Increased risk for other cancers in individuals with Ewing sarcoma and their relatives.

BackgroundThere are few reports of the association of other cancers with Ewing sarcoma in patients and their relatives. We use a resource combining statewide genealogy and cancer reporting to provide unbiased risks.MethodsUsing a combined genealogy of 2.3 million Utah individuals and the Utah Cancer Registry (UCR), relative risks (RRs) for cancers of other sites were estimated in 143 Ewing sarcoma patients using a Cox proportional hazards model with matched controls; however, risks in relatives were estimated using internal cohort-specific cancer rates in first-, second-, and third-degree relatives.ResultsCancers of three sites (breast, brain, complex genotype/karyotype sarcoma) were observed in excess in Ewing sarcoma patients. No Ewing sarcoma patients were identified among first-, second-, or third-degree relatives of Ewing sarcoma patients. Significantly increased risk for brain, lung/bronchus, female genital, and prostate cancer was observed in first-degree relatives. Significantly increased risks were observed in second-degree relatives for breast cancer, nonmelanoma eye cancer, malignant peripheral nerve sheath cancer, non-Hodgkin lymphoma, and translocation sarcomas. Significantly increased risks for stomach cancer, prostate cancer, and acute lymphocytic leukemia were observed in third-degree relatives.ConclusionsThis analysis of risk for cancer among Ewing sarcoma patients and their relatives indicates evidence for some increased cancer predisposition in this population which can be used to individualize consideration of potential treatment of patients and screening of patients and relatives

Germline PTPRD mutations in Ewing sarcoma: biologic and clinical implications.

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

C/EBPβ-1 promotes transformation and chemoresistance in Ewing sarcoma cells.

CEBPB copy number gain in Ewing sarcoma was previously shown to be associated with worse clinical outcome compared to tumors with normal CEBPB copy number, although the mechanism was not characterized. We employed gene knockdown and rescue assays to explore the consequences of altered CEBPB gene expression in Ewing sarcoma cell lines. Knockdown of EWS-FLI1 expression led to a decrease in expression of all three C/EBPβ isoforms while re-expression of EWS-FLI1 rescued C/EBPβ expression. Overexpression of C/EBPβ-1, the largest of the three C/EBPβ isoforms, led to a significant increase in colony formation when cells were grown in soft agar compared to empty vector transduced cells. In addition, depletion of C/EBPβ decreased colony formation, and re-expression of either C/EBPβ-1 or C/EBPβ-2 rescued the phenotype. We identified the cancer stem cell marker ALDH1A1 as a target of C/EBPβ in Ewing sarcoma. Furthermore, increased expression of C/EBPβ led to resistance to chemotherapeutic agents. In summary, we have identified CEBPB as an oncogene in Ewing sarcoma. Overexpression of C/EBPβ-1 increases transformation, upregulates expression of the cancer stem cell marker ALDH1A1, and leads to chemoresistance

Ewing sarcoma: a tumor with an uncanny predilection for uncommon sites

Ewing sarcoma is one of the most common bone tumors diagnosed in pediatric age group second only to osteosarcoma. The tumor is known to involve most commonly pelvic bones and long bones while extra-skeletal Ewing sarcoma accounts for 15%. Ewing sarcoma of other sites like scapula, clavicle, hands, and feet are quite rare, accounting for only 3-5%. To determine the epidemiological profile of Ewing sarcoma of unusual sites treated in our department including age at diagnosis, site of origin and mode of management along with survival data. Details of Ewing sarcoma patients reported our department in last 9 years were collected and data was analysed. We report a total of 20 cases of Ewing sarcoma in last 9 years with 4 (20%) extra skeletal Ewing sarcoma arising from soft tissues of extremities and 30% from rare sites (3 scapula, 1 clavicle, 1 phalanx, 1 calcaneum). All patients were operated after 4-7 cycles of chemotherapy with 66% limb salvage rate. With an average necrosis of 40%, with a median follow up of 36 months, the overall survival of the group was found to be 77.7%. Ewing sarcoma is one of the rare tumors of bone and soft tissue with the predilection for unusual sites, with reasonable survival outcomes in localized disease. Though scapula, clavicle, hands and feet are rare sites for Ewing sarcoma, they accounted for 30% in our department, possibly because of the referral patterns

High-content drug screening in zebrafish xenografts reveals high efficacy of dual MCL-1/BCL-XL inhibition against Ewing sarcoma

Ewing sarcoma is a pediatric bone and soft tissue cancer with an urgent need for new therapies to improve disease outcome. To identify effective drugs, phenotypic drug screening has proven to be a powerful method, but achievable throughput in mouse xenografts, the preclinical Ewing sarcoma standard model, is limited. Here, we explored the use of xenografts in zebrafish for high-throughput drug screening to discover new combination therapies for Ewing sarcoma. We subjected xenografts in zebrafish larvae to high-content imaging and subsequent automated tumor size analysis to screen single agents and compound combinations. We identified three drug combinations effective against Ewing sarcoma cells: Irinotecan combined with either an MCL-1 or an BCL-XL inhibitor and in particular dual inhibition of the anti-apoptotic proteins MCL-1 and BCL-XL, which efficiently eradicated tumor cells in zebrafish xenografts. We confirmed enhanced efficacy of dual MCL-1/BCL-XL inhibition compared to single agents in a mouse PDX model. In conclusion, high-content screening of small compounds on Ewing sarcoma zebrafish xenografts identified dual MCL-1/BCL-XL targeting as a specific vulnerability and promising therapeutic strategy for Ewing sarcoma, which warrants further investigation towards clinical application. Keywords: Anti-apoptotic protein inhibitors; Ewing sarcoma; High-content imaging; Phenotypic drug screening; Zebrafish xenograft

Ewing\u27s sarcoma/PNET of kidney in 13-year-old girl

Ewing\u27s sarcoma is the second most common primary tumour of bone in childhood. Less frequently it occurs in soft tissues. Ewing\u27s sarcoma/primitive neuroectodermal tumour (ES/PNET) is an extra ordinarily rare primary tumour in the kidney. Only very few cases of primary renal Ewing\u27s sarcoma have been reported in the literature to date. We present a case of primary right renal Ewing\u27s sarcoma in a 13-year-old girl who was diagnosed as a case of stage IV ES/PNET of kidney with metastases to lung and liver. Right nephrectomy was done followed by adjuvant radiotherapy and chemotherapy with complete response to local and distant area

Doctor of Philosophy

dissertationEwing sarcoma is a highly aggressive pediatric malignancy that is characterized by a chromosomal translocation-derived fusion protein, Ewing sarcoma (EWS)/ Friend leukemia insertion (FLI), EWS/FLI. EWS/FLI is an aberrant transcription factor and its downstream targets contribute to oncogenic transformation in Ewing sarcoma. However, the mechanisms of transcriptional regulation by EWS/FLI and the full complement of direct targets of EWS/FLI were previously unknown. The work documented in this dissertation describes a novel basis for EWS/FLI function in gene activation, and also uncovers a mechanism by which EWS/FLI directly represses a subset of critical targets genes in Ewing sarcoma. Through the identification of direct in vivo targets of EWS/FLI, we made an unexpected discovery that EWS/FLI activates some of its critical target genes, including NR0B1 and GSTM4, by binding to microsatellite repeats. These findings suggest a new paradigm for cancer-relevant gene regulation by EWS/FLI, and perhaps other ETS family members. In addition to the microsatellite repeats, EWS/FLI regulates some of its target genes through the canonical high-affinity consensus E-26 oncogene (ETS) site. We focused on one such target gene, GLI1, and uncovered a novel role for GLI1 and its downstream target, KRT17, in coordinating two cancer-relevant functions: oncogenic transformation and cellular adhesion in Ewing sarcoma. iv The functional relationship between wild-type EWS and the EWS/FLI fusion protein in Ewing sarcoma was largely unknown. Through global transcription profiling and mechanistic studies we demonstrated that EWS and EWS/FLI coregulate a subset of genes in Ewing sarcoma and that EWS functions as a cofactor of the REST transcription factor to repress neuronal differentiation genes. These data suggest that EWS, and consequently EWS/FLI, have transcriptional repressive roles in Ewing sarcoma. We next focused on identifying and characterizing the mechanism underlying EWS/FLI-mediated direct transcriptional repression. We demonstrated that EWS/FLI interacts with the nucleosome remodeling and histone deacetylase (NuRD) corepressor complex to repress critical tumor suppressor genes in Ewing sarcoma. These data identify inhibitors of the NuRD complex components as potentially effective therapeutic agents for the treatment of Ewing sarcoma. Taken together, the work presented in this dissertation advances our molecular understanding of EWS/FLI-mediated gene regulation in Ewing sarcoma

Genome-wide functional perturbation of human microsatellite repeats using engineered zinc finger transcription factors.

Repeat elements can be dysregulated at a genome-wide scale in human diseases. For example, in Ewing sarcoma, hundreds of inert GGAA repeats can be converted into active enhancers when bound by EWS-FLI1. Here we show that fusions between EWS and GGAA-repeat-targeted engineered zinc finger arrays (ZFAs) can function at least as efficiently as EWS-FLI1 for converting hundreds of GGAA repeats into active enhancers in a Ewing sarcoma precursor cell model. Furthermore, a fusion of a KRAB domain to a ZFA can silence GGAA microsatellite enhancers genome wide in Ewing sarcoma cells, thereby reducing expression of EWS-FLI1-activated genes. Remarkably, this KRAB-ZFA fusion showed selective toxicity against Ewing sarcoma cells compared with non-Ewing cancer cells, consistent with its Ewing sarcoma-specific impact on the transcriptome. These findings demonstrate the value of ZFAs for functional annotation of repeats and illustrate how aberrant microsatellite activities might be regulated for potential therapeutic applications

Immunostimulation by OX40 Ligand Transgenic Ewing Sarcoma Cells

Interleukin-2 (IL-2) transgenic Ewing sarcoma cells can induce tumor specific T and NK cell responses and reduce tumor growth in vivo and in vitro. Nevertheless, the efficiency of this stimulation is not high enough to inhibit tumor growth completely. In addition to recognition of the cognate antigen, optimal T cell stimulation requires signals from so-called co-stimulatory molecules. Several members of the tumor necrosis factor superfamily (TNFSF) have been identified as co-stimulatory molecules that can augment anti-tumor immune responses. OX40 (CD134) and OX40 ligand (OX40L = CD252; also known as tumor necrosis factor ligand family member 4) is one example for such receptor/ligand pair with co-stimulatory function. In the present investigation we generated OX40L transgenic Ewing sarcoma cells and tested their immuno-stimulatory activity in vitro. OX40L transgenic Ewing sarcoma cells showed preserved expression of Ewing sarcoma associated (anti)gens including lipase member I (LIPI), cyclin D1 (CCND1), cytochrome P450 family member 26B1 (CYP26B1) and the Ewing sarcoma breakpoint region 1-friend leukemia virus integration 1 (EWSR1-FLI1) oncogene. OX40L expressing tumor cells showed a trend for enhanced immune stimulation against Ewing sarcoma cells in combination with IL-2 and stimulation of CD137. Our data suggest that inclusion of the OX40/OX40L pathway of co-stimulation might improve immunotherapy strategies for treatment of Ewing sarcoma