Oncogenic ETS fusions promote DNA damage and proinflammatory responses via pericentromeric RNAs in extracellular vesicles.

Aberrant expression of the E26 transformation-specific (ETS) transcription factors characterizes numerous human malignancies. Many of these proteins, including EWS:FLI1 and EWS:ERG fusions in Ewing sarcoma (EwS) and TMPRSS2:ERG in prostate cancer (PCa), drive oncogenic programs via binding to GGAA repeats. We report here that both EWS:FLI1 and ERG bind and transcriptionally activate GGAA-rich pericentromeric heterochromatin. The respective pathogen-like HSAT2 and HSAT3 RNAs, together with LINE, SINE, ERV, and other repeat transcripts, are expressed in EwS and PCa tumors, secreted in extracellular vesicles (EVs), and are highly elevated in plasma of patients with EwS with metastatic disease. High human satellite 2 and 3 (HSAT2,3) levels in EWS:FLI1- or ERG-expressing cells and tumors were associated with induction of G2/M checkpoint, mitotic spindle, and DNA damage programs. These programs were also activated in EwS EV-treated fibroblasts, coincident with accumulation of HSAT2,3 RNAs, proinflammatory responses, mitotic defects, and senescence. Mechanistically, HSAT2,3-enriched cancer EVs induced cGAS-TBK1 innate immune signaling and formation of cytosolic granules positive for double-strand RNAs, RNA-DNA, and cGAS. Hence, aberrantly expressed ETS proteins derepress pericentromeric heterochromatin, yielding pathogenic RNAs that transmit genotoxic stress and inflammation to local and distant sites. Monitoring HSAT2,3 plasma levels and preventing their dissemination may thus improve therapeutic strategies and blood-based diagnostics.

TCR-transgenic T cells and YB-1-based oncolytic virotherapy improve survival in a preclinical Ewing sarcoma xenograft mouse model.

Background: Ewing sarcoma (EwS) is an aggressive and highly metastatic bone and soft tissue tumor in pediatric patients and young adults. Cure rates are low when patients present with metastatic or relapsed disease. Therefore, innovative therapy approaches are urgently needed. Cellular- and oncolytic virus-based immunotherapies are on the rise for solid cancers. Methods: Here, we assess the combination of EwS tumor-associated antigen CHM1319-specific TCR-transgenic CD8+ T cells and the YB-1-driven (i.e. E1A13S-deleted) oncolytic adenovirus XVir-N-31 in vitro and in a xenograft mouse model for antitumor activity and immunostimulatory properties. Results: In vitro both approaches specifically kill EwS cell lines in a synergistic manner over controls. This effect was confirmed in vivo, with increased survival using the combination therapy. Further in vitro analyses of immunogenic cell death and antigen presentation confirmed immunostimulatory properties of virus-infected EwS tumor cells. As dendritic cell maturation was also increased by XVir-N-31, we observed superior proliferation of CHM1319-specific TCR-transgenic CD8+ T cells only in virus-tested conditions, emphasizing the superior immune-activating potential of XVir-N-31. Conclusion: Our data prove synergistic antitumor effects in vitro and superior tumor control in a preclinical xenograft setting. Combination strategies of EwS-redirected T cells and YB-1-driven virotherapy are a highly promising immunotherapeutic approach for EwS and warrant further evaluation in a clinical setting.

Zoledronic Acid Add-on Therapy for Standard-Risk Ewing Sarcoma Patients in the Ewing 2008R1 Trial.

PURPOSE: The phase III, open-label, prospective, multicenter, randomized Ewing 2008R1 trial (EudraCT2008-003658-13) was conducted in 12 countries to evaluate the effect of zoledronic acid (ZOL) maintenance therapy compared with no add-on regarding event-free survival (EFS, primary endpoint) and overall survival (OS) in standard-risk Ewing sarcoma (EWS). PATIENTS AND METHODS: Eligible patients had localized EWS with either good histologic response to induction chemotherapy and/or small tumors (<200 mL). Patients received six cycles of VIDE induction and eight cycles of VAI (male) or eight cycles of VAC (female) consolidation. ZOL treatment started parallel to the sixth consolidation cycle. Randomization was stratified by tumor site (pelvis/other). The two-sided adaptive inverse-normal four-stage design (planned sample size 448 patients, significance level 5%, power 80%) was changed after the first interim analysis using the Müller-Schäfer method. RESULTS: Between April 2010 and November 2018, 284 patients were randomized (142 ZOL/142 no add-on). With a median follow-up of 3.9 years, EFS was not significantly different between ZOL and no add-on group in the adaptive design (HR, 0.74; 95% CI, 0.43-1.28, P = 0.27, intention-to-treat). Three-year EFS rates were 84.0% (95% CI, 77.7%-90.8%) for ZOL vs. 81.7% (95% CI, 75.2%-88.8%) for no add-on. Results were similar in the per-protocol collective. OS was not different between groups. The 3-year OS was 92.8% (95% CI, 88.4%-97.5%) for ZOL and 94.6% (95% CI, 90.9%-98.6%) for no add-on. Noticeable more renal, neurologic, and gastrointestinal toxicities were observed for ZOL (P < 0.05). Severe renal toxicities occurred more often in the ZOL arm (P = 0.003). CONCLUSIONS: In patients with standard-risk localized EWS, there is no additional benefit from maintenance treatment with ZOL.

A costimulatory chimeric antigen receptor targeting TROP2 enhances the cytotoxicity of NK cells expressing a T cell receptor reactive to human papillomavirus type 16 E7.

Immune cells modified to express a tumor-reactive T cell receptor (TCR) have shown limited efficacy as stand-alone therapy against solid tumors. Genital and oropharyngeal carcinomas induced by human papillomavirus (HPV) type 16 express constitutively its E6 and E7 oncoproteins, which makes them convenient targets for adoptive cell immunotherapy. However, viral antigen presentation by tumor cells is low and limits the anti-tumor efficacy of CD8+ T cells. To enhance the functionality of immune effector cells, we have devised a strategy combining a costimulatory chimeric antigen receptor (CAR) with a TCR. We used a clinically tested TCR specific to E7 (E7-TCR) of HPV16 and a newly constructed CAR targeting the trophoblast cell surface antigen 2 (TROP2), which carried the intracellular costimulatory domains CD28 and 4-1BB, but was devoid of the CD3ζ domain. Flow cytometry analyses showed a notable upregulation of activation markers and of cytolytic molecule release by NK-92 cells genetically engineered to express CD3, CD8 and both E7-TCR and TROP2-CAR, after co-incubation with HPV16+ cervical cancer cells. Furthermore, the E7-TCR/TROP2-CAR NK-92 cells demonstrated enhanced antigen-specific activation and augmented cytotoxicity against tumor cells compared with NK-92 cells expressing the E7-TCR alone. A costimulatory TROP2-CAR can synergistically cooperate with the E7-TCR in NK cells thereby enhancing their signaling strength and antigen-specific cytotoxicity. This approach might improve the outcome of adoptive cell immunotherapies for HPV16+ cancer patients that are currently under investigation.

The Oncolytic Adenovirus XVir-N-31 Joins Forces with CDK4/6 Inhibition Augmenting Innate and Adaptive Antitumor Immunity in Ewing Sarcoma.

PURPOSE: Ewing sarcoma (EwS) is a highly malignant pediatric tumor characterized by a non-T-cell-inflamed immune-evasive phenotype. When relapsed or metastasized, survival is poor, emphasizing the need for novel treatment strategies. Here, we analyze the novel combination approach using the YB-1-driven oncolytic adenovirus XVir-N-31 and CDK4/6 inhibition to augment EwS immunogenicity. EXPERIMENTAL DESIGN: In vitro, viral toxicity, replication, and immunogenicity were studied in several EwS cell lines. In vivo tumor xenograft models with transient humanization were applied to evaluate tumor control, viral replication, immunogenicity, and dynamics of innate as well as human T cells after treatment with XVir-N-31 combined with CDK4/6 inhibition. Furthermore, immunologic features of dendritic cell maturation and T-cell-stimulating capacities were assessed. RESULTS: The combination approach significantly increased viral replication and oncolysis in vitro, induced HLA-I upregulation, and IFNγ-induced protein 10 expression and enhanced maturation of monocytic dendritic cells with superior capacities to stimulate tumor antigen-specific T cells. These findings were confirmed in vivo showing tumor infiltration by (i) monocytes with antigen-presenting capacities and M1 macrophage marker genes, (ii) TReg suppression in spite of adenovirus infection, (iii) superior engraftment, and (iv) tumor infiltration by human T cells. Consequently, survival was improved over controls with signs of an abscopal effect after combination treatment. CONCLUSIONS: The joint forces of the YB-1-driven oncolytic adenovirus XVir-N-31 and CDK4/6 inhibition induce therapeutically relevant local and systemic antitumor effects. Innate as well as adaptive immunity against EwS is boosted in this preclinical setting, pointing toward high therapeutic potential in the clinic.

Correlation of Transcriptomics and FDG-PET SUVmax Indicates Reciprocal Expression of Stemness-Related Transcription Factor and Neuropeptide Signaling Pathways in Glucose Metabolism of Ewing Sarcoma.

BACKGROUND: In Ewing sarcoma (EwS), long-term treatment effects and poor survival rates for relapsed or metastatic cases require individualization of therapy and the discovery of new treatment methods. Tumor glucose metabolic activity varies significantly between patients, and FDG-PET signals have been proposed as prognostic factors. However, the biological basis for the generally elevated but variable glucose metabolism in EwS is not well understood. METHODS: We retrospectively included 19 EwS samples (17 patients). Affymetrix gene expression was correlated with maximal standardized uptake value (SUVmax) using machine learning, linear regression modelling, and gene set enrichment analyses for functional annotation. RESULTS: Expression of five genes correlated (MYBL2, ELOVL2, NETO2) or anticorrelated (FAXDC2, PLSCR4) significantly with SUVmax (adjusted p-value ≤ 0.05). Additionally, we identified 23 genes with large SUVmax effect size, which were significantly enriched for "neuropeptide Y receptor activity (GO:0004983)" (adjusted p-value = 0.0007). The expression of the members of this signaling pathway (NPY, NPY1R, NPY5R) anticorrelated with SUVmax. In contrast, three transcription factors associated with maintaining stemness displayed enrichment of their target genes with higher SUVmax: RNF2, E2F family, and TCF3. CONCLUSION: Our large-scale analysis examined comprehensively the correlations between transcriptomics and tumor glucose utilization. Based on our findings, we hypothesize that stemness may be associated with increased glucose uptake, whereas neuroectodermal differentiation may anticorrelate with glucose uptake.

T Cells Directed against the Metastatic Driver Chondromodulin-1 in Ewing Sarcoma: Comparative Engineering with CRISPR/Cas9 vs. Retroviral Gene Transfer for Adoptive Transfer.

Ewing sarcoma (EwS) is a highly malignant sarcoma of bone and soft tissue with early metastatic spread and an age peak in early puberty. The prognosis in advanced stages is still dismal, and the long-term effects of established therapies are severe. Efficacious targeted therapies are urgently needed. Our previous work has provided preliminary safety and efficacy data utilizing T cell receptor (TCR) transgenic T cells, generated by retroviral gene transfer, targeting HLA-restricted peptides on the tumor cell derived from metastatic drivers. Here, we compared T cells engineered with either CRISPR/Cas9 or retroviral gene transfer. Firstly, we confirmed the feasibility of the orthotopic replacement of the endogenous TCR by CRISPR/Cas9 with a TCR targeting our canonical metastatic driver chondromodulin-1 (CHM1). CRISPR/Cas9-engineered T cell products specifically recognized and killed HLA-A*02:01+ EwS cell lines. The efficiency of retroviral transduction was higher compared to CRISPR/Cas9 gene editing. Both engineered T cell products specifically recognized tumor cells and elicited cytotoxicity, with CRISPR/Cas9 engineered T cells providing prolonged cytotoxic activity. In conclusion, T cells engineered with CRISPR/Cas9 could be feasible for immunotherapy of EwS and may have the advantage of more prolonged cytotoxic activity, as compared to T cells engineered with retroviral gene transfer.

No Improvement of Survival for Alveolar Rhabdomyosarcoma Patients After HLA-Matched Versus -Mismatched Allogeneic Hematopoietic Stem Cell Transplantation Compared to Standard-of-Care Therapy.

Background: Patients with stage IV alveolar rhabdomyosarcoma (RMA) have a 5-year-survival rate not exceeding 30%. Here, we assess the role of allogeneic hematopoietic stem cell transplantation (allo-HSCT) for these patients in comparison to standard-of-care regimens. We also compare the use of HLA-mismatched vs. HLA-matched grafts after reduced vs. myeloablative conditioning regimens, respectively. Patients and Methods: In this retrospective analysis, we compare event-free survival (EFS), overall survival (OS), and toxicity of HLA-mismatched vs. -matched transplanted patients in uni- and multivariate analyses (total: n = 50, HLA-matched: n = 15, HLA-mismatched: n = 35). Here, the factors age at diagnosis, age at allo-HSCT, sex, Oberlin score, disease status at allo-HSCT, and HLA graft type are assessed. For 29 primarily transplanted patients, three matched non-transplanted patients per one transplanted patient were identified from the CWS registry. Outcomes were respectively compared for OS and EFS. Matching criteria included sex, age at diagnosis, favorable/unfavorable primary tumor site, and metastatic sites. Results: Median EFS and OS did not differ significantly between HLA-mismatched and -matched patients. In the mismatched group, incidence of acute GvHD was 0.87 (grade III-IV: 0.14) vs. 0.80 in HLA-matched patients (grade III-IV: 0.20). Transplant-related mortality (TRM) of all patients was 0.20 and did not differ significantly between HLA-mismatched and -matched groups. A proportion of 0.58 relapsed or progressed and died of disease (HLA-mismatched: 0.66, HLA-matched: 0.53) whereas 0.18 were alive in complete remission (CR) at data collection. Multivariate and competing risk analyses confirmed CR and very good partial response (VGPR) status prior to allo-HSCT as the only decisive predictor for OS (p < 0.001). Matched-pair survival analyses of primarily transplanted patients vs. matched non-transplanted patients also identified disease status prior to allo-HSCT (CR, VGPR) as the only significant predictor for EFS. Here, OS was not affected, however. Conclusion: In this retrospective analysis, only a subgroup of patients with good response at allo-HSCT survived. There was no survival benefit of allo-transplanted patients compared to matched controls, suggesting the absence of a clinically relevant graft-versus-RMA effect in the current setting. The results of this analysis do not support further implementation of allo-HSCT in RMA stage IV patients.

High-Dose Treosulfan and Melphalan as Consolidation Therapy Versus Standard Therapy for High-Risk (Metastatic) Ewing Sarcoma.

PURPOSE: Ewing 2008R3 was conducted in 12 countries and evaluated the effect of treosulfan and melphalan high-dose chemotherapy (TreoMel-HDT) followed by reinfusion of autologous hematopoietic stem cells on event-free survival (EFS) and overall survival in high-risk Ewing sarcoma (EWS). METHODS: Phase III, open-label, prospective, multicenter, randomized controlled clinical trial. Eligible patients had disseminated EWS with metastases to bone and/or other sites, excluding patients with only pulmonary metastases. Patients received six cycles of vincristine, ifosfamide, doxorubicin, and etoposide induction and eight cycles of vincristine, actinomycin D, and cyclophosphamide consolidation therapy. Patients were randomly assigned to receive additional TreoMel-HDT or no further treatment (control). The random assignment was stratified by number of bone metastases (1, 2-5, and > 5). The one-sided adaptive-inverse-normal-4-stage-design was changed after the first interim analysis via Müller-Schäfer method. RESULTS: Between 2009 and 2018, 109 patients were randomly assigned, and 55 received TreoMel-HDT. With a median follow-up of 3.3 years, there was no significant difference in EFS between TreoMel-HDT and control in the adaptive design (hazard ratio [HR] 0.85; 95% CI, 0.55 to 1.32, intention-to-treat). Three-year EFS was 20.9% (95% CI, 11.5 to 37.9) in TreoMel-HDT and 19.2% (95% CI, 10.8 to 34.4) in control patients. The results were similar in the per-protocol collective. Males treated with TreoMel-HDT had better EFS compared with controls: median 1.0 years (95% CI, 0.8 to 2.2) versus 0.6 years (95% CI, 0.5 to 0.9); P = .035; HR 0.52 (0.28 to 0.97). Patients age < 14 years benefited from TreoMel-HDT with a 3-years EFS of 39.3% (95% CI, 20.4 to 75.8%) versus 9% (95% CI, 2.4 to 34); P = .016; HR 0.40 (0.19 to 0.87). These effects were similar in the per-protocol collective. This observation is supported by comparable results from the nonrandomized trial EE99R3. CONCLUSION: In patients with very high-risk EWS, additional TreoMel-HDT was of no benefit for the entire cohort of patients. TreoMel-HDT may be of benefit for children age < 14 years.

MondoA drives malignancy in B-ALL through enhanced adaptation to metabolic stress.

Cancer cells are in most instances characterized by rapid proliferation and uncontrolled cell division. Hence, they must adapt to proliferation-induced metabolic stress through intrinsic or acquired antimetabolic stress responses to maintain homeostasis and survival. One mechanism to achieve this is reprogramming gene expression in a metabolism-dependent manner. MondoA (also known as Myc-associated factor X-like protein X-interacting protein [MLXIP]), a member of the MYC interactome, has been described as an example of such a metabolic sensor. However, the role of MondoA in malignancy is not fully understood and the underlying mechanism in metabolic responses remains elusive. By assessing patient data sets, we found that MondoA overexpression is associated with worse survival in pediatric common acute lymphoblastic leukemia (ALL; B-precursor ALL [B-ALL]). Using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) and RNA-interference approaches, we observed that MondoA depletion reduces the transformational capacity of B-ALL cells in vitro and dramatically inhibits malignant potential in an in vivo mouse model. Interestingly, reduced expression of MondoA in patient data sets correlated with enrichment in metabolic pathways. The loss of MondoA correlated with increased tricarboxylic acid cycle activity. Mechanistically, MondoA senses metabolic stress in B-ALL cells by restricting oxidative phosphorylation through reduced pyruvate dehydrogenase activity. Glutamine starvation conditions greatly enhance this effect and highlight the inability to mitigate metabolic stress upon loss of MondoA in B-ALL. Our findings give novel insight into the function of MondoA in pediatric B-ALL and support the notion that MondoA inhibition in this entity offers a therapeutic opportunity and should be further explored.

Surgical treatment of a spontaneous spinal epidural hematoma under antithrombotic treatment of a multisystem inflammatory syndrome in children associated with SARS-COV-2.

Multisystem inflammatory syndrome in children (MIS-C) is a novel syndrome of multisystemic inflammation affecting children. This case report documents an exceptional and severe complication of an epidural hematoma in a 3-year-old boy under the treatment of MIS-C. During the course of the disease, the patient suffered from a hypocoagulable state and an extensive multisegmental epidural hematoma in the cervical spinal canal. This led to severe anterior spinal cord compression and tetraparesis. Extensive emergency surgery had to be carried out to reverse rapid clinical deterioration.

Current State of Immunotherapy and Mechanisms of Immune Evasion in Ewing Sarcoma and Osteosarcoma.

We argue here that in many ways, Ewing sarcoma (EwS) is a unique tumor entity and yet, it shares many commonalities with other immunologically cold solid malignancies. From the historical perspective, EwS, osteosarcoma (OS) and other bone and soft-tissue sarcomas were the first types of tumors treated with the immunotherapy approach: more than 100 years ago American surgeon William B. Coley injected his patients with a mixture of heat-inactivated bacteria, achieving survival rates apparently higher than with surgery alone. In contrast to OS which exhibits recurrent somatic copy-number alterations, EwS possesses one of the lowest mutation rates among cancers, being driven by a single oncogenic fusion protein, most frequently EWS-FLI1. In spite these differences, both EwS and OS are allied with immune tolerance and low immunogenicity. We discuss here the potential mechanisms of immune escape in these tumors, including low representation of tumor-specific antigens, low expression levels of MHC-I antigen-presenting molecules, accumulation of immunosuppressive M2 macrophages and myeloid proinflammatory cells, and release of extracellular vesicles (EVs) which are capable of reprogramming host cells in the tumor microenvironment and systemic circulation. We also discuss the vulnerabilities of EwS and OS and potential novel strategies for their targeting.

DNA methylation-based classifier and gene expression signatures detect BRCAness in osteosarcoma.

Although osteosarcoma (OS) is a rare cancer, it is the most common primary malignant bone tumor in children and adolescents. BRCAness is a phenotypical trait in tumors with a defect in homologous recombination repair, resembling tumors with inactivation of BRCA1/2, rendering these tumors sensitive to poly (ADP)-ribose polymerase inhibitors (PARPi). Recently, OS was shown to exhibit molecular features of BRCAness. Our goal was to develop a method complementing existing genomic methods to aid clinical decision making on administering PARPi in OS patients. OS samples with DNA-methylation data were divided to BRCAness-positive and negative groups based on the degree of their genomic instability (n = 41). Methylation probes were ranked according to decreasing variance difference between two groups. The top 2000 probes were selected for training and cross-validation of the random forest algorithm. Two-thirds of available OS RNA-Seq samples (n = 17) from the top and bottom of the sample list ranked according to genome instability score were subjected to differential expression and, subsequently, to gene set enrichment analysis (GSEA). The combined accuracy of trained random forest was 85% and the average area under the ROC curve (AUC) was 0.95. There were 449 upregulated and 1,079 downregulated genes in the BRCAness-positive group (fdr < 0.05). GSEA of upregulated genes detected enrichment of DNA replication and mismatch repair and homologous recombination signatures (FWER < 0.05). Validation of the BRCAness classifier with an independent OS set (n = 20) collected later in the course of study showed AUC of 0.87 with an accuracy of 90%. GSEA signatures computed for this test set were matching the ones observed in the training set enrichment analysis. In conclusion, we developed a new classifier based on DNA-methylation patterns that detects BRCAness in OS samples with high accuracy. GSEA identified genome instability signatures. Machine-learning and gene expression approaches add new epigenomic and transcriptomic aspects to already established genomic methods for evaluation of BRCAness in osteosarcoma and can be extended to cancers characterized by genome instability.

Monocyte Maturation Mediators Upregulate CD83, ICAM-1 and MHC Class 1 Expression on Ewing's Sarcoma, Enhancing T Cell Cytotoxicity.

Ewing's sarcoma (EwS) is a pediatric solid tumor entity with low somatic mutational burden and a low rate of tumor-infiltrating T cells, indicating a low extent of immunogenicity. In EwS, immunogenicity may furthermore be significantly diminished by a predominantly M2 macrophage driven pro-tumorigenic tumor microenvironment. In the past, we demonstrated that CHM1319-specific TCR-transgenic T cells are able to control EwS growth in a preclinical mouse model as well as in a patient with metastatic disease. However, new adjuvant techniques to induce long lasting and curative CHM1319-specific TCR-transgenic T cell-mediated anti-tumor responses are needed. In this work, we sought to identify a technique to improve the cytotoxic effect of CHM1319-specific TCR-transgenic T cell by altering the immunogenic cell surface marker expression on EwS cell lines using different cytokines. We demonstrate that TNF, IL-6, IL-1ß and PGE2 cause pro-immunogenic CD83, MHC class I and II as well as ICAM-1 upregulation in EwS cell lines. This observation was associated with significantly improved recognition and killing of the tumor cells by EwS-specific CHM1319/HLA-A*02:01-restricted TCR-transgenic T cells. Conclusively, we demonstrate that the induction of an inflammatory signature renders EwS more susceptible to adoptive T cell therapy. TNF, which is upregulated during inflammatory processes, is of particular translational interest as its secretion may be induced in the patients e.g., by irradiation and hyperthermia in the clinical setting. In future clinical protocols, this finding may be important to identify appropriate conditioning regimens as well as point of time for adoptive T cell-based immunotherapy in EwS patients.

Class I histone deacetylases (HDAC) critically contribute to Ewing sarcoma pathogenesis.

BACKGROUND: Histone acetylation and deacetylation seem processes involved in the pathogenesis of Ewing sarcoma (EwS). Here histone deacetylases (HDAC) class I were investigated. METHODS: Their role was determined using different inhibitors including TSA, Romidepsin, Entinostat and PCI-34051 as well as CRISPR/Cas9 class I HDAC knockouts and HDAC RNAi. To analyze resulting changes microarray analysis, qRT-PCR, western blotting, Co-IP, proliferation, apoptosis, differentiation, invasion assays and xenograft-mouse models were used. RESULTS: Class I HDACs are constitutively expressed in EwS. Patients with high levels of individual class I HDAC expression show decreased overall survival. CRISPR/Cas9 class I HDAC knockout of individual HDACs such as HDAC1 and HDAC2 inhibited invasiveness, and blocked local tumor growth in xenograft mice. Microarray analysis demonstrated that treatment with individual HDAC inhibitors (HDACi) blocked an EWS-FLI1 specific expression profile, while Entinostat in addition suppressed metastasis relevant genes. EwS cells demonstrated increased susceptibility to treatment with chemotherapeutics including Doxorubicin in the presence of HDACi. Furthermore, HDACi treatment mimicked RNAi of EZH2 in EwS. Treated cells showed diminished growth capacity, but an increased endothelial as well as neuronal differentiation ability. HDACi synergizes with EED inhibitor (EEDi) in vitro and together inhibited tumor growth in xenograft mice. Co-IP experiments identified HDAC class I family members as part of a regulatory complex together with PRC2. CONCLUSIONS: Class I HDAC proteins seem to be important mediators of the pathognomonic EWS-ETS-mediated transcription program in EwS and in combination therapy, co-treatment with HDACi is an interesting new treatment opportunity for this malignant disease.

Ewing Sarcoma-Derived Extracellular Vesicles Impair Dendritic Cell Maturation and Function.

Ewing sarcoma (EwS) is an aggressive pediatric cancer of bone and soft tissues characterized by scant T cell infiltration and predominance of immunosuppressive myeloid cells. Given the important roles of extracellular vesicles (EVs) in cancer-host crosstalk, we hypothesized that EVs secreted by EwS tumors target myeloid cells and promote immunosuppressive phenotypes. Here, EVs were purified from EwS and fibroblast cell lines and exhibited characteristics of small EVs, including size (100-170 nm) and exosome markers CD63, CD81, and TSG101. Treatment of healthy donor-derived CD33+ and CD14+ myeloid cells with EwS EVs but not with fibroblast EVs induced pro-inflammatory cytokine release, including IL-6, IL-8, and TNF. Furthermore, EwS EVs impaired differentiation of these cells towards monocytic-derived dendritic cells (moDCs), as evidenced by reduced expression of co-stimulatory molecules CD80, CD86 and HLA-DR. Whole transcriptome analysis revealed activation of gene expression programs associated with immunosuppressive phenotypes and pro-inflammatory responses. Functionally, moDCs differentiated in the presence of EwS EVs inhibited CD4+ and CD8+ T cell proliferation as well as IFNγ release, while inducing secretion of IL-10 and IL-6. Therefore, EwS EVs may promote a local and systemic pro-inflammatory environment and weaken adaptive immunity by impairing the differentiation and function of antigen-presenting cells.

The Truncated Splice Variant of the Granulocyte-Macrophage-Colony-Stimulating Factor Receptor ß- Chain in Peripheral Blood Serves as Severity Biomarker of Respiratory Failure in Newborns.

BACKGROUND: The granulocyte-macrophage-colony-stimulating factor (GM-CSF) plays an important role in surfactant homeostasis. ßC is a subunit of the GM-CSF receptor (GM-CSF-R), and its activation mediates surfactant catabolism in the lung. ßIT is a physiological, truncated isoform of ßC and is known to act as physiological inhibitor of ßC. OBJECTIVE: The aim of this study was to determine the ratio of ßIT and ßC in the peripheral blood of newborns and its association with the degree of respiratory failure at birth. METHODS: We conducted a prospective cohort study in newborns with various degrees of respiratory impairment at birth. Respiratory status was assessed by a score ranging from no respiratory impairment (0) to invasive respiratory support (3). ßIT and ßC expression were determined in peripheral blood cells by real-time PCR. ßIT expression, defined as the ratio of ßIT and ßC, was correlated with the respiratory score. RESULTS: ßIT expression was found in all 59 recruited newborns with a trend toward higher ßIT in respiratory ill (score 2, 3) newborns than respiratory healthy newborns ([score 0, 1]; p = 0.066). Seriously ill newborns (score 3) had significantly higher ßIT than healthy newborns ([score 0], p = 0.010). Healthy preterm infants had significantly higher ßIT expression than healthy term infants (p = 0.019). CONCLUSIONS: ßIT is expressed in newborns with higher expression in respiratory ill than respiratory healthy newborns. We hypothesize that ßIT may have a protective effect in postnatal pulmonary adaptation acting as a physiological inhibitor of ßC and, therefore, maintaining surfactant in respiratory ill newborns.

Sarcoma treatment in the era of molecular medicine.

Sarcomas are heterogeneous and clinically challenging soft tissue and bone cancers. Although constituting only 1% of all human malignancies, sarcomas represent the second most common type of solid tumors in children and adolescents and comprise an important group of secondary malignancies. More than 100 histological subtypes have been characterized to date, and many more are being discovered due to molecular profiling. Owing to their mostly aggressive biological behavior, relative rarity, and occurrence at virtually every anatomical site, many sarcoma subtypes are in particular difficult-to-treat categories. Current multimodal treatment concepts combine surgery, polychemotherapy (with/without local hyperthermia), irradiation, immunotherapy, and/or targeted therapeutics. Recent scientific advancements have enabled a more precise molecular characterization of sarcoma subtypes and revealed novel therapeutic targets and prognostic/predictive biomarkers. This review aims at providing a comprehensive overview of the latest advances in the molecular biology of sarcomas and their effects on clinical oncology; it is meant for a broad readership ranging from novices to experts in the field of sarcoma.