Epigenetics in human gliomas

Cancer Letters xxx (2012) xxx–xxx

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Epigenetics in human gliomas Simone Kreth a, Niklas Thon b, Friedrich W. Kreth b,⇑ a b

Research Group of Molecular Medicine, Department of Anesthesiology, University of Munich (LMU), Germany Department of Neurosurgery, University of Munich (LMU), Germany

a r t i c l e

i n f o

Article history: Available online xxxx Keywords: Glioblastoma Epigenetic networks MicroRNAs Stereotactic biopsy

a b s t r a c t Aberrant epigenetic landscapes and their involvement in genesis and progression of tumors, as well as in treatment responses and prognosis, indicate one of the most emerging fields in cancer research. In gliomas, the most common human primary brain tumors, and in particular in glioblastoma, the most malignant and devastating brain tumor entity in adults, the elucidation of distinct patterns of aberrant DNA methylation, histone modification, and miRNA expression and their interrelationship has fundamentally changed our point of view on these highly heterogeneous tumors. In the current review article, we address the basic principles of epigenetic control in gliomas, their current and putative future role in prognostic and predictive models and possible interactions within the epigenetic network. We discuss diagnostic and therapeutic opportunities appearing at horizon of epigenetic research. Moreover, we present current and propose future clinical workflow models for molecular characterization of malignant gliomas. Ó 2012 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Diffuse gliomas represent a broad diagnostic category that also includes glioblastoma (GBM), the most common and most malignant primary brain tumor in adults. Whereas GBMs are rapidly progressive, resistant to therapy, and typically follow fatal clinical courses with median survival times in the range of 15 months, their low grade counterparts (WHO grade II gliomas) can exhibit years of clinical and radiological stability without any applied therapy [1,2]. However, even these low grade variants clearly represent malignant tumor entities, which undergo more or less delayed malignant transformation and rapid clinical decline later on [3]. Traditional molecular approaches, which have mainly focused on structural changes in genes such as point mutations, gene deletions, and rearrangements, have added important insights to both the process of glioma-genesis itself and the diagnostic, prognostic and predictive network of these tumors. Recently, for example, mutations in the IDH1/IDH2 gene encoding isocitrate dehydrogenase 1 and 2 have been identified as one of the earliest molecular event in the pathway to grade II/III astrocytomas, oligodendrogliomas, oligo-astrocytomas, and secondary GBMs but not in the development of de novo GBMs. These findings suggest that histologically differently appearing tumor subclasses might share common precursor cells and that histologically indistinguishable

tumors (e.g. de novo GBM versus secondary GBM) could be separated into biologically distinct subclasses [4–7]. The additional focus on mechanisms of regulation of gene expression at the transcriptional/translational level indicates one of the most emerging fields in cancer research: Recent research has provided evidence that malignant transformation results from a complex interplay of both genetic alterations and epigenetic changes affecting various cellular processes, including cell proliferation and invasion, DNA repair, apoptosis, angiogenesis and cell cycle regulation, finally leading to the development of a malignant phenotype [8,9]. Among the most studied epigenetic changes occurring in gliomas, DNA methylation, and histone modifications can be mentioned. Recently, non-coding RNAs have been recognized to constitute another level of epigenetic control. Identification and characterization of epigenetic alterations in human gliomas have added important parts to the puzzle of glioma-genesis and will lead to the discovery of novel prognostic and predictive biomarkers. Moreover, the reversible nature of epigenetic modifications kindled strong efforts to develop new therapeutic approaches aiming at epigenetic landmarks of human gliomas [10]. Here, we will provide a brief survey about the basic mechanisms of epigenetic control found in human glioma, their clinical significance, and the resulting therapeutical perspectives. 2. DNA methylation

⇑ Corresponding author. Address: Department of Neurosurgery, Ludwig-Maximilians-University, Campus Großhadern, Marchioninistrasse 15, D-81377 München, Germany. Tel.: +49 (0) 89 7095 3555; fax: +49 (0) 89 7095 2592. E-mail address: [email protected] (F.W. Kreth).

The most studied epigenetic modification in humans is cytosine methylation, which describes the covalent addition of a methyl group at the 50 -position of a cytosine nucleotide. In mammalian

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cells, DNA methylation predominantly occurs on a cytosine residue followed by a guanine. These CpG dinucleotides tend to cluster to so-called CpG islands being located in the promoter regions of more than half of all human genes, or to CpG island shores, which are regions of lower CpG density that lie in close proximity of CpG islands [11,12]. DNA methylation is mediated by the DNMT (DNA methyltransferase) family of enzymes that catalyze the transfer of a methyl group from S-adenosyl methionine to DNA. So far, five members of the DNMT family have been identified: DNMT1, DNMT2, DNMT3a, DNMT3b, and DNMT3L. Whereas DNMT3a and DNMT3b target unmethylated CpGs and therefore are termed de novo methyltransferases, DNMT1 preferentially targets hemimethylated DNA to maintain the methylation pattern during DNA replication; DNMT3L is required for maternal genomic imprinting processes [13,14]. Generally, methylation of CpG islands and island shores is closely associated with transcriptional inactivation. Less frequently, when occurring at gene bodies, DNA methylation is associated with transcriptional activation. In human cancer, it is widely known that global alterations of DNA methylation profiles play an important role in tumor initiation and progression [15,16]. 2.1. DNA methylation in gliomas Human glioma exhibit tumor-typical changes of methylation patterns, which are characterized by a global loss of methylation combined with focal hypermethylation [17–20]. Hypomethylation mostly takes place at DNA-repetitive regions and may promote tumor growth by activation of oncogenes and by increasing genomic instability. Hypermethylation mostly occurs at the promoter CpG island of genes that are involved in processes leading to tumor formation and progression and has been shown for a wide variety of genes associated with tumor suppression [21,22], DNA repair [23], cell cycle regulation [24], apoptosis [25,26], invasion [27,28], and migration [29], and the list of examples is constantly increasing (Table 1). Interestingly, the methylation patterns between different glioma grades appear to differ, as differences in the methylation status of various genes has been detected between gliomas WHO grade II, III, and IV [30]. Within the framework of The Cancer Genome Atlas (TCGA) project, a methylation profiling study by Noushmehr et al. identified a GBM tumor phenotype that is characterized by a concerted hypermethylation of a large number of gene loci, which has been referred to as glioma CpG island methylator phenotype (G-CIMP) [6]. G-CIMP is associated with prolonged survival and with defined gene expression profiles (proneural expression patterns) [31]. Furthermore, a close association with IDH1 mutations was found, which may be explained by metabolic alterations resulting from these mutations: IDH proteins catalyze the oxydative decarboxylation of isocitrate to a-ketoglutarate (a-KG); IDH mutations lead to changes in enzymatic activities: a-KG production is reduced, and instead, the metabolite 2-hydroxyglutarate (2-HG) is produced, which competitively inhibits the activity of enzymes regulating DNA- and histone methylation (a-KG-dependent dioxygenases), including histone demethylases [32] and the TET family of 5mC hydroxylases [33–35]. Recently, TET proteins have been identified as a new class of enzymes that are capable to alter the methylation status of the DNA by converting 5-methylcytosine (5mC) to 5hydroxymethylcytosine (5hmC). The biological function of 5hmC has not conclusively been elucidated yet. Most recent data, however, support the theory that it is an intermediate in DNA demethylation processes. In human gliomas a remarkable reduction of 5hmC as compared to normal brain and an inverse relationship between 5hmC levels and cell proliferation have been reported [36,37]. These findings unveil an additional level of regulation and demonstrate the strong interrelation of genetic and epigenetic events in glioma-genesis [38,39].

Table 1 Major epigenetic alterations found in human gliomas. DNA methylation Genes GATA4, NDRG2 MGMT p14ARF TMS1/ASC, WWOX SOCS3, PCDH-gamma-A11 Sox2

Pathway Tumor suppression DNA repair Cell cycle regulation Apoptosis Invasion Migration

[21,22] [23] [24] [25,26] [27,28] [29]

Histone modifications Global aberrations Mutations Histone deacetylases Histone demethylases Histone methyltransferases

(HDAC2, HDAC9) (JMJD1A, JMJD1B) (SET7, SETD7, MLL, MLL3, MLL4)

Altered expression levels Histone deacetylases

(HDAC1, HDAC2, HDAC3)

Modifications of individual genes Genes Effects RRP22 Repressed expression P21 Repressed expression HOXA9 Enhanced expression

[5]

[53] [54] [55]

Micro-RNA deregulation Down-regulation of tumor suppressive miRNAs mi-RNA Target gene miR-34a c-Met, Notch miR-146a Notch miR-7 EGFR miR-128 Bmi-1 miR-195 E2F3, CCND3

[79] [80] [81] [82] [83]

Up-regulation of oncogenic miRNAs miR-21 RECK, TIMP3 miR-26a pTEN, RB1 miR-10b Cell-cycle inhibitor  miR-30e IjBa miR-221/222 p27Kip1, PTPl, PUMA,

[85] [75,86] [76] [87] [88–90]

In the last decade, research on DNA methylation processes has led to the discovery of an important biomarker that has increasingly gained translational relevance: The O6-methylguanine-DNA methyltransferase (MGMT) is a DNA repair enzyme that removes alkyl adducts at the O6 position of guanine and thereby protects normal cells from carcinogens, but – unfortunately – also protects tumor cells from chemotherapy with alkylating agents such as temozolomide. MGMT expression can be epigenetically silenced by promoter methylation, a phenomenon that is found in 35–45% of malignant gliomas and in 80% of WHO grade II gliomas [40,41]. Promoter methylation status has been identified as a strong and independent predictive factor of treatment response for malignant glioma patients undergoing chemotherapy with alkylating agents. Generally, carriers of the methylated form of the MGMT promoter respond substantially better to therapy with temozolomide as compared to those with an unmethylated MGMT promoter [42,43]. Determination of the MGMT promoter methylation status has been established as an important clinical marker in neuro-oncology and is increasingly used for stratification in clinical trials. MGMT promoter methylation, however, is not unequivocally linked to a favorable treatment response. Not all patients with a methylated promoter showed treatment response after temozolomide, and a considerable number of unmethylated tumors experienced a surprisingly favorable outcome. In these patients, discordant findings of MGMT promoter methylation and mRNA MGMT expression have been demonstrated: High (low) mRNA MGMT expression was detected in approximately 25% of glioblastomas despite a methylated (unmethylated) MGMT promoter; those with low transcriptional

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S. Kreth et al. / Cancer Letters xxx (2012) xxx–xxx

activity exhibited a better treatment response, which was independent of MGMT promoter methylation. The underlying mechanisms of discordance still remain unclear. It has been hypothesized that low MGMT expression levels combined with an unmethylated promoter might result from transcript destabilization and/or transcription-repressing factors, such as miRNA regulation or histone modifications [44]. Existence of discordant findings explains that treatment decision with respect to chemotherapy cannot be based on the MGMT promoter methylation status alone. 3. Histone modifications Chromatin is the condensed form of DNA and histones within the nucleus of a cell. In eukaryotes, chromatin is composed of 147 base pairs of DNA that are tightly wrapped around an octamer of each two copies of the four core histones H2A, H2B, H3 and H4. The resulting nucleosomes are indicated as the fundamental repeating units of chromatin [45]. As each core histone possesses an amino-terminal ‘‘tail’’ which protrudes from the nucleosome, histones and particularly their tails can be subject to a remarkable number of post-translational modifications. Histone modifications comprise prominent marks like acetylation, methylation and phosphorylation, but also less studied modifications such as ubiquitylation, sumoylation, ADP ribosylation, deamination and proline isomerisation [46]. Each of these histone modifications is able to affect chromatin structure, thereby leading to alterations in DNA repair and replication as well as in gene transcription. Regarding the latter, histone modifications can broadly be categorized into active versus passive marks. In particular histone acetylation and methylation have been found to play a remarkable role in carcinogenesis [15,47]. The acetylation of lysine residues is regulated by the opposing action of histone acetyl transferases (HATs) and histone deacetylases (HDACs). Acetylation neutralizes the positive charge of lysine residue and thereby weakens the bond between DNA and histone tails. Thus, histone acetylation is linked to transcriptional activation, whereas deacetylation is generally associated with repression of transcription. Histone methylation mainly occurs on the side chains of lysines and arginines, which influences the activity of effector proteins of the transcriptional machinery. In contrast to acetylation of histones, which is generally associated with activation of gene expression, histone methylation may either activate (e.g. H3K4me2, H3K4me3) or repress (H3K9me2, H3K27me3) transcription depending on the respective methylation sites [48–50]. 3.1. Histone modifications in gliomas Recent data have mounted evidence that alterations at the histone level may also play a role in glioma-genesis. These alterations encompass a globally deregulated expression of genes involved in histone modifications as well as changes in the histone modification pattern of individual genes (Table 1). Global aberrations at the histone level result from mutations in regulatory genes, as detected in a large-scale genomic analysis of GBM samples, including HDACs (HDAC2 and HDAC9), histone demethylases (JMJD1A and JMJD1B), histone methylatransferases (SET7, SETD7, MLL, MLL3 and MLL4) [5]. Furthermore, altered expression levels of HDACs due to so far not defined reasons have been reported and have been linked to tumor recurrence and progression (HDAC1, HDAC2, and HDAC3) [51,52]. Histone modifications regulating individual genes have been reported in several studies. For example, a repressed expression of the tumor suppressor RRP22 and of the cell cycle regulator p21, and an enhanced expression of the pro-proliferative transcription factor HOXA9 have been linked to alterations in histone modification patterns [53–55]. However, the actual functional

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roles of histone modifications in gliomas and their potential to serve as biomarkers and/or therapeutic targets, still remains to be fully elucidated. 4. MicroRNAs Non-coding RNAs have recently been found to play an important role in the epigenetic regulation of gene expression [56,57]. Among these, microRNAs (miRNA) are the best characterized modulators of gene function: MiRNAs are double-stranded RNA molecules of approximately 22 nucleotides (nt) length which – in several processing steps – derive from larger precursor transcripts that are ubiquitously encoded in the human genome. By binding to specific recognition sequences within the 30 -untranslated region (30 -UTR) of target mRNAs, miRNAs are potent regulators of gene expression by either repression of translation or mRNA degradation [58,59]. Target recognition appears to be mainly mediated through basepairing of an eight nucleotide short sequence in the 50 -region of the miRNA [60]. Whereas some miRNAs regulate specific individual targets, evidence from multiple studies suggests that a single key miRNA may regulate up to several hundreds of target genes, and many types of miRNAs regulate their targets cooperatively [61,62]. Using a combination of computational prediction methods and different sequencing techniques a significant number of these regulatory molecules have been identified [63,64]. The current release of ‘‘mirbase’’, the official registry for known miRNA-genes, contains more than 17,000 mature miRNA sequences in over 140 species [65]. Recent work suggests that large parts of the transcriptome are subject to regulatory control exerted by miRNAs [66]. It is therefore not surprising that dysregulation of miRNA expression has been associated with a wide range of pathologies and their prognosis. 4.1. MiRNAs in gliomas Aberrant expression of miRNAs has been detected in many types of human tumors, including gliomas [67,68]. Recent data have shown that human tumors are characterized by globally down-regulated miRNA expression profiles due to a general defect in the miRNA production process, which promotes the view that miRNAs may mainly serve as ‘‘guardians’’ of biologic processes [69]. Nevertheless, miRNA have been demonstrated not to act only as tumor suppressors, but also – dependent on the function of the targeted mRNA – as oncogenes [70,71]. Accordingly, altered miRNA expression levels exert major impact on oncogenic processes. The reasons for the widespread differential expression of miRNAs in malignant as compared to normal cells are not fully elucidated. However, epigenetic modifications within the transcriptional regulatory sequences of miRNAs, but also genetic alterations like mutations, genomic deletions or gene amplifications, which can affect miRNA maturation and/or interactions with mRNA targets, are thought to contribute to miRNA dysregulation [72–74]. In GBM, high-throughput analyses have identified various miRNAs that are differentially expressed when compared to non-neoplastic brain tissues [75–77]. Thus, miRNAs are thought to be important mediators of multiple biological characteristics of GBM, including cell proliferation, G1/S cell cycle progression, cell survival, cell migration and cell invasion [78]. Although the exact function of altered miRNAs in glioma and of the complex network they regulate have not been elucidated yet, a growing number of studies have assigned single miRNAs to distinct functions in the processes of glioma-genesis and progression (Table 1). For example, miRNAs that are downregulated in gliomas as compared to normal brain have been found to function as tumor suppressors by directly targeting the oncogenes c-Met, Notch [79,80], Bmi-1 [76], the epidermal growth factor receptor [81], receptor tyrosine

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kinases [82], and cell cycle components [83]. Contrarily, miRNAs with enhanced expression in glioma might be designated as oncogenes; i.e. miR-21 promoting invasion by targeting regulators of matrix metalloproteinases, miR-26a targeting PTEN, and miR-10b and miR-221 targeting cell cycle inhibitors, and miR30e targeting IjBa [84–90]. 5. The ‘‘epigenetic network’’ Epigenetic regulation pathways gain further complexity by interrelationships between the epigenetic players: (i) Expression of miRNA itself can be modified by changes in chromatin structures caused by covalent modifications on histones and/or DNA methylation. For instance, it has recently been shown that miR-127 and miR124a, two putative tumor suppressor miRNAs, are methylated in tumor cells [91,92]. (ii) Tumors can use miRNAs reciprocally to target the epigenetic machinery. For example, miR-29b was found to target DNMT3A and 3B in acute myeloid leukemia, and miR-449a controls HDAC1 in prostate cancer cells. In human glioma, miR185 has recently been described to be a regulator of DNMT1, and its overexpression results in a global DNA hypomethylation [93– 95]. Furthermore, miR-101 which targets the histone methyltransferase EZH2 has been found to be down-regulated in human GBM thereby promoting tumor growth [96,97]. (iii) Histone modification enzymes may be silenced by CpG hypermethylation. For instance, silencing of the NSD1 gene, which encodes a histone methyltransferase involved in chromatin regulation, results in diminished methylation of histone residues H4-K20 and H3-K36, which in turn leads to activation of the oncogene MEIS1. This epigenetic alteration has been found to be a common event in gliomas [98]. Further studies on a genome-wide scale are needed to fully elucidate epigenetic patterns in gliomas, which will elucidate the complex pattern of the epigenetic networks of these highly heterogeneous tumors [99]. 6. Epigenetics in glioma: perspectives Enormous recent advances in high throughput analyses have opened up not only DNA sequences of individual whole genomes of human GBM samples but also complete DNA methylomes, histonomes and transcriptomes [7,5]. Based on these new technical possibilities, current brain tumor research aims at the identification and characterization of new biomarkers that may allow to exactly determine the diagnosis and to estimate prognosis and treatment response of the individual patient [100–102]. It therefore can be expected that in human gliomas, determination of MGMT promoter methylation may only be the spearhead of new evolving biomarkers based on epigenetic landmarks. Since the approval of DNA demethylating agents and histone deacetylase inhibitors for the treatment of lymphoma patients, the potential of epigenetic proteins and modifications to serve as drug targets has widely been recognized [103,104]. The underlying idea is (i) to reverse tumor specific epigenetic alterations that may essentially drive tumor growth (for example by silencing tumor suppressors, activating oncogenes, or affecting DNA repair), and (ii) to induce changes in chromatin structure permitting better access of DNA damaging drugs [105,106]. In human gliomas, several approaches aiming at the global epigenetic machinery have been made: Treatment with histone deacetylase inhibitors (HDACi) such as aliphatic acids (valproic acid, butyrate), suberoylanilide hydrocamic acid (SAHA, vorinostat), alone or in combination with radiotherapy have been suggested to exert beneficial effects in preclinical models [107–109]; furthermore, pilot studies using SAHA or valproic acid in GBM have described low toxicity and moderate response rates [110–113]. However, these therapy approaches only

represent a first step in a new direction; future approaches should enable a specific targeting of defined epigenetic landmarks. Perspectives of clinical concepts based on miRNA regulatory networks are promising, because miRNAs may prove as both suitable biomarkers and therapeutic targets in oncology: Current studies indicate the presence of miRNAs in blood circulation, which are set free from tumor cells and thus, characteristical miRNA signatures may be linked to the diagnosis and prognosis for certain tumor types [114–116]. Concerning brain tumors, a specific miRNA signature in the peripheral blood and in cerebrospinal fluid of GBM patients was reported recently [117,118]. The rapidly expanding knowledge of the substantial role of miRNA in epigenetic regulation has led to the development of therapeutic concepts based on miRNA for a range of diseases [119–121]. Basically, two approaches exist: first, suppression of disease-specific miRNAs, and second, application of specific miRNAs in excess to the diseased tissue or organism to achieve therapeutic effects by selective gen silencing [122–124]. The evolving concepts are promising; however at present all techniques (i.e. anti-miR oligonucleotides, competitive miR-inhibitors) are still in early stages and not ready to be introduced in clinical practice [125]. One general key challenge is the targeted and effective delivery of the small nucleotide agents to cells and tissues, and in gliomas, this is further complicated by the blood–brain-barrier. Since one miRNA can influence the expression of various target genes, several important aspects must be reconsidered before miRNAs may be used therapeutically: First, manipulation of one single miRNA may affect the expression of many different proteins and may potentially cause devastating side effects. Second, miRNAs can affect different cell types and tissues with occasionally contrary effects and tissue specific delivery of miRNAs or antagonists is yet an unsolved problem. 7. Clinical workflow: present and future The consideration of molecular biomarkers has improved both the diagnostic precision within the framework of the current WHO classification and the discrimination of glioma subgroups exhibiting the same WHO grade but differences in prognosis and treatment response [2]. The detection of a 1p/19 co-deletetion, for example, supports the diagnosis of an oligidendroglioma/oligoastrocytoma and has been associated with a favorable outcome after chemotherapy and/or radiation therapy [126,127]. Screening for IDH1/2 mutations is helpful to distinguish WHO grade I pilocytic astrocytomas and ependymomas (harboring no IDH mutations) from diffuse astrocytomas and to differentiate diffuse tumor infiltration from reactive gliosis. IDH mutations are associated with a better prognosis in anaplastic astrocytomas and glioblastomas [128,129]. Given the fact that complete tumor resection is seldom possible in grade II–IV gliomas and incomplete resections usually did not gain prognostic impact, the development of minimal invasive molecular characterization strategies is of utmost interest [130]. Recently, novel molecular stereotactic biopsy procedures combining histopathologic diagnosis with size-adjusted molecular genetic analyses have been shown to achieve highly reproducible and valid results at the DNA and RNA level as well; remarkably, information about MGMT promoter methylation and MGMT mRNA expression, TP53 mutational status, and LOH on 1p/19q-status could be obtained from a single 1-mm3 stereotactic tissue sample collected from an exactly defined site of the tumor space [131,132]. The introduction of the massively parallel ‘‘next generation’’ sequencing (NGS) techniques into scientific practice marks the start of a revolution in genomic research [133]; whole-genome, transcriptome- and methylome-sequencing is currently at the horizon of clinical research and could be performed even in small-sized tumor tissue samples. NGS platforms provide a comprehensive and unbiased view of the genome and epigenome and the integration

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Fig. 1. Flow chart illustrating present and future clinical workflow for glioma treatment. Tumor samples are obtained by minimally invasive stereotactic biopsy procedure; samples are used for histologic analyses and for extraction of nucleic acids. Classical workflow (1): Tumor samples (smear and paraffine embedded) are used for histopathologic diagnosis; categorized treatment concepts are applied. Modern workflow (1 + 2) implementing molecular features of the tumor (MGMT promoter methylation, LOH 1p19q, IDH1/2 mutations, further optional markers); therapeutic proceedings are adapted to the results of the analyses. Future workflow (1 + 3): Tumor samples are used for histology and next generation sequencing analyses. Whole genome-, epigenome- and transcriptome analyses allow a complete molecular characterization of the tumor. Therapy comprises an individualized concept integrating all treatment modalities.

of NGS-based data will be essential to understand and identify the dense web of connected intracellular pathways that drive formation and progression of gliomas [134,135]. With falling costs of the NGS technology and improved bioinformatic handling of the data, the simultaneous genome wide measurement of multiple epigenetic modifications in conjunction with the transcriptome and genetic variations of the same biologic sample may become a clinical standard approach in the future. In this setting, removal of tumor samples by minimal-invasive methods, such the molecular stereotactic procedure, will be of particular value. The information gained from this approach will allow creating customized therapeutic regimens for each patient based on the unique genomic signature of the individual tumor. Also, therapeutic strategies for the treatment of gliomas have to be markedly improved, which includes the development of new epigenetic therapies and the gain of knowledge how to synchronize them with other treatment modalities. A modern present clinical workflow and the anticipated future developments are schematically illustrated in Fig. 1.

8. Conclusion Currently, we are at the beginning of a comprehensive understanding of the glioma epigenome. Before introducing therapeutic concepts in the treatment of brain tumors based on epigenetic aberrations, critical aspects have yet to be prospectively addressed in clinical and basic research, and technical tools have to be further developed. Nevertheless, we envision that the unique properties of epigenetic changes in gliomas offer a highly promising future diagnostic and therapeutic potential which will certainly lead to innovative clinical concepts.

Acknowledgement Grant support from the Institutional funding of the LMU Munich. We thank Dr. S. Schütz for suggestions on the manuscript.

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Please cite this article in press as: S. Kreth et al., Epigenetics in human gliomas, Cancer Lett. (2012), http://dx.doi.org/10.1016/j.canlet.2012.04.008