Musashi1 enhances chemotherapy resistance of pediatric glioblastoma cells in vitro.

BACKGROUND: Glioblastoma (GBM) is the most aggressive form of glioma in adults and children and is associated with very poor prognosis. Pediatric tumors are biologically distinct from adult GBM and differ in response to current GBM treatment protocols. Regarding pediatric GBM, new drug combinations and the molecular background of chemotherapy effects need to be investigated, in order to increase patient survival outcome. METHODS: The expression of the RNA-binding protein Musashi1 (MSI1) in pediatric glioma samples of different WHO tumor grades was investigated on the protein (immunohistochemistry) and on the RNA level (publicly accessible RNA sequencing dataset). The impact of the chemotherapeutic temozolomide (TMZ) in combination with valproic acid (VPA) was tested in two pediatric glioblastoma-derived cell lines. The supportive effect of MSI1 expression against this treatment was investigated via transient knockdown and protein overexpression. RESULTS: MSI1 expression correlates with pediatric high-grade glioma (HGG). The combination of TMZ with VPA significantly increases the impact of drug treatment on cell viability in vitro. MSI1 was found to promote drug resistance to the combined treatment with TMZ and VPA. CONCLUSION: MSI1 expression is a potential marker for pediatric HGG and increases chemoresistance. Inhibition of MSI1 might lead to an improved patient outcome and therapy response.

Occurrence of high-grade glioma in Noonan syndrome: Report of two cases.

Noonan syndrome (NS) is an autosomal dominant disorder commonly caused by PTPN11 germline mutations. Patients are characterized by short stature, congenital heart defects, facial dysmorphism, and increased risk of malignancies including brain tumors. Commonly associated brain tumors are dysembryoplastic neuroepithelial tumor and low-grade glioma. We report two cases of anaplastic astrocytoma with PTPN11-related NS. We conducted a systematic search of medical databases looking for other reported cases of high-grade glioma associated with NS and identified 24 cases of brain tumors, all of which were low-grade glial or glioneuronal tumors except for one case of medulloblastoma.

Use of nonacog beta pegol during surgery in persons with hemophilia B: a case series.

Background: Hemophilia B is a coagulation disorder that puts patients at an increased risk of bleeding. Factor (F) IX replacement therapy is traditionally used in such cases to maintain hemostasis. Nonacog beta pegol (N9-GP; Refixia) is a glycoPEGylated, extended half-life, recombinant FIX product that has demonstrated safety and efficacy when used to manage persons with hemophilia B. Key clinical question: Given the limited real-world evidence, we aimed to explore the role of N9-GP in maintaining hemostasis in persons with hemophilia B undergoing surgery. Clinical approach: In this case series, we report real-world clinical experience with N9-GP to maintain hemostasis in persons with hemophilia B undergoing major and minor surgeries. Conclusion: The majority of cases presented in this case series had an excellent or very good hemostatic response, with no reported surgical complications related to the use of N9-GP.

MSI1 Promotes the Expression of the GBM Stem Cell Marker CD44 by Impairing miRNA-Dependent Degradation.

The stem cell marker Musashi1 (MSI1) is highly expressed during neurogenesis and in glioblastoma (GBM). MSI1 promotes self-renewal and impairs differentiation in cancer and non-malignant progenitor cells. However, a comprehensive understanding of its role in promoting GBM-driving networks remains to be deciphered. We demonstrate that MSI1 is highly expressed in GBM recurrences, an oncologist's major defiance. For the first time, we provide evidence that MSI1 promotes the expression of stem cell markers like CD44, co-expressed with MSI1 within recurrence-promoting cells at the migrating front of primary GBM samples. With GBM cell models of pediatric and adult origin, including isolated primary tumorspheres, we show that MSI1 promotes stem cell-like characteristics. Importantly, it impairs CD44 downregulation in a 3'UTR- and miRNA-dependent manner by controlling mRNA turnover. This regulation is disturbed by the previously reported MSI1 inhibitor luteolin, providing further evidence for a therapeutic target potential of MSI1 in GBM treatment.

The use of Do-Not-Resuscitate-Order equivalents in pediatric palliative care medicine in Germany.

BACKGROUND: In the last years, the structure of Pediatric Palliative Care in Germany has developed more and more. Since 2007, there is a legal claim for a specialized palliative care in German which also applies to children. Therefore, the need of an advance care planning for children is frequently discussed. In Germany, a written advance is judicially approved only when the person concerned is of full age, intentions to change this legal ground are going on. Nevertheless in many institutions involved in pediatric palliative care standard forms similar to an advance patient directive are used, especially since 2009 when a Do-Not-Resuscitate-Order (DNR-Order) equivalent for children was published on which many German pediatric medical societies had agreed. METHODS: To get an overview which DNR-Order equivalents are actually used in pediatric palliative care in Germany we sent a questionnaire with 10 items to 174 institutions that are involved in pediatric oncological palliative care between August 2012 and October 2013. RESULTS: Only 46.9% of replying institutions used the DNR-Order equivalent for children approved by many German pediatric societies. When asked for optimizing such an advance patient directive for minors it was mostly suggested to include always a protocol of the consenting talk, an individualized treatment algorithm of all therapeutic options (not only emergency measures) in the palliative setting, and a more detailed information about the patient's current palliative situation. CONCLUSIONS: All collected data were summarized within a suggestion for a new advance pediatric oncological care planning standard form for minors in Germany.

Snail homolog 1 is involved in epithelial-mesenchymal transition-like processes in human glioblastoma cells.

Despite advancements in neurosurgery, chemotherapy and radiation therapy, the outcome of patients with glioblastoma remains poor. The migration of tumor cells from the primary tumor site with subsequent invasion of these cells into the surrounding normal brain tissue is frequently responsible for relapse and treatment failure. The present study hypothesized that snail homolog 1 (SNAI1), a factor critically involved in the epithelial-mesenchymal transition (EMT) of human carcinoma cells, may also contribute to an invasive EMT-like phenotype of glioblastoma cells. The majority of glioblastoma cell lines investigated in the present study expressed SNAI1 at basal levels. The present study overexpressed SNAI1 in glioblastoma cell lines by lentiviral transfer of human SNAI1 complementary DNA. In addition, the inhibition of SNAI1 expression was achieved by lentiviral transfer of a short hairpin RNA specific for SNAI1. SNAI1 overexpression increased proliferation of one of the cell lines, U251MG, but exhibited only a weak effect on the migration and invasion of glioblastoma cells. However, downregulation of SNAI1 significantly decreased the invasive capacity of all investigated cell lines. In parallel, regained expression of E-cadherin, a marker that is usually lost during EMT, was observed subsequent to SNAI1 knockdown in the glioblastoma cell lines U87MG and U251MG. The data of the present study suggest that certain key genes of the EMT in carcinoma are also involved in the migration and invasion of human glioblastoma cells.

Lenalidomide in an in vitro Dendritic Cell Model for Malignant Gliomas.

Malignant gliomas are tumors with a very unfavorable prognosis. They are characterized by rapid proliferation and invasion in the surrounding healthy tissue. Complete resection of the tumor is still the most important therapeutic option. Despite a variety of therapy modifications in the last years, long term survivors are still rare. Dendritic cell vaccination (DCV) might offer a new therapy option for the treatment of malignant gliomas. Hereby, tumorlysate pulsed dendritic cells (TPDC) can prime T cells to generate anti-tumor immune responses. Lenalidomide is an immune-modulatory piperidine-dione that has demonstrated activity especially in the treatment of hematopoietic malignancies. Here, we tested the combination of DCV and lenalidomide in an in vitro model for immunotherapy of malignant gliomas. No changes of T or NK cell subsets were observed when lenalidomide was used. In addition, interferon gamma enzyme linked immunospot (ELISPOT) showed no effects after priming of autologous peripheral blood mononuclear cells (PBMC) with TPDC and challenge with tumor cells. Although analyses of supernatants did not show higher amounts of interferon gamma and tumor necrosis factor alpha in the presence of lenalidomide, enhanced immune reaction by lenalidomide was detectable by granzyme B ELISPOT. Significantly higher numbers of spots were detected after challenge of TPDCprimed PBMC with tumor cells in the case that lenalidomide was present in the culture medium during priming. Our data suggest that the combination of DCV with lenalidomide might improve immunotherapy for malignant gliomas.

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 have been identified as co-stimulatory molecules that can augment antitumor immune responses. OX40 (CD134) and OX40 ligand (OX40L = CD252; also known as tumor necrosis factor ligand family member 4) is one example of such receptor/ligand pair with co-stimulatory function. In the present investigation, we generated OX40L transgenic Ewing sarcoma cells and tested their immunostimulatory activity in vitro. OX40L transgenic Ewing sarcoma cells showed preserved expression of Ewing sarcoma-associated (anti)gens including lipase member I, 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 the treatment of Ewing sarcoma.

Indoleamine-2,3-dioxygenase in an immunotherapy model for Ewing sarcoma.

BACKGROUND/AIM: Interleukin-2 (IL2) transgenic Ewing sarcoma cells reduce tumor growth in vivo and in vitro. In the present study we analyzed the expression of immune suppressive indoleamine-2,3-dioxygenase (IDO) in this model. MATERIALS AND METHODS: Expression of IDO was analyzed by polymerase chain reaction. The impact of the cluster of differentiation 137 (CD137)/CD137 ligand (CD137L) co-stimulatory system on expression of IDO and different cytokines was analyzed both in vivo and in vitro. RESULTS: Tumors that developed in vivo in the presence of IL2 transgenic tumor cells expressed IDO. The presence of CD137L transgenic tumor cells led to down-regulation of IDO. Further in-vitro analysis of this phenomenon indicated that IDO was expressed in tumor cells as a consequence of interferon-gamma produced by lymphocytes in response to IL2. Depending on the concentration of IL2, stimulation of CD137 increased or reduced cytokine production in lymphocytes. CONCLUSION: Our data indicate that the CD137/CD137L pathway can modulate the immune response against Ewing sarcoma cells.