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The outcome of patients with anaplastic gliomas varies considerably. Whether a molecular classification of anaplastic gliomas based on large-scale genomic or epigenomic analyses is superior to histopathology for reflecting distinct biological groups, predicting outcomes and guiding therapy decisions has yet to be determined. Epigenome-wide DNA methylation analysis, using a platform which also allows the detection of copy-number aberrations, was performed in a cohort of 228 patients with anaplastic gliomas (astrocytomas, oligoastrocytomas, and oligodendrogliomas), including 115 patients of the NOA-04 trial. We further compared these tumors with a group of 55 glioblastomas. Unsupervised clustering of DNA methylation patterns revealed two main groups correlated with IDH status: CpG island methylator phenotype (CIMP) positive (77.5 %) or negative (22.5 %). CIMP(pos) (IDH mutant) tumors showed a further separation based on copy-number status of chromosome arms 1p and 19q. CIMP(neg) (IDH wild type) tumors showed hallmark copy-number alterations of glioblastomas, and clustered together with CIMP(neg) glioblastomas without forming separate groups based on WHO grade. Notably, there was no molecular evidence for a distinct biological entity representing anaplastic oligoastrocytoma. Tumor classification based on CIMP and 1p/19q status was significantly associated with survival, allowing a better prediction of outcome than the current histopathological classification: patients with CIMP(pos) tumors with 1p/19q codeletion (CIMP-codel) had the best prognosis, followed by patients with CIMP(pos) tumors but intact 1p/19q status (CIMP-non-codel). Patients with CIMP(neg) anaplastic gliomas (GBM-like) had the worst prognosis. Collectively, our data suggest that anaplastic gliomas can be grouped by IDH and 1p/19q status into three molecular groups that show clear links to underlying biology and a significant association with clinical outcome in a prospective trial cohort.

// Maximilian Georg Schliesser 1,3 , Rainer Claus 6,24 , Thomas Hielscher 7 , Christiane Grimm 1,3 , Dieter Weichenhan 6 , Jonas Blaes 3 , Benedikt Wiestler 1,3,25 , Peter Hau 10 , Johannes Schramm 11 , Felix Sahm 2,4 , Elisa K. Weis 1,3 , Markus Weiler 1,3,8 , Constance Baer 6 , Friederike Schmidt-Graf 8,12 , Gabriele Schackert 13 , Manfred Westphal 14 , Anne Hertenstein 1,3 , Patrick Roth 8,15 , Norbert Galldiks 16 , Christian Hartmann 2,4,17 , Torsten Pietsch 19 , Joerg Felsberg 20,22 , Guido Reifenberger 20,22 , Michael Christoph Sabel 21 , Frank Winkler 1,3 , Andreas von Deimling 2,4 , Christoph Meisner 9 , Peter Vajkoczy 23 , Michael Platten 1,5,8 , Michael Weller 8,15 , Christoph Plass 6 and Wolfgang Wick 1,3,8 1 Department of Neurology, Heidelberg University Hospital and German Cancer Consortium, Clinical Cooperation Units, Germany 2 Department of Neuropathology, Heidelberg University Hospital and German Cancer Consortium, Clinical Cooperation Units, Germany 3 Clinical Cooperation Unit of Neurooncology, German Cancer Research Center (DKFZ), Heidelberg, Germany 4 Clinical Cooperation Unit of Neuropathology, German Cancer Research Center (DKFZ), Heidelberg, Germany 5 Clinical Cooperation Unit of Neuroimmunology and Brain Tumor Immunology, German Cancer Research Center (DKFZ), Heidelberg, Germany 6 Division of Epigenomics and Cancer Risk Factors, German Cancer Research Center (DKFZ), Heidelberg, Germany 7 Division of Biostatistics, German Cancer Research Center (DKFZ), Heidelberg, Germany 8 Department of General Neurology, University Hospital Tubingen, Germany 9 Department of Biostatistics, University Hospital Tubingen, Germany 10 Neurology Clinic, Regensburg University, Regensburg, Germany 11 Neurosurgery Clinic, University of Bonn Medical Center, TU Munich, Munich, Germany 12 Neurology Clinic, TU Munich, Munich, Germany 13 Neurosurgery Clinic, Dresden University Medical Center, Germany 14 Neurosurgery Clinic, University Clinic Hamburg, Eppendorf, Germany 15 Department of Neurology, University Hospital Zurich, Zurich, Switzerland 16 Neurology Clinic, Cologne University, Cologne, Germany 17 Department for Neuropathology, Institute of Pathology, Medical University of Hannover, Hannover, Germany 19 Department of Neuropathology, University of Bonn Medical Center, Bonn, Germany 20 Department of Neuropathology, Heinrich-Heine-University, Germany 21 Department of Neurosurgery, Heinrich-Heine-University, Germany 22 DKTK, Partner Site Essen/Dusseldorf, Dusseldorf, Germany 23 Neurosurgery Clinic, Charite, Berlin, Germany 24 Department Hematology, Oncology and Stem Cell Transplantation, University Hospital Freiburg, Germany 25 Department of Diagnostic and Interventional Neuroradiology, Klinikum rechts der Isar der Technischen Universitat Munchen, Munich, Germany Correspondence to: Wolfgang Wick, email: // Keywords : anaplastic glioma, miR-155, IDH, NFκB, NOA-04 Received : May 09, 2016 Accepted : September 13, 2016 Published : November 18, 2016 Abstract 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.

Transient or lasting increases in glucocorticoids accompany deficits in hippocampus-dependent memory formation. Recent data indicate that the formation and consolidation of declarative and spatial memory are mechanistically related to different patterns of hippocampal network oscillations. These include gamma oscillations during memory acquisition and the faster ripple oscillations (approximately 200 Hz) during subsequent memory consolidation. We therefore analysed the effects of acutely applied glucocorticoids on network activity in mouse hippocampal slices. Evoked field population spikes and paired-pulse responses were largely unaltered by corticosterone or cortisol, respectively, despite a slight increase in maximal population spike amplitude by 10 microm corticosterone. Several characteristics of sharp waves and superimposed ripple oscillations were affected by glucocorticoids, most prominently the frequency of spontaneously occurring sharp waves. At 0.1 microm, corticosterone increased this frequency, whereas maximal (10 microm) concentrations led to a reduction. In addition, gamma oscillations became slightly faster and less regular in the presence of high doses of corticosteroids. The present study describes acute effects of glucocorticoids on sharp wave-ripple complexes and gamma oscillations in mouse hippocampal slices, revealing a potential background for memory deficits in the presence of elevated levels of these hormones.

Glioblastoma (World Health Organization [WHO] grade IV) is the most common intrinsic brain tumor. The prognosis for patients suffering from this disease remains dismal. Age in particular is a strong negative predictor for survival, resulting in a population-based median survival of elderly patients (ie, >65 y) of 70% of WHO grades II and III gliomas and secondary glioblastomas.13 In primary glioblastomas, IDH mutations are rare. Importantly, IDH mutations are associated with a significantly longer survival time compared with IDH wild-type tumors. Among grades III and IV gliomas pooled, IDH has a stronger prognostic impact than WHO grade. In the same study, the authors also found that in patients age ≥60 years with anaplastic astrocytoma and glioblastoma, IDH1 mutation was found in only 7.5% of the patients (11/146).14 Analysis of the glioblastoma epigenome revealed a distinct hypermethylator phenotype, the glioma cytosine–phosphate–guanine (CpG) island methylator phenotype (G-CIMP), which confers a good prognosis.15 Tumors positive for G-CIMP usually harbor IDH mutations, hence they are common among grades II and III gliomas and rare in primary glioblastomas. Genome- and epigenome-wide analyses of glioblastoma samples further revealed frequent mutations in the histone 3.3 gene (H3F3A) at K27 or G34.16 Just like IDH mutations, both K27M and G34R/V mutations are associated with a distinct epigenetic signature and possibly cell of origin each. In this study, tumors carrying G34 mutations show a favorable clinical course.17 Importantly, H3F3A and IDH mutations are mutually exclusive, suggesting that mutations in either of these genes represent different gliomagenic pathways. While the aforementioned alterations are prognostic, Agnihotri et al18 recently reported on epigenetic inactivation of alkylpurine DNA N-glycosylase (APNG) as a predictive biomarker for benefit from TMZ treatment. APNG is a DNA base excision repair enzyme, which catalyzes removal of N3-methyladenine and N7-methylguanine from DNA, both of which can be caused by TMZ. APNG expression is regulated through promoter methylation, and in patients with an unmethylated MGMT promoter receiving TMZ, the subset of APNG-negative tumors was reported to have a better prognosis. Peroxiredoxin 1 (PRDX1) is another interesting candidate, especially in the group of anaplastic astrocytomas. The PRDX1 promoter was found to be frequently hypermethylated in oligodendroglial tumors and secondary glioblastomas carrying a deletion of 1p/19q, leading to epigenetic downregulation of PRDX1 expression.19 In this study, silencing of PRDX1 in Hs683 glioma cells sensitized these cells both to TMZ and to RT in vitro. The objective of our present study was to determine the prevalence and impact of recently defined biomarkers associated with survival, heretofore established in younger patients, in an elderly collective. We hypothesized that these biomarkers might help to explain the heterogeneity in survival seen in the NOA-08 population. However, as our study revealed that they are virtually absent in elderly patients, we assume that relevant molecular differences exist among malignant gliomas of different age groups, which warrant further studies.

Neurons form transiently stable assemblies that may underlie cognitive functions, including memory formation. In most brain regions, coherent activity is organized by network oscillations that involve sparse firing within a well-defined minority of cells. Despite extensive work on the underlying cellular mechanisms, a fundamental question remains unsolved: how are participating neurons distinguished from the majority of nonparticipators? We used physiological and modeling techniques to analyze neuronal activity in mouse hippocampal slices during spontaneously occurring high-frequency network oscillations. Network-entrained action potentials were exclusively observed in a defined subset of pyramidal cells, yielding a strict distinction between participating and nonparticipating neurons. These spikes had unique properties, because they were generated in the axon without prior depolarization of the soma. GABA A receptors had a dual role in pyramidal cell recruitment. First, the sparse occurrence of entrained spikes was accomplished by intense perisomatic inhibition. Second, antidromic spike generation was facilitated by tonic effects of GABA in remote axonal compartments. Ectopic spike generation together with strong somatodendritic inhibition may provide a cellular mechanism for the definition of oscillating assemblies.

Electrical synapses, particularly gap junctions composed of connexin (Cx) 36, have been suggested to synchronize neuronal network oscillations. Recently, we generated Cx30.2-deficient mice which express beta-galactosidase under control of Cx30.2 gene regulatory elements. In the central nervous system beta-galactosidase activity representing Cx30.2 expression was restricted to NeuN-positive cells, thus identifying Cx30.2 as new neuronal connexin. In the hippocampus, co-immunofluorescence analyses revealed beta-galactosidase/Cx30.2 expression in GABAergic inhibitory interneurons such as parvalbumin- and somatostatin-positive basket, axo-axonic, bistratified or oriens lacunosum-moleculare cells. approximately 94% of the Cx30.2 expressing parvalbumin-positive interneurons also expressed Cx36. Performing field potential recordings from hippocampal slices we found no differences in basal excitation and excitation-inhibition balance between Cx30.2+/+ and Cx30.2LacZ/LacZ)mice. Furthermore, frequency and power of gap junction dependent gamma and ripples oscillations were similar in these animals. This suggests that the lack of Cx30.2 in interneurons can be largely compensated by other connexins, most likely Cx36.