A Novel Type of IDH-wildtype Glioma Characterized by Gliomatosis Cerebri-like Growth Pattern, TERT Promoter Mutation, and Distinct Epigenetic Profile.

Diffuse gliomas in adults encompass a heterogenous group of central nervous system neoplasms. In recent years, extensive (epi-)genomic profiling has identified several glioma subgroups characterized by distinct molecular characteristics, most importantly IDH1/2 and histone H3 mutations. A group of 16 diffuse gliomas classified as "adult-type diffuse high-grade glioma, IDH-wildtype, subtype F (HGG-F)" was identified by the DKFZ v12.5 Brain Tumor Classifier . Histopathologic characterization, exome sequencing, and review of clinical data was performed in all cases. Based on unsupervised t -distributed stochastic neighbor embedding and clustering analysis of genome-wide DNA methylation data, HGG-F shows distinct epigenetic profiles separate from established central nervous system tumors. Exome sequencing demonstrated frequent TERT promoter (12/15 cases), PIK3R1 (11/16), and TP53 mutations (5/16). Radiologic characteristics were reminiscent of gliomatosis cerebri in 9/14 cases (64%). Histopathologically, most cases were classified as diffuse gliomas (7/16, 44%) or were suspicious for the infiltration zone of a diffuse glioma (5/16, 31%). None of the cases demonstrated microvascular proliferation or necrosis. Outcome of 14 patients with follow-up data was better compared to IDH-wildtype glioblastomas with a median progression-free survival of 58 months and overall survival of 74 months (both P <0.0001). Our series represents a novel type of adult-type diffuse glioma with distinct molecular and clinical features. Importantly, we provide evidence that TERT promoter mutations in diffuse gliomas without further morphologic or molecular signs of high-grade glioma should be interpreted in the context of the clinicoradiologic presentation as well as epigenetic profile and may not be suitable as a standalone marker for glioblastoma, IDH-wildtype.

Feasibility of ApoC1 serum levels as tumor biomarker in glioblastoma patients: a pilot study.

Apolipoprotein C1 (ApoC1) has been detected immunohistochemically in glioblastoma tissue, probably expressed by activated monocytes and microglia. The present study was conceived to determine whether the amount of intratumoral ApoC1 expression leads to measurable changes of serum levels after glioblastoma resection or during recurrence. 176 blood samples from 70 glioblastoma patients were collected perioperatively and during subsequent therapy. ApoC1 serum levels were determined using an enzyme linked immunosorbent assay (ELISA). High absorption values due to lipemic or hemolytic serum were removed from the final dataset using a stem and leaf plot. Samples were grouped according to the treatment stage to compare mean ApoC1 serum levels. The number of patients with falling or increasing perioperative values was assessed. 167 ApoC1 serum values from 68 glioblastoma patients were amenable to statistical evaluation. Mean ApoC1 serum level was 91.9 µg/ml (n = 167, sd = 36.0). In samples from patients undergoing first glioblastoma resection, the mean preoperative value was significantly higher (94.8 µg/ml, n = 37, sd = 29.5) than after surgery (77.4 µg/ml, n = 41, sd = 23.2, p = 0.009). Individually, falling ApoC1 levels were detected in 25 and rising levels in 9 patients (p = 0.0061). Single absolute serum levels of ApoC1 do not allow an estimation of glioblastoma activity or tumor response. Although pathophysiologically of interest, ApoC1 serum levels did not qualify as a potential biomarker in glioblastoma management. Our results do not seem to encourage larger, multicenter studies.

The Mean ApoC1 Serum Level in Postoperative Samples from Neurosurgical Patients Is Lower than in Preoperative Samples and during Chemotherapy.

Serum levels of apolipoprotein ApoC1 have been described in a number of systemic tumor entities as potential biomarkers, but little is known about ApoC1 in neurosurgical patients. A total of 230 serum samples from 96 patients were analyzed using an ELISA technique. Patient diagnoses comprised 70 glioblastomas WHO IV°, 10 anaplastic astrocytomas III°, one anaplastic oligodendroglioma III°, one oligodendroglioma II°, one diffuse astrocytoma II°, one pilocytic astrocytoma I°, and a single case of a spindle cell tumor without WHO grading, as well as 11 spinal interventions. The mean ApoC1 level of the 230 samples was 132.03 µg/mL (median 86.83, SD 292.91). In the 176 glioblastoma samples, the mean ApoC1 level was 130.0 µg/mL (median 86.23, SD 314.9), which was neither different from the whole group nor from patients with spinal interventions (215.1 µg/mL, median 63.6, SD 404.9). In the postoperative samples, the mean ApoC1 level was significantly lower (85.81 µg/mL) than in the preoperative samples (129.64 µg/mL) and in samples obtained during adjuvant chemotherapy (168.44 µg/mL). While absolute ApoC1 serum levels in a patient do not allow for the distinction between neurosurgical histological entities, future analyses will examine whether the time course of ApoC1 in an individual patient can be related to certain treatment stages.

Recurrent fusions in PLAGL1 define a distinct subset of pediatric-type supratentorial neuroepithelial tumors.

Ependymomas encompass a heterogeneous group of central nervous system (CNS) neoplasms that occur along the entire neuroaxis. In recent years, extensive (epi-)genomic profiling efforts have identified several molecular groups of ependymoma that are characterized by distinct molecular alterations and/or patterns. Based on unsupervised visualization of a large cohort of genome-wide DNA methylation data, we identified a highly distinct group of pediatric-type tumors (n = 40) forming a cluster separate from all established CNS tumor types, of which a high proportion were histopathologically diagnosed as ependymoma. RNA sequencing revealed recurrent fusions involving the pleomorphic adenoma gene-like 1 (PLAGL1) gene in 19 of 20 of the samples analyzed, with the most common fusion being EWSR1:PLAGL1 (n = 13). Five tumors showed a PLAGL1:FOXO1 fusion and one a PLAGL1:EP300 fusion. High transcript levels of PLAGL1 were noted in these tumors, with concurrent overexpression of the imprinted genes H19 and IGF2, which are regulated by PLAGL1. Histopathological review of cases with sufficient material (n = 16) demonstrated a broad morphological spectrum of tumors with predominant ependymoma-like features. Immunohistochemically, tumors were GFAP positive and OLIG2- and SOX10 negative. In 3/16 of the cases, a dot-like positivity for EMA was detected. All tumors in our series were located in the supratentorial compartment. Median age of the patients at the time of diagnosis was 6.2 years. Median progression-free survival was 35 months (for 11 patients with data available). In summary, our findings suggest the existence of a novel group of supratentorial neuroepithelial tumors that are characterized by recurrent PLAGL1 fusions and enriched for pediatric patients.

Quality assurance in neuropathology: Experiences from the round robin trials on IDH mutation and MGMT promoter methylation testing launched by the Quality Assurance Initiative Pathology (QuIP) in 2018 and 2019.

We here report on the first neuropathological round robin trials initiated by the Quality Assurance Initiative Pathology (QuIP) in Germany in the years 2018 and 2019. Testing services as external laboratory controls were offered for IDH1-R132H immunohistochemistry in 2018 followed by a molecular trial for IDH1 and IDH2 mutations in 2019 including the rare mutational variants. Also in 2019, a trial on MGMT promoter methylation testing was offered. On a national scale, trial offers were well received with around 40 participating institutions. The international announcement of the molecular IDH1/IDH2 mutational trial achieved only moderate European outspread. Success rates in all three trials were excellent (IDH1-R132H immunohistochemistry 2018: 94%, 18 out of 20 possible points required; IDH1/IDH2 mutational status 2019: 100%, 19 out of 20 possible points required; MGMT promoter methylation 2019: 94%, 19 out of 20 possible points required) indicating that quality standards are high in the broad majority of the institutions. Trial participation also involved filling in a questionnaire asking for background information on local testing procedures. We here present a first assessment of the information collected providing unique insights in the landscape of molecular testing in neuropathology. Derived from this information we identify future challenges and provide an outlook on the development of quality assurance in the field of neuropathology.

Encephalitis with radial perivascular emphasis: Not necessarily associated with GFAP antibodies.

OBJECTIVE: Autoimmune steroid-responsive meningoencephalomyelitis with linear perivascular gadolinium enhancement in brain MRI is regarded as glial fibrillary acidic protein (GFAP) astrocytopathy characterized by anti-GFAP antibodies (ABs). We questioned whether anti-GFAP ABs are necessarily associated with this syndrome. METHODS: Two patients with a strikingly similar disease course suggestive of autoimmune GFAP astrocytopathy are reported. Clinical examination, MRI, laboratory, and CSF analysis were performed. Neuropathologic examination of brain tissue was obtained from one patient. Serum and CSF were additionally tested using mouse brain slices, microglia-astrocyte cocultures, and a GFAP-specific cell-based assay. RESULTS: Both patients presented with subacute influenza-like symptoms and developed severe neurocognitive and neurologic deficits and impaired consciousness. MRIs of both patients revealed radial perivascular gadolinium enhancement extending from the lateral ventricles to the white matter suggestive of autoimmune GFAP astrocytopathy. Both patients responded well to high doses of methylprednisolone. Only one patient had anti-GFAP ABs with a typical staining pattern of astrocytes, whereas serum and CSF of the other patient were negative and showed neither reactivity to brain tissue nor to vital or permeabilized astrocytes. Neuropathologic examination of the anti-GFAP AB-negative patient revealed infiltration of macrophages and T cells around blood vessels and activation of microglia without obvious features of clasmatodendrosis. CONCLUSIONS: The GFAP-AB negative patient had both a striking (para)clinical similarity and an immediate response to immunotherapy. This supports the hypothesis that the clinical spectrum of steroid-responsive meningoencephalomyelitis suggestive of autoimmune GFAP astrocytopathy may be broader and may comprise also seronegative cases.

Publisher Correction: NRG1 type I dependent autoparacrine stimulation of Schwann cells in onion bulbs of peripheral neuropathies.

Michael W. Sereda was incorrectly associated with the Department of Cellular Neurophysiology, Hanover Medical School, Carl-Neuberg-Str. 1, 30625 Hanover, Germany. The correct affiliations for Michael W. Sereda are Department of Neurogenetics, Max-Planck-Institute of Experimental Medicine, Hermann-Rein-Str. 3, 37075 Göttingen, Germany and Department of Clinical Neurophysiology, University Medical Center Göttingen, Robert-Koch-Str. 40, 37075 Göttingen, Germany.

NRG1 type I dependent autoparacrine stimulation of Schwann cells in onion bulbs of peripheral neuropathies.

In contrast to acute peripheral nerve injury, the molecular response of Schwann cells in chronic neuropathies remains poorly understood. Onion bulb structures are a pathological hallmark of demyelinating neuropathies, but the nature of these formations is unknown. Here, we show that Schwann cells induce the expression of Neuregulin-1 type I (NRG1-I), a paracrine growth factor, in various chronic demyelinating diseases. Genetic disruption of Schwann cell-derived NRG1 signalling in a mouse model of Charcot-Marie-Tooth Disease 1A (CMT1A), suppresses hypermyelination and the formation of onion bulbs. Transgenic overexpression of NRG1-I in Schwann cells on a wildtype background is sufficient to mediate an interaction between Schwann cells via an ErbB2 receptor-MEK/ERK signaling axis, which causes onion bulb formations and results in a peripheral neuropathy reminiscent of CMT1A. We suggest that diseased Schwann cells mount a regeneration program that is beneficial in acute nerve injury, but that overstimulation of Schwann cells in chronic neuropathies is detrimental.

Loss of IBA1-Expression in brains from individuals with obesity and hepatic dysfunction.

Microglia, the brain's resident immune cells, exhibit constitutive expression of the ionized calcium binding adaptor molecule 1 (IBA1), a cytoplasmic protein with actin and calcium-binding functions involved in membrane ruffling. Microglia are long-lived cells that exhibit a senescent morphology (dystrophy) with aging, which may be indicative of cell dysfunction. It has been reported that dystrophy of IBA1-positive microglia is exacerbated in obese humans. Our own preliminary studies of microglia in the medial temporal lobe of obese subjects have revealed another microglial abnormality, which is the loss of IBA1 immunoreactivity that can create large areas in the brain seemingly devoid of all microglial cells. Here, we systematically compared microglial appearance in human hippocampi derived from obese individuals compared to controls (nobese = 33, nnon-obese = 30). In both groups, we found areas that were negative for IBA1, but contained P2YR12 and glutathione-peroxidase 1 (GPX)-positive microglia. The number and extent of IBA1-negative regions was increased in obese cases. Since some cases of non-obese individuals also exhibited loss of IBA-1 immunoreactivity, we searched for possible confounders and found that hepatic dysfunction strongly impacts the distribution of microglial cells: By computational analysis of scanned IBA1-stained sections, we detected increased Mean Empty Space distances (p = 0.016) and IBA1-negative areas (p = 0.090) which were independent from the cause of liver dysfunction, but also from aging. Thus, we report on a novel type of microglia pathological change, i.e. localized loss of IBA1 that is linked, at least in part, to obesity and hepatic dysfunction.

Qualitative and Quantitative Analysis of Cardiac Progenitor Cells in Cases of Myocarditis and Cardiomyopathy.

We aimed to identify and quantify CD117+ and CD90+ endogenous cardiac progenitor cells (CPC) in human healthy and diseased hearts. We hypothesize that these cells perform a locally acting, contributing function in overcoming medical conditions of the heart by endogenous means. Human myocardium biopsies were obtained from 23 patients with the following diagnoses: Dilatative cardiomyopathy (DCM), ischemic cardiomyopathy (ICM), myocarditis, and controls from healthy cardiac patients. High-resolution scanning microscopy of the whole slide enabled a computer-based immunohistochemical quantification of CD117 and CD90. Those signals were evaluated by Definiens Tissue Phenomics® Technology. Co-localization of CD117 and CD90 was determined by analyzing comparable serial sections. CD117+/CD90+ cardiac cells were detected in all biopsies. The highest expression of CD90 was revealed in the myocarditis group. CD117 was significantly higher in all patient groups, compared to healthy specimens (*p < 0.05). The highest co-expression was found in the myocarditis group (6.75 ± 3.25 CD90+CD117+ cells/mm2) followed by ICM (4 ± 1.89 cells/mm2), DCM (1.67 ± 0.58 cells/mm2), and healthy specimens (1 ± 0.43 cells/mm2). We conclude that the human heart comprises a fraction of local CD117+ and CD90+ cells. We hypothesize that these cells are part of local endogenous progenitor cells due to the co-expression of CD90 and CD117. With novel digital image analysis technologies, a quantification of the CD117 and CD90 signals is available. Our experiments reveal an increase of CD117 and CD90 in patients with myocarditis.

Stroke-induced blood-brain barrier breakdown along the vascular tree - No preferential affection of arteries in different animal models and in humans.

Stroke-induced blood-brain barrier breakdown promotes complications like cerebral edema and hemorrhagic transformation, especially in association with therapeutical recanalization of occluded vessels. As arteries, capillaries and veins display distinct functional and morphological characteristics, we here investigated patterns of blood-brain barrier breakdown for each segment of the vascular tree in rodent models of embolic, permanent, and transient middle cerebral artery occlusion, added by analyses of human stroke tissue. Twenty-four hours after ischemia induction, loss of blood-brain barrier function towards FITC-albumin was equally observed for arteries, capillaries, and veins in rodent brains. Noteworthy, veins showed highest ratios of leaky vessels, whereas capillaries exhibited the most and arteries the least widespread perivascular tracer extravasation. In contrast, human autoptic stroke tissue exhibited pronounced extravasations of albumin around arteries and veins, while the pericapillary immunoreactivity appeared only faint. Although electron microscopy revealed comparable alterations of the arterial and capillary endothelium throughout the applied animal models, structural loss of arterial smooth muscle cells was only observed in the translationally relevant model of embolic middle cerebral artery occlusion. In light of the so far available concepts of stroke treatment, the consideration of a differential vascular pathophysiology along the cerebral vasculature is likely to allow development of novel effective treatment strategies.

Up-regulation of neurofilament light chains is associated with diminished immunoreactivities for MAP2 and tau after ischemic stroke in rodents and in a human case.

As stroke therapies are still limited to a minority of patients, efforts have been intensified to an improved understanding of pathophysiological processes during ischemia formation, potentially allowing the development of specific therapeutic interventions. In this context, cytoskeletal elements became evident as key players during the transition process towards long-lasting tissue damage. This study focused on ischemia-related alterations of the cytoskeleton with a special focus on microtubule-associated proteins and neurofilament light chains (NF-L). Immunohistochemical analyses were applied to brain sections of mice and rats after experimental stroke and to autoptic samples from a stroke patient. To consider translational aspects, a thromboembolic model of stroke in rats, closely mimicking the human situation, was used in addition to the filament-based model of focal cerebral ischemia in mice. One day after ischemia onset, immunoreactivity of microtubule-associated protein tau and microtubule-associated protein-2 (MAP2) was reduced in ischemic areas. These findings were consistently present in the ischemia-affected striatum and the neocortex. In a quite opposite fashion, ischemic areas displayed NF-L-immunoreactivity in neuropathologically altered fibers, local agglomerations probably related to degraded cell bodies and neocortical pyramidal cells. Notably, up-regulation of NF-L was also confirmed in infarcted tissue from a human brain sample. Furthermore, analyses of rodent brain tissue revealed corkscrew curl-like fibers as a special feature of MAP2 in the ischemia-affected hippocampus. In conclusion, this study provides evidence for an opposite reaction of microtubule-associated proteins and neurofilaments after focal cerebral ischemia. Accordingly, cytoskeletal elements appear as a promising target for stroke treatment.

WHO grade related expression of TRAIL-receptors and apoptosis regulators in meningioma.

BACKGROUND AND AIMS: The expression of the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptors and key regulators of the extrinsic apoptosis pathway correlate with clinical features and the WHO grade of malignancy in some tumor entities. Expression of pro-apoptotic TRAIL receptors and executioners of apoptosis are a prerequisite for TRAIL-based therapies as a promising future targeted therapy. METHODS: Human meningioma tissues (n=24 WHO grade I, n=7 WHO grade II, n=6 WHO grade III) were immunohistochemically analyzed for the expression of TRAIL-R1, TRAIL-R2, TRAIL-R3, TRAIL-R4, caspase-8, cFLIP, Bcl-2, Bcl-XL, Mcl-1, Bax, and Bak. Staining intensities were quantified by an automated software-based algorithm. RESULTS: While TRAIL-R1 and TRAIL-R3 were nearly absent in meningiomas, TRAIL-R2 and TRAIL-R4 were abundantly expressed. However, only TRAIL-R4 expression correlated with the WHO grade of malignancy. Bcl-2 showed a non-significant upregulation in WHO grade III meningiomas. Bcl-XL and Mcl-1 expression was significantly higher in WHO grade II compared to grade I. Bcl-XL and TRAIL-R4 expression correlated with the mitotic activity (Ki67) of the tumor. Furthermore, TRAIL-R2 expression correlated with TRAIL-R4. Bak expression correlated with both, Bcl-XL and Mcl-1 expression. The expression patterns did neither correlate with the progression-free nor with the overall survival of the meningioma patients. CONCLUSIONS: Apoptosis-inducing TRAIL-R2 and all key executioners of the extrinsic apoptosis pathway are abundantly expressed in meningioma. For some regulators of apoptosis with opposite functions, the expression of the pro-apoptotic protein significantly correlated with the expression level of the respective anti-apoptotic binding partner, possibly resulting in a steady-state of apoptosis. TRAIL-R2 might serve as a novel therapeutic target in meningioma.

Bortezomib sensitizes primary meningioma cells to TRAIL-induced apoptosis by enhancing formation of the death-inducing signaling complex.

A meningioma is the most common primary intracranial tumor in adults. Here, we investigated the therapeutic potential of the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in 37 meningiomas. Freshly isolated primary meningioma cells were treated with TRAIL with or without different sensitizing protocols, and apoptotic cell death was then quantified. Mechanisms of TRAIL sensitization were determined by a combination of Western blotting, flow cytometry, receptor complex immunoprecipitation, and siRNA-mediated knockdown experiments. Tumor necrosis factor-related apoptosis-inducing ligand receptor expression was analyzed using immunohistochemistry and quantified by an automated software-based algorithm. Primary tumor cells from 11 (29.7%) tumor samples were sensitive to TRAIL-induced apoptosis, 12 (32.4%) were intermediate TRAIL resistant, and 14 (37.8%) were completely TRAIL resistant. We tested synergistic apoptosis-inducing cotreatment strategies and determined that only the proteasome inhibitor bortezomib potently enhanced expression of the TRAIL receptors TRAIL-R1 and/or TRAIL-R2, the formation of the TRAIL death-inducing signaling complex, and activation of caspases; this treatment resulted in sensitization of all TRAIL-resistant meningioma samples to TRAIL-induced apoptosis. Bortezomib pretreatment induced NOXA expression and downregulated c-FLIP, neither of which caused the TRAIL-sensitizing effect. Native TRAIL receptor expression could not predict primary TRAIL sensitivity. This first report on TRAIL sensitivity of primary meningioma cells demonstrates that TRAIL/bortezomib cotreatment may represent a novel therapeutic option for meningiomas.

A second-generation microRNA-based assay for diagnosing tumor tissue origin.

BACKGROUND: Cancers of unknown primary origin (CUP) constitute 3%-5% (50,000 to 70,000 cases) of all newly diagnosed cancers per year in the United States. Including cancers of uncertain primary origin, the total number increases to 12%-15% (180,000 to 220,000 cases) of all newly diagnosed cancers per year in the United States. Cancers of unknown/uncertain primary origins present major diagnostic and clinical challenges because the tumor tissue of origin is crucial for selecting optimal treatment. MicroRNAs are a family of noncoding, regulatory RNA genes involved in carcinogenesis. MicroRNAs that are highly stable in clinical samples and tissue specific serve as ideal biomarkers for cancer diagnosis. Our first-generation assay identified the tumor of origin based on 48 microRNAs measured on a quantitative real-time polymerase chain reaction platform and differentiated 25 tumor types. METHODS: We present here the development and validation of a second-generation assay that identifies 42 tumor types using a custom microarray. A combination of a binary decision-tree and a k-nearest-neighbor classifier was developed to identify the tumor of origin based on the expression of 64 microRNAs. RESULTS: Overall assay sensitivity (positive agreement), measured blindly on a validation set of 509 independent samples, was 85%. The sensitivity reached 90% for cases in which the assay reported a single answer (>80% of cases). A clinical validation study on 52 true CUP patients showed 88% concordance with the clinicopathological evaluation of the patients. CONCLUSION: The abilities of the assay to identify 42 tumor types with high accuracy and to maintain the same performance in samples from patients clinically diagnosed with CUP promise improved utility in the diagnosis of cancers of unknown/uncertain primary origins.

Accurate classification of metastatic brain tumors using a novel microRNA-based test.

BACKGROUND: Identification of the tissue of origin of a brain metastatic tumor is vital to its management. Carcinoma of unknown primary (CUP) is common in oncology, representing 3%-5% of all invasive malignancies. We aimed to validate a recently developed microRNA-based quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) test for identifying the tumor tissue of origin, first in a consecutive cohort of metastatic tumors of known origin and then in a cohort of CUP cases resected from the central nervous system (CNS). PATIENTS AND METHODS: One hundred two resected CNS metastatic tumors with known origin, previously classified based on the patient's clinical history and pathological data, as well as a second cohort of resected CNS tumors from 57 patients originally diagnosed as CUP were studied. A qRT-PCR diagnostic assay that measures the expression level of 48 microRNAs was used to classify the tissue of origin of these metastatic tumors. RESULTS: In this blinded study, the test predictions correctly identified the reference diagnosis of the samples of known origin, excluding samples from prostate origin, in 84% of cases. In the second CUP patient cohort, the test prediction was in agreement with the diagnosis that was later confirmed clinically or with pathological evaluation in 80% of cases. CONCLUSION: In a cohort of brain and spinal metastases, a previously developed test based on the expression of 48 microRNAs allowed accurate identification of the tumor tissue of origin in the majority of cases. The high accuracy of this test in identifying the tissue of origin of metastases of unknown primary is demonstrated for the first time and may have broad clinical application.

Gene regulation by methylation.

Epigenetic gene regulation of specific genes strongly affects clinical outcome of malignant glioma. MGMT is the best studied gene for the connection of promoter methylation and clinical course in glioblastoma. While MGMT promoter methylation analysis currently does not alter treatment of glioblastoma patients, mainly because of a lack of convincing therapy to radiotherapy and concomitant administration of alkylating drugs, there is increasing interest on the part of patients and physicians in having this molecular parameter assessed. This chapter gives a short overview of the physiological characteristics of the epigenome in normal cells and tissues and the changes in epigenetic gene regulation following malignant transformation. It discusses the technical aspects, advantages, and shortcomings of currently used approaches for single-gene and genome-wide methylation analyses. Finally, an outlook is given on potential therapeutic avenues and targets to overcome tumor-suppressor gene silencing by aberrant promoter methylation in gliomas.