoma secretome - Implications for tumor:tumor microenvironment crosstalk [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5836. doi:10.1158/1538-7445.AM2017-5836

Targeted therapies are increasingly being evaluated for patients with Ewing sarcoma (EWS). Optimal strategies for quantifying key signaling proteins in EWS remain unclear. We sought to quantify tumor expression of signaling pathway proteins in EWS using 3 methodologies. A total of 46 blocks of formalin-fixed paraffin-embedded tissue were obtained from 40 patients with EWS. Tumor was evaluated for the expression of proteins in the insulin-like growth factor type 1 receptor (IGF-1R), epithelial growth factor receptor (EGFR), and mTOR pathways using standard immunohistochemical analysis (IHC), automated quantitative analysis (AQUA) immunohistochemical analysis, and mass spectrometry quantification. The mean age at diagnosis was 14 years (range, 1 to 49 y). About 67.5% were male and 57.5% had localized disease. Samples displayed a wide range of expression by AQUA: mean (range) IGF-1R=10,702 (393 to 14,424); EGFR=2750 (672 to 9798); and phosphatase and tensin homolog (PTEN)=2250 (251 to 6557). Mean IGF-1R expression by AQUA did not differ between standard IHC expression categories (low IHC=11,255; medium IHC=11,070; high IHC=11,023; P=0.98). Mean PTEN expression by AQUA was higher in the medium and high IHC categories (low IHC=1229; medium IHC=2715; high IHC=2940; P=0.064). Only 2 samples expressed EGFR by standard IHC. Mass spectrometry trended toward correlation with standard IHC but did not yield interpretable results in the majority of samples. This study demonstrates that the relative quantification of signaling protein expression in EWS is dependent on the methodology used. Optimization and validation of these tools are necessary before clinical application for risk stratification of patients or measurement of biomarker expression.

Author(s): Monument, Michael J; Johnson, Kirsten M; McIlvaine, Elizabeth; Abegglen, Lisa; Watkins, W Scott; Jorde, Lynn B; Womer, Richard B; Beeler, Natalie; Monovich, Laura; Lawlor, Elizabeth R; Bridge, Julia A; Schiffman, Joshua D; Krailo, Mark D; Randall, R Lor; Lessnick, Stephen L | Abstract: BackgroundThe genetics involved in Ewing sarcoma susceptibility and prognosis are poorly understood. EWS/FLI and related EWS/ETS chimeras upregulate numerous gene targets via promoter-based GGAA-microsatellite response elements. These microsatellites are highly polymorphic in humans, and preliminary evidence suggests EWS/FLI-mediated gene expression is highly dependent on the number of GGAA motifs within the microsatellite.ObjectivesHere we sought to examine the polymorphic spectrum of a GGAA-microsatellite within the NR0B1 promoter (a critical EWS/FLI target) in primary Ewing sarcoma tumors, and characterize how this polymorphism influences gene expression and clinical outcomes.ResultsA complex, bimodal pattern of EWS/FLI-mediated gene expression was observed across a wide range of GGAA motifs, with maximal expression observed in constructs containing 20-26 GGAA motifs. Relative to white European and African controls, the NR0B1 GGAA-microsatellite in tumor cells demonstrated a strong bias for haplotypes containing 21-25 GGAA motifs suggesting a relationship between microsatellite function and disease susceptibility. This selection bias was not a product of microsatellite instability in tumor samples, nor was there a correlation between NR0B1 GGAA-microsatellite polymorphisms and survival outcomes.ConclusionsThese data suggest that GGAA-microsatellite polymorphisms observed in human populations modulate EWS/FLI-mediated gene expression and may influence disease susceptibility in Ewing sarcoma.

A goal of the COG Ewing Sarcoma Biology Committee is enabling identification of reliable biomarkers that can predict treatment response and outcome through the use of prospectively collected tissues and correlative studies in concert with COG therapeutic studies. In this report, we aim to provide a concise review of the most well-characterized prognostic biomarkers in Ewing Sarcoma (ES), and to provide recommendations concerning design and implementation of future biomarker studies. Of particular interest and potentially high clinical relevance are studies of cell cycle proteins, sub-clinical disease, and copy number alterations. We discuss findings of particular interest from recent biomarker studies and examine factors important to the success of identifying and validating clinically relevant biomarkers in ES. A number of promising biomarkers have demonstrated prognostic significance in numerous retrospective studies and now need to be validated prospectively in larger cohorts of equivalently treated patients. The eventual goal of refining the discovery and use of clinically relevant biomarkers is the development of patient specific ES therapeutic modalities.

Outcomes for patients with metastatic and recurrent Ewing sarcoma remain poor and a better understanding of the biology of this malignancy is critical to the development of prognostic biomarkers and novel therapies. Therefore, the Children’s Oncology Group (COG) has created tissue banking protocols designed to collect high quality, clinically annotated, tumor specimens that can be distributed to researchers to perform basic science and correlative investigation. Data from the COG Ewing sarcoma tissue banking protocols AEWS02B1 and its successor study AEWS07B1 were reviewed in this study. Six-hundred and thirty five patients were enrolled on AEWS02B1 and 396 patients have had tissue submitted to AEWS07B1. The average age of participation was 13.2 years. About 86% were less than 19 years old and only 6% were greater than 21 years of age at diagnosis. When compared to SEER data, approximately 18% of all cases and only 8% of all patients >20 years old diagnosed with Ewing sarcoma annually in the United States have had tumor banked. The majority of participants submitted formalin fixed, paraffin embedded, primary tumor and blood samples. In total, fresh frozen tissue was submitted for only 29% of cases. Only seven metastatic tumor samples have been collected. Although the COG has been successful in collecting tumor samples from patients newly diagnosed with Ewing sarcoma, fresh frozen tumor specimens from primary and metastatic disease are critically needed, especially from young adult patients, in order to conduct high quality basic science and translational research investigation with a goal of developing better treatments.

Despite intensive treatment with chemotherapy, radiotherapy and surgery, over 70% of patients with metastatic Ewing's Sarcoma Family of Tumors (EFT) will die of their disease. We hypothesize that properly characterized laboratory models reflecting the drug resistance of clinical tumors will facilitate the application of new therapeutic agents to EFT. To determine resistance patterns, we studied newly established EFT cell lines derived from different points in therapy: two established at diagnosis (CHLA-9, CHLA-32), two after chemotherapy and progressive disease (CHLA-10, CHLA-25), and two at relapse after myeloablative therapy and autologous bone marrow transplantation (post-ABMT) (CHLA-258, COG-E-352). The new lines were compared to widely studied EFT lines TC-71, TC-32, SK-N-MC, and A-673. These lines were extensively characterized with regard to identity (short tandem repeat (STR) analysis), p53, p16/14 status, and EWS/ETS breakpoint and target gene expression profile. The DIMSCAN cytotoxicity assay was used to assess in vitro drug sensitivity to standard chemotherapy agents. No association was found between drug resistance and the expression of EWS/ETS regulated genes in the EFT cell lines. No consistent association was observed between drug sensitivity and p53 functionality or between drug sensitivity and p16/14 functionality across the cell lines. Exposure to chemotherapy prior to cell line initiation correlated with drug resistance of EFT cell lines in 5/8 tested agents at clinically achievable concentrations (CAC) or the lower tested concentration (LTC): (cyclophosphamide (as 4-HC) and doxorubicin at CAC, etoposide, irinotecan (as SN-38) and melphalan at LTC; P

Purpose of the Study: Ewing sarcoma (ES) is the second most common bone cancer in children. Despite improved survival for patients with localized disease, the outcome for patients with metastases remains dismal. In order to improve outcomes for ES patients, there is an imminent need to further understand the molecular and cellular biology driving ES metastasis. CXCR4 is the most commonly expressed chemokine receptor in human cancer and high expression contributes to metastasis. We have recently demonstrated that CXCR4 is dynamic and that its expression is induced in response to microenvironmental cues leading to transition of cells from CXCR4- to CXCR4+ states. In the current study we investigated the potential contribution of epigenetic regulation to the CXCR4 cell-state transitions in response to changes in the microenvironment. Experimental Procedures: Immunohistochemistry of 93 diagnostic tumor sections was performed and frequency of CXCR4+ cells assessed. Chromatin Immunoprecipitation (ChIP) PCR was used to analyze histone modifications at the CXCR4 locus in ES cell lines. mRNA levels were quantified using qRT-PCR. Chemotactic migration and invasion to CXCL12 was evaluated using the x-CELLigence system in the presence and absence of the bromodomain inhibitor JQ1. Data Summary: CXCR4+ tumor cells were identified in 31 cases. Frequency ranged from Conclusions: In summary, we have demonstrated that stress-dependent induction of CXCR4 in ES is, in part, epigenetically regulated. In particular, the CXCR4 promoter in ES cells resides in a bivalent state, which permits rapid and reversible transitions between CXCR4- and CXCR4+ cell states. Further, enhancer-dependent regulation of CXCR4 transcription opens the door to the potential of bromodomain inhibitors, such as JQ1, as novel therapeutics that could be used to antagonize metastasis-promoting cell state transitions. Citation Format: Melanie A. Krook, Raj Patel, David Lucas, Rashmi Chugh, Elizabeth R. Lawlor. Epigenetic plasticity of the CXCR4 locus contributes to cell state transitions in Ewing sarcoma. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Pediatric Cancer Research: From Mechanisms and Models to Treatment and Survivorship; 2015 Nov 9-12; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(5 Suppl):Abstract nr A19.

Objective To evaluate our own experience with primary Ewing sarcoma family tumors (ESFTs) of the kidney and to review cases of this in published reports. Materials and Methods Institutional cases of renal ESFT were identified in our pathology database. The retrieved records were reviewed for relevant data. Published cases of renal ESFTs were identified from the National Library of Medicine Medline database and restricted to English language studies. The factors associated with initial surgical management (diagnostic biopsy vs surgical resection) were analyzed using chi-square analysis. Results We diagnosed and treated 10 cases of renal ESFT from 2002 to 2011 and identified an additional 97 published cases describing this tumor. A review of these 107 cases revealed that renal ESFTs more often presented with distant metastases than did ESFTs of the bone or soft tissue. Moreover, patients rarely received preoperative (neoadjuvant) chemotherapy, the current standard of care for ESFT, often because of early total tumor resection without diagnostic biopsy. Younger patients and patients with distant metastases were more likely to undergo diagnostic biopsy as initial management (P

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.

Rhabdomyosarcoma, consisting of alveolar (aRMS) and embryonal (eRMS) subtypes, is the most common type of sarcoma in children. Currently, there are no targeted drug therapies available for rhabdomyosarcoma. In searching for new molecular therapeutic targets, we carried out genome-wide small interfering RNA (siRNA) library screens targeting human phosphatases (n = 206) and kinases (n = 691) initially against an aRMS cell line, RH30. Sixteen phosphatases and 50 kinases were identified based on growth inhibition after 72 hours. Inhibiting polo-like kinase 1 (PLK1) had the most remarkable impact on growth inhibition (∼80%) and apoptosis on all three rhabdomyosarcoma cell lines tested, namely, RH30, CW9019 (aRMS), and RD (eRMS), whereas there was no effect on normal muscle cells. The loss of PLK1 expression and subsequent growth inhibition correlated with decreased p-CDC25C and Cyclin B1. Increased expression of WEE 1 was also noted. The induction of apoptosis after PLK1 silencing was confirmed by increased p-H2AX, propidium iodide uptake, and chromatin condensation, as well as caspase-3 and poly(ADP-ribose) polymerase cleavage. Pediatric Ewing's sarcoma (TC-32), neuroblastoma (IMR32 and KCNR), and glioblastoma (SF188) models were also highly sensitive to PLK1 inhibition. Finally, based on cDNA microarray analyses, PLK1 mRNA was overexpressed (>1.5 fold) in 10 of 10 rhabdomyosarcoma cell lines and in 47% and 51% of primary aRMS (17 of 36 samples) and eRMS (21 of 41 samples) tumors, respectively, compared with normal muscles. Similarly, pediatric Ewing's sarcoma, neuroblastoma, and osteosarcoma tumors expressed high PLK1. We conclude that PLK1 could be a promising therapeutic target for the treatment of a wide range of pediatric solid tumors including rhabdomyosarcoma. [Mol Cancer Ther 2009;8(11):3024–35]

The Ewing Sarcoma Family Tumors (ESFT) consist of the classical pathologic entities of Ewing Sarcoma and peripheral Primitive Neuroectodermal Tumor. Occurring largely in the childhood through young adult years, these tumors have an unsurpassed propensity for metastasis and have no defined cell of origin. The biology of these aggressive malignancies centers around EWS/FLI1 and related EWS/ETS chimeric transcription factors, which are largely limited to this tumor class. Much progress has been made in the identification of a network of loci whose expression is modulated by EWS/FLI1 and its congeners. To date, little progress has been made in reconstructing the sequence of direct and indirect events that produce this network of modulated loci. The recent identification of GLI1 as an upregulated target of EWS/ETS transcription factors suggests a target which may be a more central mediator in the ESFT signaling network. In this paper, we further define the relationship of EWS/FLI1 expression and GLI1 upregulation in ESFT. This relationship is supported with data from primary tumor specimens. It is consistently observed across multiple ESFT cell lines and with multiple means of EWS/FLI1 inhibition. GLI1 inhibition affects tumor cell line phenotype whether shRNA or endogenous or pharmacologic inhibitors are employed. As is seen in model transformation systems, GLI1 upregulation by EWS/FLI1 appears to be independent of Hedgehog stimulation. Consistent with a more central role in ESFT pathogenesis, several known EWS/FLI1 targets appear to be targeted through GLI1. These findings further establish a central role for GLI1 in the pathogenesis of Ewing Tumors.

Background Some human cancers demonstrate cellular hierarchies in which tumor-initiating cancer stem cells generate progeny cells with reduced tumorigenic potential. This cancer stem cell population is proposed to be a source of therapy-resistant and recurrent disease. Ewing sarcoma family tumors (ESFT) are highly aggressive cancers in which drug-resistant, relapsed disease remains a significant clinical problem. Recently, the cell surface protein CD133 was identified as a putative marker of tumor-initiating cells in ESFT. We evaluated ESFT tumors and cell lines to determine if high levels of CD133 are associated with drug resistance. Methods Expression of the CD133-encoding PROM1 gene was determined by RT-PCR in ESFT tumors and cell lines. CD133 protein expression was assessed by western blot, FACS and/or immunostaining. Cell lines were FACS-sorted into CD133+ and CD133- fractions and proliferation, colony formation in soft agar, and in vivo tumorigenicity compared. Chemosensitivity was measured using MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxy-methoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) assays. Results PROM1 expression was either absent or extremely low in most tumors. However, PROM1 was highly over-expressed in 4 of 48 cases. Two of the 4 patients with PROM1 over-expressing tumors rapidly succumbed to primary drug-resistant disease and two are long-term, event-free survivors. The expression of PROM1 in ESFT cell lines was similarly heterogeneous. The frequency of CD133+ cells ranged from 2-99% and, with one exception, no differences in the chemoresistance or tumorigenicity of CD133+ and CD133- cell fractions were detected. Importantly, however, the STA-ET-8.2 cell line was found to retain a cellular hierarchy in which relatively chemo-resistant, tumorigenic CD133+ cells gave rise to relatively chemo-sensitive, less tumorigenic, CD133- progeny. Conclusions Up to 10% of ESFT express high levels of PROM1. In some tumors and cell lines the CD133+ fraction is relatively more drug-resistant, while in others there is no apparent difference between CD133+ and CD133- cells. These studies reveal heterogeneity in PROM1/CD133 expression in ESFT tumors and cell lines and confirm that high levels of PROM1 expression are, in at least some cases, associated with chemo-resistant disease. Further studies are required to elucidate the contribution of PROM1/CD133 expressing cells to therapeutic resistance in a large, prospective cohort of primary ESFT.

The Children's Oncology Group (COG) is a multi-institutional cooperative group dedicated to childhood cancer research that has helped to increase the survival of children with cancer through clinical trials. These clinical trials include a standardized regimen of imaging examinations performed prior to, during, and following therapy. This article presents imaging guidelines developed by a multidisciplinary group from the COG Bone Tumor Committee. These guidelines provide both required and recommended studies. Recommended examinations may become required in the future. These guidelines should be considered a work in progress that will evolve with advances in imaging and childhood cancer research.

Normal development is governed by covalent modifications to chromatin that are mediated by the reciprocal actions of Polycomb group (PcG) and Trithorax (TrxG) proteins, which promote gene repression and activation, respectively. We recently reported that HOX genes, key developmental transcription factors that are regulated by PcG and TrxG during embryogenesis, are abnormally expressed in Ewing sarcoma (ES). In particular, we found that the posterior HOXD genes, including HOXD13, are markedly overexpressed, and that the promoters of these genes are aberrantly marked with the TrxG-dependent histone mark, H3K4me3. The TrxG proteins MLL and menin cooperate to drive HOX gene expression and promote tumorigenesis in leukemia. We therefore hypothesized that MLL and menin might also contribute to ES pathogenesis. Gene and protein expression were determined by microarray, q-RT-PCR and western blot of ES cell lines and tumors. Lentiviral shRNA constructs were used to knock down HOXD13, menin and MLL. Proliferation, survival, and tumorigenicity of genetically modified cells were assessed in vitro and in vivo. Inhibition of TrxG function was achieved by exposing cells to MI-503, a small molecule inhibitor of menin-MLL interactions. Our results confirm that menin, MLL and HOXD13 are all highly expressed by ES relative to non-malignant tissues and stem cells. Moreover, loss of function studies implicate each as a tumor promoting oncogene. Specifically, knockdown of HOXD13, menin or MLL inhibited cell proliferation and cell death was also induced by loss of either MLL or HOXD13. Colony formation in soft agar was likewise inhibited by MLL and HOXD13 knockdown and tumor formation in vivo was significantly delayed in ES cells with reduced expression of HOXD13. Importantly, knockdown of MLL led to reduced expression of HOXD13 further implicating MLL as a key mediator of HOXD13 over-expression in ES and supporting the hypothesis that, like MLL-fusion-dependent leukemias, HOX deregulation and tumorigenicity in ES are dependent on the TrxG complex. To further test this hypothesis we exposed ES cells to MI-503, an inhibitor of menin-MLL protein-protein interaction that disrupts the TrxG complex. Exposure of ES cells to MI-503 resulted in a dose-dependent inhibition of cell proliferation and marked inhibition of colony formation in soft agar. In contrast, MI-NC, a control compound with similar structure that lacks affinity for the menin-MLL interaction had no effect on ES cell growth or viability. Together these data for the first time demonstrate a key role for menin and MLL in ES pathogenesis and highlight a novel opportunity for therapeutic intervention in these aggressive tumors. Citation Format: Laurie K. Svoboda, Cassondra Cramer, Ashley Harris, Natashay Bailey, Tomek Cierpicki, Jolanta Grembecka, Elizabeth Lawlor. The Trithorax proteins menin and MLL promote Ewing sarcoma tumorigenicity. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 490. doi:10.1158/1538-7445.AM2015-490

Potassium channels regulate a variety of biological processes and dysfunction of these channels contributes to cancer initiation and progression. We recently showed that the KCNA5-encoded channel, Kv1.5, is a target of polycomb-group (PcG)-dependent repression in aggressive pediatric solid tumors and that this epigenetic silencing of the Kv1.5 channel contributes to cell survival under hypoxic stress. In human cancer, the promoters of PcG target genes are often subject to aberrant DNA hypermethylation. Therefore, in the current study we investigated whether the KCNA5 locus is hypermethylated in Ewing sarcoma. The methylation status of KCNA5 was determined in tumor samples, non-transformed cells and a panel of cancer cell lines using a custom Illumina GoldenGate® methylation array and by MethyLight. Promoter methylation and expression of KCNA5 were measured by MethyLight and qRT-PCR, respectively, before and after exposure to decitabine. Proliferation was assessed in untreated and decitabine treated cells in the presence or absence of the Kv1.5 channel blocker diphenyl phosphine oxide-1 (DPO-1). Methylation array studies of normal tissues and Ewing sarcoma tumors and cell lines showed that the KCNA5 promoter is hypermethylated in Ewing sarcoma compared to normal tissues. This hypermethylation was confirmed by MethyLight analysis in an independent set of primary tumors and cell lines. These studies also showed that methylation was relatively higher in cell lines than primary tumors. Exposure of Ewing sarcoma cells to decitabine led to demethylation of the KCNA5 promoter and directly correlated with increased expression of the KCNA5 transcript. Decitabine treatment did not affect the viability of Ewing sarcoma cell lines; however it led to a decreased proliferation. Significantly, pharmacologic inhibition of the Kv1.5 channel by DPO-1 partially reversed the decitabine-induced reduction in cell proliferation in three different cell lines (TC-71, A673 and A4573). Together these studies show that the KCNA5 promoter is hypermethylated in Ewing sarcoma and that inactivation of the Kv1.5 channel promotes cell proliferation. This work identifies DNA methylation as another epigenetic mechanism that regulates KCNA5 expression in cancer and further implicates epigenetic repression of the Kv1.5 potassium ion channel in tumor pathogenesis. Citation Format: Katherine Ryland, Allegra Hawkins, Daniel J. Weisenberger, Scott Borinstein, Peter W. Laird, Jeffrey R. Martens, Elizabeth R. Lawlor. The KCNA5 promoter is hypermethylated in Ewing sarcoma and silencing contributes to cell proliferation. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3288. doi:10.1158/1538-7445.AM2015-3288

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.

10045 Background: Targeted therapies are increasingly being evaluated for patients with Ewing sarcoma (EWS). Optimal strategies for quantifying key signaling proteins from paraffin-embedded material remain unclear. We sought to quantify tumor expression of IGF-1R, EGFR, and PTEN in EWS using two different methodologies. Methods: Decalcified paraffin-embedded tissue was obtained from 40 patients with EWS. 28 samples were obtained at time of diagnosis, 6 after initial chemotherapy, and 6 at time of relapse. Tumor was evaluated for the expression of IGF-1R, PTEN, and EGFR using standard immunohistochemistry (IHC) and automated quantitative analysis (AQUA) immunohistochemistry. Standard IHC results were categorized as low, medium and high expression based on a pathologist’s review. Expression by AQUA was measured as a continuous variable in arbitrary units. One sided ANOVA analysis was used to compare the mean AQUA expression for each analyte between categories of expression by IHC. Results: The mean age of patients was 15 years (range 1-49 yrs) at time of diagnosis. 68% were male and 66% had localized disease. Samples displayed a wide range of expression by AQUA: mean IGF-1R = 10702 (range 393 - 14424); EGFR = 2750 (range 672 – 9798); and PTEN = 2250 (range 251 – 6557). Mean IGF-1R expression by AQUA did not differ significantly between standard IHC expression categories (mean IGF-1R expression by AQUA for low IHC = 11255, medium IHC = 11070, high IHC = 11023; p = 0.98). Mean PTEN expression by AQUA was higher in the medium and high IHC categories (mean PTEN expression by AQUA for low IHC = 1229, medium IHC = 2715, high IHC = 2940; p = 0.064). Only two samples expressed EGFR by standard IHC. These samples qualitatively had higher AQUA expression levels, but there were too few samples for a reliable determination of statistical significance. Conclusions: AQUA provides a more dynamic range for each marker, though there was poor correlation with standard IHC results. Larger sample sets are needed to determine the optimal approach for quantifying signaling proteins in Ewing sarcoma.

Ewing's sarcoma (ES) is a highly malignant bone and soft tissue tumor that primarily affects children and young adults. These tumors are characterized by chromosomal translocations, most commonly t(11;22)(q24;q12), which generates the EWS-FLI1 fusion oncogene. Despite its oncogenic potential, the fusion protein induces cell cycle arrest or apoptosis in most non-transformed cells unless mutations in tumor suppressors such as p16 are also present. However, only a minority of patients (∼25%) have such mutations at diagnosis, suggesting other mechanisms for cellular tolerance must be present in the cell of origin. Currently, the cell of origin of these tumors is controversial, with data supporting both a mesenchymal and/or neural crest origin. To assess the potential to model ES using mouse neural crest stem cells (NCSCs) and elucidate the mechanism(s) of EWS-FLI1 tolerance, primary fetal and adult mouse NCSCs were transduced with a V5-tagged EWS-FLI1 2a eGFP construct. Both wild-type fetal and postnatal NCSCs tolerated EWS-FLI1 expression, with 40-60% of the stem cell-derived neurospheres positive for GFP. There was no difference in neurosphere forming ability between cells infected with EWS-FLI1 or an empty vector, suggesting EWS-FLI1 expression was not toxic to these cells. EWS-FLI1 expression was maintained as the stem cells self-renewed, with GFP-positive primary neurospheres giving rise to GFP-positive secondary and tertiary neurospheres but there was no difference in the frequency of GFP-positive and -negative secondary neurospheres. EWS-FLI1 expression was confirmed by quantitative PCR and the V5 tag was localized to the nucleus by immunocytochemistry, consistent with transcription factor function. EWS-FLI1-mediated gene expression analysis is currently ongoing. Interestingly, the tolerance of EWS-FLI1 was dramatically reduced in Bmi-1 deficient NCSCs. Bmi-1 is known to function cooperatively with multiple other oncogenes to mediate transformation but its role in tolerance has not been studied. In the absence of Bmi-1 only 8% of the fetal neurospheres were positive for GFP, a large decrease compared to 40% of the wild-type neurospheres. Taken together, these data are consistent with a neural crest origin for ES and demonstrate a critical role for Bmi-1-mediated tolerance to EWS-FLI1. Work is ongoing to determine whether EWS-FLI1 can transform these cells and to assess the full utility of this novel system to model ES and genetically assess important regulators of EWS-FLI1 function. Citation Format: Jack T. Mosher, Victor S. Chen, Elizabeth R. Lawlor. Modeling Ewing's sarcoma and tolerance of EWS-FLI1 with neural crest stem cells. [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 5033. doi:10.1158/1538-7445.AM2013-5033

Background: Ewing sarcoma (ES) is the second most common malignant bone tumor in pediatric patients. While intensification of chemotherapy has improved the outlook for patients with localized disease, little progress has been made for those with metastatic or relapsed disease. Prognosis for these patients remains dismal with fewer than 20% surviving. Moving forward, we need to develop molecularly targeted therapy for patients with high-risk disease. The mechanisms underlying metastatic relapse in ES remain unknown but gene encoding the chemokine receptor CXCR4 has been shown to be upregulated in metastatic ES. The CXCL12-CXCR4 axis contributes to tumor metastasis in other sarcomas but its function in ES is not yet known. Objective: In the current study we are investigating whether CXCR4 is a mediator of ES cell metastasis and may be useful as a biomarker of high-risk disease. Results: Using qRT-PCR and flow cytometry we have shown that expression of CXCR4 transcript and protein varies significantly across ES cell lines. Importantly, the frequency of CXCR4+ cells and CXCR4 expression levels were found to be highly dynamic and were altered under different experimental conditions. Specifically, both serum deprivation and hypoxia induced upregulation of CXCR4 in TC32, TC71, and A673 ES cells. CXCR4+ cell frequency was found to be consistently highest in TC32 where the frequency ranged from 35% in ambient conditions to 62% in serum deprivation. When sorted into CXCR4+ and CXCR4- populations TC32 populations returned to their baseline state of 40% CXCR4+ cells within 3 weeks indicating that CXCR4+ cells can change into CXCR4- cells and vice versa. Interestingly, CXCR4 transcript was identified to be increased in slowly proliferating (PKH-dye-retaining) TC71 and A673 cells compared to more rapidly dividing counterparts. These data implicate CXCR4 as a potential marker of putative cancer stem cells. Finally, initial immunohistochemical studies have demonstrated highly variable patterns of CXCR4 expression in primary ES tumor samples. Summary: ES cells express CXCR4 both in primary tumors and in cultured cell lines. However, the expression is both variable and highly dynamic. Specifically, we have found that ES cells can switch back and forth between CXCR4- and CXCR4+ states in response to environmental cues including serum deprivation and hypoxia. Ongoing work is now testing the hypothesis that CXCR4+ ES cells are more migratory than CXCR4- cells and may thus be a source of metastasis. To further test this hypothesis we are using correlative studies of clinically annotated tumor microarrays to determine if a high frequency of CXCR4+ cells at diagnosis is associated with an increased risk of metastatic relapse. Citation Format: Melanie A. Krook, Lauren A. Nicholls, Natashay J. Bailey, Amy Han, Chris Scannell, Dafydd Thomas, Elizabeth R. Lawlor. Elucidating the role of CXCR4 in Ewing sarcoma. [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 5032. doi:10.1158/1538-7445.AM2013-5032

Cancer genomics can help uncover genes that may serve as indicators of tumor susceptibility, progression, or treatment outcome, as exemplified by studies of adult tumors for which large cohorts are readily available. Childhood cancers comprise a large number of low-incidence histogenetically and molecularly distinct neoplasms, rendering population-based genomic studies unrealistic for these diseases. In recent years, several small-size cohort childhood cancer genomic studies have been published. We report here a novel analytical approach, named intercohort co-analysis or Ican, whereby gene expression significance is inferred from gene expression levels in biologically relevant normal tissues, and gene-disease association significance is assessed in each individual cohort. Next, only genes found to be consistently significant in all cohorts are considered as putative markers of specific disease states. We tested Ican in Ewing's sarcoma (ESFT), a family of tumors that lacks a reliable molecular prognostic or metastasis indicator. We report here that this approach revealed a high intercohort gene expression significance variability, and identified CXCR4 and CXCR7, two chemokine receptors known to correlate to metastasis and/or to poor prognosis mostly in adult cancers, as significantly and consistently associated with ESFT subsets. Accordingly, we show for the first time that CXCR4 expression marks metastatic ESFT and that of CXCR7 serves as a predictor of adverse patient survival. We submit these findings as a proof-of-principle that Ican of publicly available gene expression profiles can yield genes with predictive value for the progression or outcome of low-incidence diseases. We further submit that this approach is poised to gain momentum as many journals now request the mandatory contribution of gene expression profiles to public repositories. These policies, along with the growing number of cancer gene expression profiling investigations, we predict, will swiftly drive upward the number of publicly available low-incidence disease expression datasets, and consequently, of Ican applications. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3410.

BACKGROUND: Ewing sarcoma family of tumors (EFT) represents the second most common primary malignant bone tumor in children and adolescents. The majority of EFTs harbor a translocation (t11;22)(q24;q12) resulting in the expression of the EWS-FLI1 chimeric oncoprotein. We hypothesized that microarray gene expression profiling, in combination with next-generation sequencing technology, could be used to identify EFT-specific genes and transcripts including large intergenic noncoding (linc)RNAs. Next-generation sequencing, coupled with target enrichment for validation, can also identify driver mutations of metastasis and treatment resistance in high-risk cases. RESULTS: Genome-wide expression profiles of childhood sarcoma and normal tissues were analyzed using both Partek Genomics Suite as well as our own customized software, Genetrix. These are compared with Helicos single molecule sequencing data. The results show that both known genes and candidate lincRNAs strongly associate with EFT and can be used to distinguish EFT from other childhood tumors. Moreover, many lincRNAs are seen on both the poly-A selected track and on the random hexamer primed total RNA expression profile, indicating that the transcripts are multi-exonic and poly-adenylated. Importantly, regions of EFT-associated lincRNA expression may indicate regions that are deregulated by EWS-FLI1 in these tumors. We also focused on lincRNAs with differential expression in an EFT cell line pair (CHLA-9 and −10) derived, respectively, from patient-matched primary and metastatic tumors. RNA-seq determined ∼160,000 regions representing ∼16MB of genomic sequence as being at least 3-fold differentially expressed between the two cell lines. A higher threshold of at least 10-fold expression difference still revealed ∼20,000 regions representing ∼2MB of genomic sequence. In addition, the proportion of differentially-expressed intergenic transcripts was higher in CHLA-9 (18.1%) versus CHLA-10 (11.2%). Annotated transcripts with at least 10-fold expression in CHLA-10 are enriched in plasma membrane and adhesion-related functions, and this is consistent with metastatic behavior. Genes most highly expressed in CHLA-10 include ANXA5, FABP3, and HIST1H1A, whereas genes with much lower expression include CXCL14, MCTP2, and TRIM22. These genes, along with lincRNAs, may play important roles in disease progression and drug resistance. CONCLUSIONS: We have identified several large intergenic noncoding (linc)RNAs that are highly and differentially expressed by EFT. We hypothesize that these lincRNAs may be novel therapeutic targets in EFT. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4087.

Ewing's sarcoma family tumors (ESFT) are aggressive bone and soft tissue tumors that are most commonly diagnosed in childhood and adolescence. Unfortunately, drug-resistant relapse is not uncommon and is associated with very poor outcomes. No clinical or pathologic features currently exist that reliably predict chemosensitivity or relapse in newly diagnosed patients. In this study we used whole genome expression microarray profiling to identify potential markers of drug resistance and relapse in ESFT. Affymetrix human exon (HuEx) arrays were used to profile a highly drug resistant case of primary, localized ESFT before and after induction therapy. Comparison of gene summarized data from pre- and post-therapy samples with 10 chemosensitive tumors identified 309 genes to be differentially expressed in the resistant case at diagnosis (fold change>2). Of the 309 differentially expressed genes at diagnosis, 130 demonstrated an even greater change in expression post-induction therapy suggesting that these genes might be markers of the most highly drug-resistant cells. Intriguingly, two stem cell associated genes, PROM1 (prominin 1) and LGR5 (leucine-rich-repeat containing G-protein-coupled receptor 5), were included in the list of putative chemoresistance markers. Expression of PROM1 and LGR5 were 18-fold and 17-fold higher, respectively, at diagnosis and 31-fold and 21-fold higher post-induction therapy in the drug resistant case. To determine if either gene is associated with chemoresistant disease or relapse in ESFT we next evaluated whether their expression correlated with outcome in a large cohort of institutional and Children's Oncology Group patients. Clinical correlates analysis of HuEx gene expression data obtained from 53 tumors revealed no correlation between PROM1 expression and outcome. In contrast, increased expression of LGR5 was associated with worse event-free (p=0.008) and overall survival (p=0.046). Variable but detectable expression of LGR5 in ESFT tumors and cell lines was validated using quantitative RT-PCR. Finally, LGR5 expression in ESFT cells was compared to human embryonic and somatic stem cells and fibroblasts. Significantly, LGR5 was found to be highly expressed by neural crest stem cells but not by bone-marrow derived mesenchymal stem cells, differentiated neural crest cells, or embryonic fibroblasts. These data suggest that LGR5 might be useful as a predictor of outcome in ESFT and implicate a primitive neural crest stem cell phenotype in the developmental origins of drug-resistant and relapsed disease. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4640.