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Simple Summary: Temozolomide (TMZ) is the first-line drug in the treatment of glioblastoma. Due to the formation of the DNA damage O6-methylguanine, it is toxic to cancer cells, resulting in the induction of apoptosis. However, the damage also induces cellular senescence. Here, we show that not apoptosis, but cellular senescence is the main response triggered by TMZ-induced DNA damage. We analyzed the senescent cells in detail and show that they are characterized by a high level of non-repaired DNA double-strand breaks that trigger the DNA damage response. The primary damage, O6-methylguanine, is the upstream trigger, but it is not required for maintaining the senescent state. Cells which acquired temozolomide resistance also became resistant to the induction of senescence. Comparing specimens from primary and recurrent glioblastoma, we show that recurrences contain a higher proportion of senescent cells than the primary tumor, indicating that induction of senescence also occurs upon treatment in vivo, which likely plays a role in therapy resistance. First-line drug in the treatment of glioblastoma, the most severe brain cancer, is temozolomide (TMZ), a DNA-methylating agent that induces the critical damage O6-methylguanine (O6MeG). This lesion is cytotoxic through the generation of mismatch repair-mediated DNA double-strand breaks (DSBs), which trigger apoptotic pathways. Previously, we showed that O6MeG also induces cellular senescence (CSEN). Here, we show that TMZ-induced CSEN is a late response which has similar kinetics to apoptosis, but at a fourfold higher level. CSEN cells show a high amount of DSBs, which are located outside of telomeres, a high level of ROS and oxidized DNA damage (8-oxo-guanine), and sustained activation of the DNA damage response and histone methylation. Despite the presence of DSBs, CSEN cells are capable of repairing radiation-induced DSBs. Glioblastoma cells that acquired resistance to TMZ became simultaneously resistant to TMZ-induced CSEN. Using a Tet-On glioblastoma cell system, we show that upregulation of MGMT immediately after TMZ completely abrogated apoptosis and CSEN, while induction of MGMT long-term (>72 h) after TMZ did not reduce apoptosis and CSEN. Furthermore, upregulation of MGMT in the senescent cell population had no impact on the survival of senescent cells, indicating that O6MeG is required for induction, but not for maintenance of the senescent state. We further show that, in recurrent GBM specimens, a significantly higher level of DSBs and CSEN-associated histone H3K27me3 was observed than in the corresponding primary tumors. Overall, the data indicate that CSEN is a key node induced in GBM following chemotherapy. [ABSTRACT FROM AUTHOR]

Elderly patients represent a growing proportion of individuals with glioblastoma, who however, are often excluded from clinical trials owing to poor expected prognosis. We aimed at identifying age-related molecular differences that would justify and guide distinct treatment decisions in elderly glioblastoma patients. The combined DNA methylome (450 k) of four IDH wild-type glioblastoma datasets, comprising two clinical trial cohorts, was interrogated for differences based on the patients' age, DNA methylation (DNAm) age acceleration (DNAm age "Horvath-clock" minus patient age), DNA methylation-based tumor classification (Heidelberg), entropy, and functional methylation of DNA damage response (DDR) genes. Age dependent methylation included 19 CpGs (p-value ≤ 0.1, Bonferroni corrected), comprising a CpG located in the ELOVL2 gene that is part of a 13-gene forensic age predictor. Most of the age related CpGs (n = 16) were also associated with age acceleration that itself was associated with a large number of CpGs (n = 50,551). Over 70% age acceleration-associated CpGs (n = 36,348) overlapped with those associated with the DNA methylation based tumor classification (n = 170,759). Gene set enrichment analysis identified associated pathways, providing insights into the biology of DNAm age acceleration and respective commonalities with glioblastoma classification. Functional methylation of several DDR genes, defined as correlation of methylation with gene expression (r ≤ −0.3), was associated with age acceleration (n = 8), tumor classification (n = 12), or both (n = 4), the latter including MGMT. DNAm age acceleration was significantly associated with better outcome in both clinical trial cohorts, whereof one comprised only elderly patients. Multivariate analysis included treatment (RT, RT/TMZ→TMZ; TMZ, RT), MGMT promoter methylation status, and interaction with treatment. In conclusion, DNA methylation features of age acceleration are an integrative part of the methylation-based tumor classification (RTK I, RTK II, MES), while patient age seems hardly reflected in the glioblastoma DNA methylome. We found no molecular evidence justifying other treatments in elderly patients, not owing to frailty or co-morbidities. [ABSTRACT FROM AUTHOR]

The invasive behavior of glioblastoma, the most aggressive primary brain tumor, is considered highly relevant for tumor recurrence. However, the invasion zone is difficult to visualize by Magnetic Resonance Imaging (MRI) and is protected by the blood brain barrier, posing a particular challenge for treatment. We report biological features of invasive growth accompanying tumor progression and invasion based on associated metabolic and transcriptomic changes observed in patient derived orthotopic xenografts (PDOX) in the mouse and the corresponding patients' tumors. The evolution of metabolic changes, followed in vivo longitudinally by 1H Magnetic Resonance Spectroscopy (1H MRS) at ultra-high field, reflected growth and the invasive properties of the human glioblastoma transplanted into the brains of mice (PDOX). Comparison of MRS derived metabolite signatures, reflecting temporal changes of tumor development and invasion in PDOX, revealed high similarity to spatial metabolite signatures of combined multi-voxel MRS analyses sampled across different areas of the patients' tumors. Pathway analyses of the transcriptome associated with the metabolite profiles of the PDOX, identified molecular signatures of invasion, comprising extracellular matrix degradation and reorganization, growth factor binding, and vascular remodeling. Specific analysis of expression signatures from the invaded mouse brain, revealed extent of invasion dependent induction of immune response, recapitulating respective signatures observed in glioblastoma. Integrating metabolic profiles and gene expression of highly invasive PDOX provided insights into progression and invasion associated mechanisms of extracellular matrix remodeling that is essential for cell–cell communication and regulation of cellular processes. Structural changes and biochemical properties of the extracellular matrix are of importance for the biological behavior of tumors and may be druggable. Ultra-high field MRS reveals to be suitable for in vivo monitoring of progression in the non-enhancing infiltration zone of glioblastoma. [ABSTRACT FROM AUTHOR]

Glioblastoma (GBM) is the most aggressive brain tumor type in adults. GBM is heterogeneous, with a compact core lesion surrounded by an invasive tumor front. This front is highly relevant for tumor recurrence but is generally non-detectable using standard imaging techniques. Recent studies demonstrated distinct metabolic profiles of the invasive phenotype in GBM. Magnetic resonance (MR) of hyperpolarized 13C-labeled probes is a rapidly advancing field that provides real-time metabolic information. Here, we applied hyperpolarized 13C-glucose MR to mouse GBM models. Compared to controls, the amount of lactate produced from hyperpolarized glucose was higher in the compact GBM model, consistent with the accepted "Warburg effect". However, the opposite response was observed in models reflecting the invasive zone, with less lactate produced than in controls, implying a reduction in aerobic glycolysis. These striking differences could be used to map the metabolic heterogeneity in GBM and to visualize the infiltrative front of GBM. [ABSTRACT FROM AUTHOR]

Glioblastoma are notorious for their highly invasive growth, diffusely infiltrating adjacent brain structures that precludes complete resection, and is a major obstacle for cure. To characterize this “invisible” tumor part, we designed a high resolution multimodal imaging approach assessing in vivo the metabolism of invasively growing glioma xenografts in the mouse brain. Animals were subjected longitudinally to magnetic resonance imaging (MRI) and 1H spectroscopy (MRS) at ultra high field (14.1 Tesla) that allowed the measurement of 16 metabolic biomarkers to characterize the metabolic profiles. As expected, the neuronal functionality was progressively compromised as indicated by decreasing N‐acetyl aspartate, glutamate and gamma‐aminobutyric acid and reduced neuronal TCA cycle (−58%) and neurotransmission (−50%). The dynamic metabolic changes observed, captured differences in invasive growth that was modulated by re‐expression of the tumor suppressor gene WNT inhibitory factor 1 (WIF1) in the orthotopic xenografts that attenuates invasion. At late stage mice were subjected to 13C MRS with infusion of [1,6‐13C]glucose and 18FDG positron emission tomography (PET) to quantify cell‐specific metabolic fluxes involved in glucose metabolism. Most interestingly, this provided the first in vivo evidence for significant glucose oxidation in glioma cells. This suggests that the infiltrative front of glioma does not undergo the glycolytic switch per se, but that environmental triggers may induce metabolic reprograming of tumor cells. [ABSTRACT FROM AUTHOR]

The O(6)-methylguanine-DNA methyltransferase (MGMT) gene is located at chromosome 10q26 and codes for a DNA repair enzyme that--if active--can counteract the effects of alkylating chemotherapy. Malignant gliomas often have the MGMT gene inactivated due to aberrant methylation of its promoter region. The assessment of the MGMT promoter methylation status has become of clinical relevance as a molecular marker associated with response to alkylating chemotherapy and prolonged survival of glioblastoma patients. MGMT promoter methylation testing is also on the merge of being used as a marker for patient selection within clinical trials, e.g., the current CENTRIC trial that is specifically focusing on patients with MGMT promoter-methylated glioblastomas. In anaplastic gliomas, MGMT promoter methylation is a favorable prognostic marker independent of the type of therapy, i.e., radio- or chemotherapy. This occurrence might be associated with the high incidence of other prognostically favorable molecular markers in these tumors, such as IDH1 mutation, 1p/19q deletion or yet to be identified novel aberrations. A variety of different methods are being used to assess MGMT promoter methylation in clinical samples, which may give rise to inter-laboratory variations in test results. Immunohistochemical determination of MGMT protein expression has not proven reliable for diagnostic purposes. This brief review article aims to summarize the main aspects of MGMT promoter methylation testing in contemporary neuro-oncology, in particular its value as a clinically useful molecular marker, putting it into the context of other molecular markers of clinical use in gliomas of adult patients. [ABSTRACT FROM AUTHOR]

BACKGROUND: Cilengitide is a selective integrin inhibitor that is well tolerated and has demonstrated biologic activity in patients with recurrent malignant glioma. The primary objectives of this randomized phase 2 trial were to determine the safety and efficacy of cilengitide when combined with radiation and temozolomide for patients with newly diagnosed glioblastoma multiforme and to select a dose for comparative clinical testing. METHODS: In total, 112 patients were accrued. Eighteen patients received standard radiation and temozolomide with cilengitide in a safety run-in phase followed by a randomized phase 2 trial with 94 patients assigned to either a 500 mg dose group or 2000 mg dose group. The trial was designed to estimate overall survival benefit compared with a New Approaches to Brain Tumor Therapy (NABTT) Consortium internal historic control and data from the published European Organization for Research and Treatment of Cancer (EORTC) trial EORTC 26981. RESULTS: Cilengitide at all doses studied was well tolerated with radiation and temozolomide. The median survival was 19.7 months for all patients, 17.4 months for the patients in the 500 mg dose group, 20.8 months for patients in the 2000 mg dose group, 30 months for patients who had methylated O6-methylguanine-DNA methyltransferase (MGMT) status, and 17.4 months for patients who had unmethylated MGMT status. For patients aged ≤70 years, the median survival and survival at 24 months was superior to what was observed in the EORTC trial (20.7 months vs 14.6 months and 41% vs 27%, respectively; P = .008). CONCLUSIONS: Cilengitide was well tolerated when combined with standard chemoradiation and may improve survival for patients newly diagnosed with glioblastoma multiforme regardless of MGMT methylation status. The authors concluded that, from an efficacy and safety standpoint, future trials of this agent in this population should use the 2000 mg dose. Cancer 2012. © 2012 American Cancer Society. [ABSTRACT FROM AUTHOR]

The methylation status of the O-methylguanine-DNA methyltransferase ( MGMT) gene is an important predictive biomarker for benefit from alkylating agent therapy in glioblastoma. Recent studies in anaplastic glioma suggest a prognostic value for MGMT methylation. Investigation of pathogenetic and epigenetic features of this intriguingly distinct behavior requires accurate MGMT classification to assess high throughput molecular databases. Promoter methylation-mediated gene silencing is strongly dependent on the location of the methylated CpGs, complicating classification. Using the HumanMethylation450 (HM -450K) BeadChip interrogating 176 CpGs annotated for the MGMT gene, with 14 located in the promoter, two distinct regions in the CpG island of the promoter were identified with high importance for gene silencing and outcome prediction. A logistic regression model (MGMT-STP27) comprising probes cg1243587 and cg12981137 provided good classification properties and prognostic value (kappa = 0.85; log-rank p < 0.001) using a training-set of 63 glioblastomas from homogenously treated patients, for whom MGMT methylation was previously shown to be predictive for outcome based on classification by methylation-specific PCR. MGMT-STP27 was successfully validated in an independent cohort of chemo-radiotherapy-treated glioblastoma patients ( n = 50; kappa = 0.88; outcome, log-rank p < 0.001). Lower prevalence of MGMT methylation among CpG island methylator phenotype (CIMP) positive tumors was found in glioblastomas from The Cancer Genome Atlas than in low grade and anaplastic glioma cohorts, while in CIMP-negative gliomas MGMT was classified as methylated in approximately 50 % regardless of tumor grade. The proposed MGMT-STP27 prediction model allows mining of datasets derived on the HM -450K or HM-27K BeadChip to explore effects of distinct epigenetic context of MGMT methylation suspected to modulate treatment resistance in different tumor types. [ABSTRACT FROM AUTHOR]

Background: Several studies have established Glioblastoma Multiforme (GBM) prognostic and predictive models based on age and Karnofsky Performance Status (KPS), while very few studies evaluated the prognostic and predictive significance of preoperative MR-imaging. However, to date, there is no simple preoperative GBM classification that also correlates with a highly prognostic genomic signature. Thus, we present for the first time a biologically relevant, and clinically applicable tumor Volume, patient Age, and KPS (VAK) GBM classification that can easily and non-invasively be determined upon patient admission. Methods: We quantitatively analyzed the volumes of 78 GBM patient MRIs present in The Cancer Imaging Archive (TCIA) corresponding to patients in The Cancer Genome Atlas (TCGA) with VAK annotation. The variables were then combined using a simple 3-point scoring system to form the VAK classification. A validation set (N = 64) from both the TCGA and Rembrandt databases was used to confirm the classification. Transcription factor and genomic correlations were performed using the gene pattern suite and Ingenuity Pathway Analysis. Results: VAK-A and VAK-B classes showed significant median survival differences in discovery (P = 0.007) and validation sets (P = 0.008). VAK-A is significantly associated with P53 activation, while VAK-B shows significant P53 inhibition. Furthermore, a molecular gene signature comprised of a total of 25 genes and microRNAs was significantly associated with the classes and predicted survival in an independent validation set (P = 0.001). A favorable MGMT promoter methylation status resulted in a 10.5 months additional survival benefit for VAK-A compared to VAK-B patients. Conclusions: The non-invasively determined VAK classification with its implication of VAK-specific molecular regulatory networks, can serve as a very robust initial prognostic tool, clinical trial selection criteria, and important step toward the refinement of genomics-based personalized therapy for GBM patients. [ABSTRACT FROM AUTHOR]

Malignant gliomas are currently diagnosed based on morphological criteria and graded according to the World Health Organization classification of primary brain tumors. This algorithm of diagnosis and classification provides clinicians with an estimated prognosis of the natural course of the disease. It does not reflect the expected response to specific treatments beyond surgery (eg, radiotherapy or alkylating chemotherapy). Clinical experience has revealed that gliomas sharing similar histomorphological criteria might indeed have different clinical courses and exhibit highly heterogenous responses to treatments. This was very impressively demonstrated first for oligodendrogliomas. The presence or lack of combined deletions of the chromosomal segments 1p/19q was associated with different benefit from radiotherapy and chemotherapy. We review current molecular markers for malignant gliomas and discuss their current and future impact on clinical neuro-oncology. [ABSTRACT FROM AUTHOR]

Glioblastoma (GBM) is a morphologically heterogeneous tumor type with a median survival of only 15 months in clinical trial populations. However, survival varies greatly among patients. As part of a central pathology review, we addressed the question if patients with GBM displaying distinct morphologic features respond differently to combined chemo-radiotherapy with temozolomide. Morphologic features were systematically recorded for 360 cases with particular focus on the presence of an oligodendroglioma-like component and respective correlations with outcome and relevant molecular markers. GBM with an oligodendroglioma-like component (GBM-O) represented 15% of all confirmed GBM (52/339) and was not associated with a more favorable outcome. GBM-O encompassed a pathogenetically heterogeneous group, significantly enriched for IDH1 mutations (19 vs. 3%, p = 0.003) and EGFR amplifications (71 vs. 48%, p = 0.04) compared with other GBM, while co-deletion of 1p/19q was found in only one case and the MGMT methylation frequency was alike (47 vs. 46%). Expression profiles classified most of the GBM-O into two subtypes, 36% (5/14 evaluable) as proneural and 43% as classical GBM. The detection of pseudo-palisading necrosis (PPN) was associated with benefit from chemotherapy ( p = 0.0002), while no such effect was present in the absence of PPN ( p = 0.86). In the adjusted interaction model including clinical prognostic factors and MGMT status, PPN was borderline nonsignificant ( p = 0.063). Taken together, recognition of an oligodendroglioma-like component in an otherwise classic GBM identifies a pathogenetically mixed group without prognostic significance. However, the presence of PPN may indicate biological features of clinical relevance for further improvement of therapy. [ABSTRACT FROM AUTHOR]

Glioblastoma is the most common malignant primary brain tumor in adults. Its often rapid clinical course, with many medical and psychosocial challenges, requires a multidisciplinary management. Modern multimodality treatment and care improve patients' life expectancy and quality of life. This review covers major aspects of care of glioblastoma patients with a focus on the management of common symptoms and complications. We aim to provide a guide for clinicians confronted with glioblastoma patients in their everyday practice. Ann Neurol 2011; [ABSTRACT FROM AUTHOR]

Medulloblastomas (MB) are the most common malignant brain tumors in childhood. Alkylator-based drugs are effective agents in the treatment of patients with MB. In several tumors, including malignant glioma, elevated O-methylguanine-DNA methyltransferase (MGMT) expression levels or lack of MGMT promoter methylation have been found to be associated with resistance to alkylating chemotherapeutic agents such as temozolomide (TMZ). In this study, we examined the MGMT status of MB and central nervous system primitive neuroectodermal tumor (PNET) cells and two large sets of primary MB. In seven MB/PNET cell lines investigated, MGMT promoter methylation was detected only in D425 human MB cells as assayed by the qualitative methylation-specific PCR and the more quantitative pyrosequencing assay. In D425 human MB cells, MGMT mRNA and protein expression was clearly lower when compared with the MGMT expression in the other MB/PNET cell lines. In MB/PNET cells, sensitivity towards TMZ and 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU) correlated with MGMT methylation and MGMT mRNA expression. Pyrosequencing in 67 primary MB samples revealed a mean percentage of MGMT methylation of 3.7-92% (mean: 13.25%, median: 10.67%). Percentage of MGMT methylation and MGMT mRNA expression as determined by quantitative RT-PCR correlated inversely ( n = 46; Pearson correlation r = 0.14, P = 0.01). We then analyzed MGMT mRNA expression in a second set of 47 formalin-fixed paraffin-embedded primary MB samples from clinically well-documented patients treated within the prospective randomized multicenter trial HIT'91. No association was found between MGMT mRNA expression and progression-free or overall survival. Therefore, it is not currently recommended to use MGMT mRNA expression analysis to determine who should receive alkylating agents and who should not. [ABSTRACT FROM AUTHOR]