Background O6-methylguanine-DNA-methyltransferase (MGMT) promoter methylation status is a predictive biomarker in glioblastoma. We investigated whether this marker furthermore defines a molecularly distinct tumor subtype with clinically different outcome. Methods We analyzed copy number variation (CNV) and methylation profiles of 1095 primary and 92 progressive isocitrate dehydrogenase wildtype glioblastomas, including paired samples from 49 patients. DNA mutation data from 182 glioblastoma samples of The Cancer Genome Atlas (TCGA) and RNA expression from 107 TCGA and 55 Chinese Glioma Genome Atlas samples were analyzed. Results Among untreated glioblastomas, MGMT promoter methylated (mMGMT) and unmethylated (uMGMT) tumors did not show different CNV or specific gene mutations, but a higher mutation count in mMGMT tumors. We identified 3 methylation clusters. Cluster 1 showed the highest average methylation and was enriched for mMGMT tumors. Seventeen genes including gastrulation brain homeobox 2 (GBX2) were found to be hypermethylated and downregulated on the mRNA level in mMGMT tumors. In progressive glioblastomas, platelet derived growth factor receptor alpha (PDGFRA) and GLI2 amplifications were enriched in mMGMT tumors. Methylated MGMT tumors gain PDGFRA amplification of PDGFRA, whereas uMGMT tumors with amplified PDGFRA frequently lose this amplification upon progression. Glioblastoma patients surviving <6 months and with mMGMT harbored less frequent epidermal growth factor receptor (EGFR) amplifications, more frequent TP53 mutations, and a higher tumor necrosis factor-nuclear factor-kappaB (TNF-NFκB) pathway activation compared with patients surviving >12 months. Conclusions MGMT promoter methylation status does not define a molecularly distinct glioblastoma subpopulation among untreated tumors. Progressive mMGMT glioblastomas and mMGMT tumors of patients with short survival tend to have more unfavorable molecular profiles.

Background: Checkpoint inhibitors for cancer immunotherapy have been shown to unleash a subdued endogenous T-cell response against mutated tumor antigens, so-called neoepitopes. However, tumors with a low burden of non-synonymous mutations - including gliomas - are poorly presented to the immune system, and therefore unlikely to respond to checkpoint inhibition alone. It is therefore important to identify the signature of relevant immunogenic neoepitopes and the T cell receptors (TCRs) recognizing these epitopes to employ a more specific immunotherapy. Limited availability of tumor-infiltrating lymphocytes (TILs) in turn prompts the identification of neoepitope-specific T-cells in peripheral blood. Aim: This study aimed at establishing a platform for the identification of immunogenic neoepitopes and corresponding neoepitope-specific T cells in the peripheral blood of glioma patients. Methods and Results: Putative neoepitopes were predicted from next generation sequencing mutanome data of tumor samples from three glioma patients. Patient-specific neoepitopes were synthesized as codon-optimized tandem mini gene vector (TMG) optimized for neoepitope presentation, with up to 10 neoeptiopes per TMG. Autologous PBMC-derived dendritic cells were transfected with in vitro transcribed TMG RNA or loaded with peptides to test T-cell neoepitope-reactivity in co-culture assays. On probing the reactivity of T-cells from peripheral blood, we found patient-specific reactivity against neoepitopes. Conclusion and scope: We investigated the T-cell reactivity towards patient specific neoepitopes in the peripheral blood of 3 glioma patients and the phenotypic identity of these neoepitope-reactive T-cells. We are currently probing CD8+ T-cell subpopulations to characterize the neoepitope-reactive T-cell phenotype. Ongoing studies aim to further characterize the neoepitope-specific T-cells with respect to their TCR sequences and expand this platform to a larger cohort of glioma patients to provide a proof-of-principle for a minimally invasive approach to identify tumor-reactive T-cells for personalized glioma immunotherapy.

BACKGROUND: Malignant gliomas belong to the most aggressive neoplasms in humans. The interactions and intercellular communication between malignant and non-malignant cells in the tumor microenvironment are tumor-promoting and critically to improve the understanding of the disease. N-myc downstream regulated gene 1 (NDRG1) is a stress inducible gene and key determinant of resistance towards alkylating chemotherapy in glioblastoma. NDRG1 promotes invasive growth and acts antiapoptotic in hepatocellular carcinoma. In human pancreatic cancer cells NDRG1 suppresses chemokine expression, decreases macrophage infiltration and microvascular density. Further understanding the influence of NDRG1 on the tumor microenvironment may lead to new therapeutic approaches in glioma. METHODS: To analyze the NDRG1 effects on the glioma microenvironment, we used human and murine NDRG1 knockdown (KD) cells. In orthotopic xenograft experiments control and KD microenvironments were compared for infiltrating immune cells dependent on NDRG1. Interaction and signaling mechanisms were evaluated in vitro using western blots, cytokine and migration assays and treatment with the NF-kB inhibitor JSH-23 as well as SGK1 inhibitor EMD638683 to assess the influence of NDRG1. Results: Orthotopic NDRG1 KD tumors were significantly larger compared to control tumors. Ex vivo flow cytometry analyses of the tumor microenvironment revealed a significant increase in peripheral macrophages, dendritic cells and monocytic myeloid derived suppressor cells in NDRG1 KD tumors. In vitro cytokine array analyses showed an increase of the chemokine CCL2 in NDRG1 KD cells compared to control cells. This was confirmed by ELISA. Macrophages showed an increased migration rate in the NDRG1 knockdown environment. To analyze molecular mechanisms CCL2 secretion was blocked with a NF-κB inhibitor (JSH-23) and stimulated with a SGK1 inhibitor (EMD638683). We found that NDRG1 induced NF-κB signaling attenuation is mediated through decreases in IKK α expression and IκBα phosphorylation. CONCLUSION: We identified CCL2 as a NDRG1-induced NF-κB target gene that triggers macrophage recruitment. Our findings support an immunomodulatory effect of NDRG1 on the tumor microenvironment and provide novel insights into the biological relevance of the tumor stroma and the function of NDRG1 with important implications for the development of cancer therapeutics.

Background: Dysregulation of p53 pathway is common in glioma. Frequent causes are p53 mutations or overexpression of the oncogene MDM2 in p53 wildtype tumors. Idasanutlin is a MDM2 inhibitor, which is currently under investigation for multiple myeloma and acute myeloid leukemia, but of principal interest also for other p53 mutated solid tumors. Therefore, preclinical data and molecular understanding about treatment effects and resistance mechanisms are important. Methods: Treatment effects of idasanutlin monotherapy and combination with radiotherapy were analyzed on cell viability, proliferation and clonogenicity in p53 wildtype cell lines (U87MG, A172) and glioma-initiating cells (GICs, S24, T1) at radiation doses of 2 and 4 Gray (photons). Toxicity assays were performed on freshly isolated murine astrocytes and cerebellum granule neurons. Idasanutlin resistant cells were generated by treatment of U87MG cells with increasing doses of idasanutlin for 3 months. Resistance mechanisms were analyzed by microarray analysis and confirmed via qRT-PCR, immuno blots and treatment with the nuclear factor (NF)kB inhibitor JSH-23 and transient knockdown of insulin-like growth factor binding protein (IGFBP)1 via siRNA. Results: Idasanutlin reduced cell viability and clonogenicity in p53 wildtype glioblastoma cell lines and GICs. Combined treatment with radiotherapy showed synergistic effects on clonogenicity and proliferation at clinically relevant doses without toxicity on normal brain cells. Long-term treatment with idasanutlin resulted in resistance against the drug in U87MG cells and led to a more clonogenic, invasive and proliferative phenotype of the cells. Microarray analysis revealed an activation of NFkB and ERK1/2 pathway and an upregulation of IGFBP1 expression in idasanutlin resistant cells. While radiotherapy did not overcome resistance conferred by idasanutlin, inhibition of NFkB pathway by JSH23 treatment and transient knockdown of IGFBP1 by siRNA partly restored sensitivity towards idasanutlin. Transient knockdown of IGFBP1 additionally reduced the invasiveness of idasanutlin resistant cells. Conclusions: Combination of idasanutlin treatment and radiotherapy showed synergistic effects on glioblastoma cells in a clinically relevant setting. However, long-term treatment with idasanutlin resulted in resistance against the drug mediated by activation of NFkB pathway and upregulation of IGFBP1. Targeting these pathways may provide strategies to overcome idasanutlin resistance.

Influencing cancer metabolism by lifestyle changes is an attractive strategy as - if effective - exercise-induced problems may be less severe than those induced by classical anti-cancer therapies. Pursuing this idea, clinical trials evaluated the benefit of e.g. different diets such as the ketogenic diet, intermittent caloric restriction and physical exercise (PE) in the primary and secondary prevention of different cancer types. PE proved to be beneficial in the context of breast and colon cancer.Glioblastoma has a dismal prognosis, with an average overall survival of about one year despite maximal safe resection, concomitant radiochemotherapy with temozolomide followed by adjuvant temozolomide therapy. Here, we focused on the influence of PE as an isolated and adjuvant treatment in murine GB therapy.PE did not reduce toxic side effects of chemotherapy in mice administered in a dose escalating scheme as shown before for starvation. Although regular treadmill training on its own had no obvious beneficial effects, its combination with temozolomide was beneficial in the treatment of glioblastoma-bearing mice. As PE might partly act through the induction of reactive oxygen species, dihydroartemisinin - an approved anti-malarial drug which induces oxidative stress in glioma cells - was further evaluated in vitro and in vivo. Dihydroartemisinin showed anti-glioma activity by promoting autophagy, reduced the clonogenic survival and proliferation capacity of glioma cells, and prolonged the survival of tumor bearing mice. Using the reactive oxygen species scavenger n-acetyl-cysteine these effects were in part reversible, suggesting that dihydroartemisinin partly acts through the generation of reactive oxygen species.

Neurological complications of cancer and of anticancer treatments can be substantially disabling to patients, especially with classic chemotherapies. As a rare but important complication, targeted therapies might also result in similar unwanted effects, partly because inhibition of VEGF is a common downstream effect. Therapeutic antibodies, such as the CD20-depleting antibody rituximab, and underlying haematological malignancies, can induce long-lasting cellular immunosuppression, predisposing patients to opportunistic CNS infections, such as progressive multifocal leukoencephalopathy, where treatment-induced recovery can result in severe reconstitution of immune inflammatory syndromes of the central nervous system. Immune-related neurological adverse events, particularly from immune-activating checkpoint inhibitors, occur as a result of immune activation, resulting in organ-specific autoimmune-like disease. The prevalence of immune-related neurological adverse events might only be about 1%-a low prevalence compared with toxicities in other organs-but it constitutes new patterns of neurological toxic forms, which could result in considerable morbidity and fatal outcomes. Clinicians should be aware of treatment-associated neurotoxicity, and consider discontinuation of the drug with parallel supportive measures to help patients.

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The outcome of patients with anaplastic gliomas varies considerably depending on single molecular markers, such as mutations of the isocitrate dehydrogenase (IDH) genes, as well as molecular classifications based on epigenetic or genetic profiles. Remarkably, 98% of the RNA within a cell is not translated into proteins. Of those, especially microRNAs (miRNAs) have been shown not only to have a major influence on physiologic processes but also to be deregulated and prognostic in malignancies.To find novel survival markers and treatment options we performed unbiased DNA methylation screens that revealed 12 putative miRNA promoter regions with differential DNA methylation in anaplastic gliomas. Methylation of these candidate regions was validated in different independent patient cohorts revealing a set of miRNA promoter regions with prognostic relevance across data sets. Of those, miR-155 promoter methylation and miR-155 expression were negatively correlated and especially the methylation showed superior correlation with patient survival compared to established biomarkers.Functional examinations in malignant glioma cells further cemented the relevance of miR-155 for tumor cell viability with transient and stable modifications indicating an onco-miRNA activity. MiR-155 also conferred resistance towards alkylating temozolomide and radiotherapy as consequence of nuclear factor (NF)κB activation.Preconditioning glioma cells with an NFκB inhibitor reduced therapy resistance of miR-155 overexpressing cells. These cells resembled tumors with a low methylation of the miR-155 promoter and thus mir-155 or NFκB inhibition may provide treatment options with a special focus on patients with IDH wild type tumors.

TPS2084 Background: Patients with newly diagnosed glioblastoma without O6-methyl guanine O6-methylatransferase (MGMT) promoter methylation do not benefit from alkylating therapy. Clinical trials aiming at replacing temozolomide (TMZ) with targeted agents in unselected patient populations have failed to demonstrate any benefit. Advances in the understanding of glioblastoma at a molecular level along with technological progress have led to the identification of key genetic alterations in a timely manner allowing for treatment decisions in newly diagnosed patients. In this setting, these alterations not only refine the sub-classification of glioblastoma, but also allow for subset specific treatments, with contemporary analyses allowing for differentiating prognostic and predictive biomarkers. Methods: N2M2 is an open label phase I/II umbrella trial for patients with newly diagnosed glioblastoma without MGMT promoter methylation to show safety, feasibility and preliminary efficacy (decision for future randomized phase II/III) of treatment with targeted compounds in addition to radiotherapy based on thorough molecular characterization. N2M2 is formally divided into a DISCOVERY and a TREATMENT aspect. DISCOVERY includes the use of panel, whole exome and transcriptome sequencing, methylome analysis using Illumina EPIC arrays, and gene expression arrays to find new, unexpected targets and get a comprehensive view on affected pathways, with dedicated bioinformatics evaluation (GUIDE), a Molecular Tumor Board and a timely initiation (≤ 4 weeks) of the postoperative treatment. Stratification for TREATMENT takes place in seven subtrials according to the best matching molecular alteration. For the phase I parts, a maximum of 9 patients will be enrolled in each cohort. A Bayesian criterion is used for continuous monitoring of toxicity. In the phase II trials, progression-free survival at six months is used as endpoint for efficacy. Patients with no matching alteration receive the standard chemoradiotherapy with TMZ and serve as contemporary, non-randomized controls with an estimated PFS6 of 40%.

Bevacizumab is frequently used in patients with progressive glioblastoma raising questions regarding frequency of treatments, dosage, duration of therapy and the possibility of tapering and discontinuation for selected patient groups. We retrospectively assessed the safety and outcome of tapering and discontinuation of bevacizumab therapy for reasons other than disease progression and toxicity in 19 patients with progressive glioblastoma receiving bevacizumab for at least 6 months. In 10 of the 19 patients tapering bevacizumab resulted in complete discontinuation and reinitiation after disease progression during halted treatment. As a comparison group 33 patients with bevacizumab for at least 6 months continuously dosed at 10 mg/kg every 2 weeks were selected. Age and Karnofsky performance status at start of bevacizumab were similar in both groups. Influenced by the selection process, progression-free survival (PFS) and overall survival (OS) were longer in the group receiving a tapered and discontinued bevacizumab regimen (PFS 22.7 versus 11.2 months, HR 0.33, p-value = 0.01; OS 29.9 versus 15.5 months, HR 0.22, p-value = 0.001) with a median time of discontinuation of 4.5 months (range: 1.9–44.2 months). Stable disease or partial response according to RANO at ≥3 months was achieved in 89 % of patients with reinitiated bevacizumab therapy after discontinuation. These data indicate that tapering and discontinuation of bevacizumab therapy for other reasons than progression is feasible without an increased risk for tumor rebound or unresponsiveness to reinitiated bevacizumab therapy.

BACKGROUND:The purpose of this study was to determine the relevance of clinical data, apparent diffusion coefficient (ADC), and relative cerebral blood volume (rCBV) from dynamic susceptibility contrast (DSC) perfusion and the volume transfer constant (ktrans) from dynamic contrast-enhanced (DCE) perfusion for predicting overall survival (OS) and progression-free survival (PFS) in newly diagnosed treatment-naïve glioblastoma patients.METHODS:Preoperative MR scans including standardized contrast-enhanced T1 (cT1), T2 - fluid-attenuated inversion recovery (FLAIR), ADC, DSC, and DCE of 125 patients with subsequent histopathologically confirmed glioblastoma were performed on a 3 Tesla MRI scanner. ADC, DSC, and DCE parameters were analyzed in semiautomatically segmented tumor volumes on contrast-enhanced (CE) cT1 and hyperintense signal changes on T2 FLAIR (ED). Univariate and multivariable Cox regression analyses including age, sex, extent of resection (EOR), and KPS were performed to assess the influence of each parameter on OS and PFS.RESULTS:Univariate Cox regression analysis demonstrated a significant association of age, KPS, and EOR with PFS and age, KPS, EOR, lower ADC, and higher rCBV with OS. Multivariable analysis showed independent significance of male sex, KPS, EOR, and increased rCBVCE for PFS, and age, sex, KPS, and EOR for OS.CONCLUSIONS:MRI parameters help to predict OS in a univariate Cox regression analysis, and increased rCBVCE is associated with shorter PFS in the multivariable model. In summary, however, our findings suggest that the relevance of MRI parameters is outperformed by clinical parameters in a multivariable analysis, which limits their prognostic value for survival prediction at the time of initial diagnosis.

Molecular targets for the pathological vasculature are the vascular endothelial growth factor (VEGF)/VEGF receptor axis, integrins, angiopoietins, and platelet-derived growth factor receptor (PDGFR), as well as several intracellular or downstream effectors like protein kinase C beta and mammalian target of rapamycin (mTOR). Besides hypoxic damage or tumor cell starvation, preclinical models imply vessel independent tumor regression and suggest differential effects of anti-angiogenic treatments on tumorous and nontumorous precursor cells or the immune system. Despite compelling preclinical data and positive data in other cancers, the outcomes of clinical trials with anti-angiogenic agents in gliomas by and large have been disappointing and include VEGF blockage with bevacizumab, integrin inhibition with cilengitide, VEGF receptor inhibition with sunitinib or cediranib, PDGFR inhibition with imatinib or dasatinib, protein kinase C inhibition with enzastaurin, and mTOR inhibition with sirolimus, everolimus, or temsirolimus. Importantly, there is a lack of real understanding for this negative data. Anti-angiogenic therapies have stimulated the development of standardized imaging assessment and the integration of functional MRI sequences into daily practice. Here, we delineate directions in the identification of molecularly or image-based defined subgroups, anti-angiogenic cotreatment for immunotherapy, and the potential of ongoing trials or modified targets to change the game.

Loss of the tumor suppressor phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is a prerequisite for tumor cell-specific expression of vascular endothelial growth factor receptor (VEGFR)-2 in glioblastoma defining a subgroup prone to develop evasive resistance towards antiangiogenic treatments. Immunohistochemical analysis of human tumor tissues showed VEGFR-2 expression in glioma cells in 19% of specimens examined, mainly in the infiltration zone. Glioma cell VEGFR-2 positivity was restricted to PTEN-deficient tumor specimens. PTEN overexpression reduced VEGFR-2 expression in vitro, as well as knock-down of raptor or rictor. Genetic interference with VEGFR-2 revealed proproliferative, antiinvasive and chemoprotective functions for VEGFR-2 in glioma cells. VEGFR-2-dependent cellular effects were concomitant with activation of 'kappa-light-chain-enhancer' of activated B-cells, protein kinase B, and N-myc downstream regulated gene 1. Two-photon in vivo microscopy revealed that expression of VEGFR-2 in glioma cells hampers antiangiogenesis. Bevacizumab induces a proinvasive response in VEGFR-2-positive glioma cells. Patients with PTEN-negative glioblastomas had a shorter survival after initiation of bevacizumab therapy compared with PTEN-positive glioblastomas. Conclusively, expression of VEGFR-2 in glioma cells indicates an aggressive glioblastoma subgroup developing early resistance to temozolomide or bevacizumab. Loss of PTEN may serve as a biomarker identifying those tumors upfront by routine neuropathological methods.

BACKGROUND AND PURPOSE:MR imaging in neuro-oncology is challenging due to inherent ambiguities in proton signal behavior. Sodium-MR imaging may substantially contribute to the characterization of tumors because it reflects the functional status of the sodium-potassium pump and sodium channels. MATERIALS AND METHODS:Sodium-MR imaging data of patients with treatment-naïve glioma WHO grades I-IV (n = 34; mean age, 51.29 ± 17.77 years) were acquired by using a 7T MR system. For acquisition of sodium-MR images, we applied density-adapted 3D radial projection reconstruction pulse sequences. Proton-MR imaging data were acquired by using a 3T whole-body system. RESULTS:We demonstrated that the initial sodium signal of a treatment-naïve brain tumor is a significant predictor of isocitrate dehydrogenase (IDH) mutation status (P < .001). Moreover, independent of this correlation, the Cox proportional hazards model confirmed the sodium signal of treatment-naïve brain tumors as a predictor of progression (P = .003). Compared with the molecular signature of IDH mutation status, information criteria of model comparison revealed that the sodium signal is even superior to IDH in progression prediction. In addition, sodium-MR imaging provides a new approach to noninvasive tumor classification. The sodium signal of contrast-enhancing tumor portions facilitates differentiation among most glioma types (P < .001). CONCLUSIONS:The information of sodium-MR imaging may help to classify neoplasias at an early stage, to reduce invasive tissue characterization such as stereotactic biopsy specimens, and overall to promote improved and individualized patient management in neuro-oncology by novel imaging signatures of brain tumors.

Armin Biller, Jens Kleesiek, Jan Oliver Neumann, Felix Sahm, Anne Dorothea Hertenstein, and Armin Michael Nagel Neuroradiology, University of Heidelberg, Heidelberg, Badem-Württemberg, Germany, Radiology, German Cancer Research Center DKFZ, Heidelberg, BademWürttemberg, Germany, University of Heidelberg, Baden-Württemberg, Germany, Neuropathology, Baden-Württemberg, Germany, Medical Physics in Radiology, German Cancer Research Center DKFZ, Baden-Württemberg, Germany

3,4-dimethoxycinnamonyl-anthranilic acid (tranilast) is an orally available anti-allergic drug with structural and functional homologies to immunosuppressive catabolites of the essential amino acid tryptophan and broad anti-inflammatory properties. It has recently been shown to be effective in animal models of multiple sclerosis and rheumatoid arthritis, two autoimmune diseases that are mediated by auto-aggressive Th1-polarized CD4+ T lymphocytes. Here we demonstrate potent suppressive effects of tranilast on the function of naïve human CD4+ T cells. Tranilast inhibited inhibits activation and proliferation of purified CD4+ T cells stimulated through the T cell receptor with an EC50 of less than 10μM, a concentration that is well below plasma levels achieved after oral administration of approved doses of 200–600mg in humans. The antiproliferative effects were less potent on naïve CD8+ T cells. Suppression of CD4+ and CD8+ T cell proliferation was associated with an inhibition of T cell activation. Cytokine analyses of naïve CD4+ T cells revealed that tranilast interferes with the production of cyto- and chemokines driven by signal transducer and activator of transcription 1 (STAT1), notably chemokine (C-X-C motif) ligands (CXCL) 9 and 10. Tranilast limited STAT1 phosphorylation in activated T cells and supplementation of CXCL9 or CXCL10 reversed the anti-proliferative effects of tranilast. These data imply CXCL9 and CXCL10 as novel therapeutic targets of tranilast in Th1-mediated autoimmune diseases and identify phospho-STAT1 and its target chemokines CXCL9 and CXCL10 as potential markers for monitoring the bioactivity of tranilast in humans.