Andrea Luecke

BACKGROUND: Novel treatment strategies are needed to cure disseminated Ewing sarcoma. Primitive neuroectodermal features and a mesenchymal stem cell origin are both compatible with aberrant expression of the ganglioside antigen G(D2) and led us to explore G(D2) immune targeting in this cancer. METHODS: We investigated G(D2) expression in Ewing sarcoma by immunofluorescence staining. We then assessed the antitumour activity of T cells expressing a chimeric antigen receptor specific for G(D2) against Ewing sarcoma in vitro and in vivo. RESULTS: Surface G(D2) was detected in 10 out of 10 Ewing sarcoma cell lines and 3 out of 3 primary cell cultures. Moreover, diagnostic biopsies from 12 of 14 patients had uniform G(D2) expression. T cells specifically modified to express the G(D2)-specific chimeric receptor 14. G2a-28ζ efficiently interacted with Ewing sarcoma cells, resulting in antigen-specific secretion of cytokines. Moreover, chimeric receptor gene-modified T cells from healthy donors and from a patient exerted potent, G(D2)-specific cytolytic responses to allogeneic and autologous Ewing sarcoma, including tumour cells grown as multicellular, anchorage-independent spheres. G(D2)-specific T cells further had activity against Ewing sarcoma xenografts. CONCLUSION: G(D2) surface expression is a characteristic of Ewing sarcomas and provides a suitable target antigen for immunotherapeutic strategies to eradicate micrometastatic cells and prevent relapse in high-risk disease.

. Here we determined tumour phylogenies at diagnosis and throughout chemotherapy and immunotherapy by multiregion sequencing of 160 tumours from 65 patients. Treatment-naive SCLC exhibited clonal homogeneity at distinct tumour sites, whereas first-line platinum-based chemotherapy led to a burst in genomic intratumour heterogeneity and spatial clonal diversity. We observed branched evolution and a shift to ancestral clones underlying tumour relapse. Effective radio- or immunotherapy induced a re-expansion of founder clones with acquired genomic damage from first-line chemotherapy. Whereas TP53 and RB1 alterations were exclusively part of the common ancestor, MYC family amplifications were frequently not constituents of the founder clone. At relapse, emerging subclonal mutations affected key genes associated with SCLC biology, and tumours harbouring clonal CREBBP/EP300 alterations underwent genome duplications. Gene-damaging TP53 alterations and co-alterations of TP53 missense mutations with TP73, CREBBP/EP300 or FMN2 were significantly associated with shorter disease relapse following chemotherapy. In summary, we uncover key processes of the genomic evolution of SCLC under therapy, identify the common ancestor as the source of clonal diversity at relapse and show central genomic patterns associated with sensitivity and resistance to chemotherapy.

Novel treatment strategies for Ewing sarcoma aim to eliminate residual tumor cells that have maintained the capacity to reinitiate tumor growth after intensive conventional therapy. Preclinical models that more closely mimic in vivo tumor growth than standard monolayer cultures are needed. Sphere formation under anchorage-independent, serum-free conditions has been proposed to enrich for cells with tumor-initiating, stem cell-like properties in various solid cancers. In the present study, we assessed the phenotype and functional stem cell characteristics of Ewing sarcoma spheres. Spheres were generated under serum-free culture conditions from four Ewing sarcoma cell lines and four relapse tumor biopsies. Standard monolayer cultures were established as controls. Median levels of surface expression of the Ewing sarcoma marker CD99 as well as the supposed stem cell marker CD133 and the neural crest marker CD57 were comparable between spheres and monolayers. Ewing sarcoma spheres from individual tumors failed to continuously self-renew by secondary sphere formation. They contained variable proportions of side populations (SPs). Sphere culture did not enhance the in vivo tumorigenicity of Ewing sarcoma cells in a murine xenograft model. We conclude that sphere formation under serum-free conditions is not a reliable tool to enrich for cells with stem cell characteristics in Ewing sarcoma. By mimicking the anchorage-independent, multicellular growth of Ewing sarcoma micrometastases, in vitro sphere growth may still add value as a preclinical tool to evaluate the efficacy of novel therapeutics.

This paper aims to present a snapshot of some of the best practices in the promotion and use of renewable energy, and provide practical examples of the development of renewable energy markets that countries in Latin America and the Caribbean can replicate. This brief study provides an overview of some of the most widely used renewable energy technologies. It also examines current and potential renewable energy markets in Latin America and the Caribbean, economic development benefits of expanding renewable energy markets, policy tools and mechanisms that have been used to build and promote renewable energy in the United States, and the role governments and the private sector can play. This paper was presented at the Fifth Americas Competiveness Forum for the Inter-American Development Bank and Compete Caribbean Santo Domingo, Dominican Republic, October 5-7, 2011.

Abstract The outcome of disseminated Ewing sarcoma remains poor despite intensive multimodal treatment regimens. The disease often responds well to chemotherapy, but systemic relapses occur in the majority of patients. Targeting of residual disease by cellular immunotherapy may sustain remission and improve outcome. Specifically, Ewing sarcoma cells have been shown to be exquisitely sensitive to targeting by activated NK cells. To further explore the value of cellular strategies, preclinical models are needed that mimic the anchorage-independent, multicellular growth of Ewing sarcoma micrometastases. Here, we generated Ewing sarcoma spheres from cell lines (VH-64, TC-32, TC-71, A4573) and from four low-passage cell cultures established from Ewing sarcoma biopsies at primary diagnosis or at relapse under serum-free growth conditions. Standard monolayer cultures were used for comparisons. Phenotypic analysis revealed considerable heterogeneity among individual Ewing sarcomas and between spheres and monolayers. While the Ewing sarcoma marker CD99 as well as CD133 were expressed at comparable densities, spheres had significantly higher expression of the neural crest marker CD57 (HNK-1) and of MHC class I than monolayers, whereas CD117 (c-kit) expression was lower. Side populations characterized by Hoechst dye exclusion and previously associated with cancer stem cell function were identified in one of two primary sphere cultures and in VH-64 spheres but were absent or reduced in monolayer cultures. However, cells resuspended from spheres did not form subcutaneous tumors in immunodeficient (NOD/scid) mice at higher efficiencies than monolayer cultures, arguing against higher tumorigenicity of sphere-cultured cells. VH64 spheres were significantly more resistant towards doxorubicine than monolayers, and resuspended cells from sphere cultures remained less susceptible to lysis than monolayer cultures, but among primary tumor cells, consistent differences in chemosensitivity were not observed between the two culture systems. In vitro activated allogeneic NK cells were uniformly capable to lyse single cells derived from both monolayer and sphere cultures from established cell lines and primary cell cultures. Moreover, NK cells efficiently eliminated intact Ewing sarcoma spheres. Thus, cultured Ewing sarcoma cells are highly heterogenous. Their phenotype, function and susceptibility to both chemo- and immunotherapy differs among individual cell lines and primary cultures and under variable in vitro growth conditions. Activated NK cells efficiently target Ewing sarcoma cells both as monolayers and as spheres. The sphere model may provide a useful tool to analyze the contribution of micrometastatic architecture and serum-free niches to immune evasion. Experiments with autologous NK cells and with NK cells engineered to express tumor antigen-specific chimeric receptors are ongoing. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2503. doi:1538-7445.AM2012-2503