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Background Glioblastoma (GBM) is an aggressive brain cancer that typically results in death in the first 15 months after diagnosis. There have been limited advances in finding new treatments for GBM. In this study, we investigated molecular differences between patients with extremely short (≤ 9 months, Short term survivors, STS) and long survival (≥ 36 months, Long term survivors, LTS). Methods Patients were selected from an in-house cohort (GLIOTRAIN-cohort), using defined inclusion criteria (Karnofsky score > 70; age Results Transcriptomic analysis of tumour samples identified cilium gene signatures as enriched in LTS. Moreover, Immunohistochemical analysis confirmed the presence of cilia in the tumours of LTS. Notably, reverse phase protein array analysis (RPPA) demonstrated increased phosphorylated GAB1 (Y627), SRC (Y527), BCL2 (S70) and RAF (S338) protein expression in STS compared to LTS. Next, we identified 25 unique master regulators (MR) and 13 transcription factors (TFs) belonging to ontologies of integrin signalling and cell cycle to be upregulated in STS. Conclusion Overall, comparison of STS and LTS GBM patients, identifies novel biomarkers and potential actionable therapeutic targets for the management of GBM. Graphical abstract

Glioblastoma is the deadliest brain cancer. One of the main reasons for poor outcome resides in therapy resistance, which adds additional challenges in finding an effective treatment. Small protein kinase inhibitors are molecules that have become widely studied for cancer treatments, including glioblastoma. However, none of these drugs have demonstrated a therapeutic activity or brought more benefit compared to the current standard procedure in clinical trials. Hence, understanding the reasons of the limited efficacy and drug resistance is valuable to develop more effective strategies toward the future. To gain novel insights into the method of action and drug resistance in glioblastoma, we established in parallel two patient-derived glioblastoma 2D and 3D organotypic multicellular spheroids models, and exposed them to a prolonged treatment of three weeks with temozolomide or either the two small protein kinase inhibitors enzastaurin and imatinib. We coupled the phenotypic evidence of cytotoxicity, proliferation, and migration to a novel kinase activity profiling platform (QuantaKinome™) that measured the activities of the intracellular network of kinases affected by the drug treatments. The results revealed a heterogeneous inter-patient phenotypic and molecular response to the different drugs. In general, small differences in kinase activation were observed, suggesting an intrinsic low influence of the drugs to the fundamental cellular processes like proliferation and migration. The pathway analysis indicated that many of the endogenously detected kinases were associated with the ErbB signaling pathway. We showed the intertumoral variability in drug responses, both in terms of efficacy and resistance, indicating the importance of pursuing a more personalized approach. In addition, we observed the influence derived from the application of 2D or 3D models in in vitro studies of kinases involved in the ErbB signaling pathway. We identified in one 3D sample a new resistance mechanism derived from imatinib treatment that results in a more invasive behavior. The present study applied a new approach to detect unique and specific drug effects associated with pathways in in vitro screening of compounds, to foster future drug development strategies for clinical research in glioblastoma.

BACKGROUND Major obstacles that have impeded the development of effective new therapies for GBM include inter- and intratumoral heterogeneity, the blood-brain-barrier and use of sub-optimal cell line-based preclinical models. Taking these hurdles into account, we have set up a patient-derived GBM drug-screening platform. We optimized protocols to improve cell culture success rate and retrospectively assessed the predictive power of our assay for patient response to TMZ. A large panel of GBM cells was screened for sensitivity to available oncological agents. Drugs of interest were selected based on favorable physicochemical properties for BBB crossing and potent activity in (a subset of) GBM cultures. Finally, we determined the success rate of performing a small-scale screen with 20 selected agents within 4 weeks of receiving tumor tissue. RESULTS By combining both CUSA and tissue piece-derived dissociation protocols, culture success increased to 95%, ensuring representation of the near-complete spectrum of GBM subtypes. Single-cell sequencing studies confirmed heterogeneity in our low-passage cell cultures. In vitro screening of TMZ on a large cohort (n = 55) identified 3 response categories (responders/intermediates/non-responders) for which Cox regression analysis revealed significantly different overall survival curves of corresponding patients. Screening of 107 FDA-approved anticancer agents on 45 GBM cultures underscored the tremendous intertumoral heterogeneity in drug sensitivities. We identified 20 potent agents each effective at clinically-achievable concentrations in (a subset of) GBM cultures and having favorable BBB penetration properties (CNS-MPO score). Screening of these agents on a per patient basis within 4 weeks of receiving tissue was successful in 18 out of 24 (75%) tested tumors. In the remaining cases the tumor cells grew very slowly and longer culture times were required. CONCLUSION Our drug screening platform offers a tool to predict TMZ response and assess sensitivity to candidate treatments, either for GBM subsets or on a per patient basis.

Glioblastoma (GBM) remains one of the most difficult tumors to treat. The mean overall survival rate of 15 months and the 5-year survival rate of 5% have not significantly changed for almost 2 decades. Despite progress in understanding the pathophysiology of the disease, no new effective treatments to combine with radiation therapy after surgical tumor debulking have become available since the introduction of temozolomide in 1999. One of the main reasons for this is the scarcity of compounds that cross the blood–brain barrier (BBB) and reach the brain tumor tissue in therapeutically effective concentrations. In this review, we focus on the role of the BBB and its importance in developing brain tumor treatments. Moreover, we discuss drug repurposing, a drug discovery approach to identify potential effective candidates with optimal pharmacokinetic profiles for central nervous system (CNS) penetration and that allows rapid implementation in clinical trials. Additionally, we provide an overview of repurposed candidate drug currently being investigated in GBM at the preclinical and clinical levels. Finally, we highlight the importance of phase 0 trials to confirm tumor drug exposure and we discuss emerging drug delivery technologies as an alternative route to maximize therapeutic efficacy of repurposed candidate drug.

Background Glioblastoma (GBM) is the most aggressive primary brain tumor. Its cellular composition is very heterogeneous, with cells exhibiting stem-cell characteristics (GSCs) that co-determine therapy resistance and tumor recurrence. Bone Morphogenetic Protein (BMP)-4 promotes astroglial and suppresses oligodendrocyte differentiation in GSCs, processes associated with superior patient prognosis. We characterized variability in cell viability of patient-derived GBM cultures in response to BMP4 and, based on single-cell transcriptome profiling, propose predictive positive and early-response markers for sensitivity to BMP4. Methods Cell viability was assessed in 17 BMP4-treated patient-derived GBM cultures. In two cultures, one highly-sensitive to BMP4 (high therapeutic efficacy) and one with low-sensitivity, response to treatment with BMP4 was characterized. We applied single-cell RNA-sequencing, analyzed the relative abundance of cell clusters, searched for and identified the aforementioned two marker types, and validated these results in all 17 cultures. Results High variation in cell viability was observed after treatment with BMP4. In three cultures with highest sensitivity for BMP4, a substantial new cell subpopulation formed. These cells displayed decreased cell proliferation and increased apoptosis. Neuronal differentiation was reduced most in cultures with little sensitivity for BMP4. OLIG1/2 levels were found predictive for high sensitivity to BMP4. Activation of ribosomal translation (RPL27A, RPS27) was up-regulated within one day in cultures that were very sensitive to BMP4. Conclusion The changes in composition of patient-derived GBM cultures obtained after treatment with BMP4 correlate with treatment efficacy. OLIG1/2 expression can predict this efficacy, and upregulation of RPL27A and RPS27 are useful early-response markers.

A better understanding of transcriptional evolution of IDH-wild-type glioblastoma may be crucial for treatment optimization. Here, we perform RNA sequencing (RNA-seq) (n = 322 test, n = 245 validation) on paired primary-recurrent glioblastoma resections of patients treated with the current standard of care. Transcriptional subtypes form an interconnected continuum in a two-dimensional space. Recurrent tumors show preferential mesenchymal progression. Over time, hallmark glioblastoma genes are not significantly altered. Instead, tumor purity decreases over time and is accompanied by co-increases in neuron and oligodendrocyte marker genes and, independently, tumor-associated macrophages. A decrease is observed in endothelial marker genes. These composition changes are confirmed by single-cell RNA-seq and immunohistochemistry. An extracellular matrix-associated gene set increases at recurrence and bulk, single-cell RNA, and immunohistochemistry indicate it is expressed mainly by pericytes. This signature is associated with significantly worse survival at recurrence. Our data demonstrate that glioblastomas evolve mainly by microenvironment (re-)organization rather than molecular evolution of tumor cells.

INTRODUCTION Little progress has been made in the development of effective new therapies for glioblastoma (GBM) the past decades. Fresh patient-derived GBM cell culture models have become the gold standard for GBM drug discovery and development. One of the major obstacles in identifying novel candidate drugs against GBM remains the blood-brain barrier (BBB). Therefore, it is crucial to select drugs with favourable physicochemical properties to cross BBB and reach the tumour tissue in therapeutically effective concentrations. In current drug repurposing approach, we evaluated available anti-cancer agents in our patient-derived drug screening platform against GBM. METHODS The FDA-approved Oncology Drug Set II library was tested on 45 primary GBM cell cultures. We developed a drug shortlisting pipeline combining efficacy data with pharmacodynamic and pharmacokinetic characteristics of each compound. The therapeutic efficacy of the selected agent was assessed in an orthotopic mouse PDX model, while penetration into the CNS by LC/MS/MS. RESULTS Omacetaxine mepesuccinate (OMA) was ranked as one of the most promising candidates applying our drug selection approach. In vitro, OMA revealed anti-tumour activity at IC50 values well-below reported Cmax plasma values in approximately 80% of GBM cultures. NanoString nCounter analysis, revealed DNA damage repair as the main pathway involved in OMA’s anti-tumour effect. Activation of caspase 3/7 activity and decrease of glioma cell invasiveness were also linked to its anti-tumour effect. In vivo, 1mg/kg dose of OMA was found to reach the brain tumour tissue in concentrations similar to the reported IC50 values in vitro. No adverse reactions were noted and a survival benefit was observed in a proportion of the treated mice. CONCLUSIONS At 1 mg/kg, OMA reaches the tumour brain tissue in therapeutically effective concentrations in mice while a moderate therapeutic benefit was observed. Additional in vivo experiments are ongoing investigating higher dosages of OMA and longer exposure.

BACKGROUND Although prognosis is poor in high-grade glioma (HGG) patients, some become a long-term survivor (LTS). Health-related quality of life (HRQOL) and cognition are seen as indicators for quality of survival in HGG but data in LTS and their caregivers are scarce. We aim to investigate cognition and HRQOL in LTS with evaluation by their proxies. MATERIAL AND METHODS We included LTS with initial HGG-diagnosis between 1999–2014. Cognition was assessed with: Boston Naming Test, shortened Token Test, verbal (Category and Letter) Fluency, Diagnostic Instrument of Mild Aphasia, Hopkins Verbal Learning Test and Trail Making Test (A, B, B/A) and HRQOL with: EORTC Quality of Life Questionnaire (C30) and Brain Cancer Module (BN20). 15 Proxies evaluated patients’ HRQOL with: C30, BN20 and they filled in a burden scale: Caregiver Strain Index (CSI). RESULTS 21 patients with grade III or IV glioma and 7 years (range 5–20 years) median survival after diagnosis participated. Compared to normative data patients deviated significantly on HVLT (direct recall, delayed recall, recognition true positives), TMT (A, B and B/A), BNT and CF (p12 years survival compared to patients with CONCLUSION Most LTS suffer from cognitive impairments. Despite worse prognosis and cognitive impairments, at group level patients report relatively intact QoL, which is supported by their proxies. However, at the individual level severity of impairments is variable and high caregiver strain is reported in one-third of the proxies. Objective cognitive functioning was only modestly associated with subjective experience in patients. A patient-tailored approach is recommended to investigate individual impairments and needs for rehabilitation and support for patients and also for their carers.

Background: Glioblastoma is the most prevalent and severe type of malignant brain tumor in adults. Although the genetic make-up initiating glioblastoma is increasingly better understood, a better understanding in the mechanisms that drive its evolution, heterogeneity and therapy resistance may reveal new directions for therapy development. To get better insights into glioblastoma evolution, we analyzed and de-convoluted transcriptomes of primary and recurrent glioblastoma resections. Material and Methods: Matching primary and secondary resections from n=185 glioblastoma patients were collected as part of EORTC Study 1542. The study was extended with tumor pairs from n=51 patients from the international GLASS study. The datasets were subjected to differential and deconvolution analysis using in-house algorithms. Results: When mapping the tumor samples into a reduced Glioblastoma Intrinsic Transcriptional Subtype space, we visualized subtype traversal, indicating that the CL subtype most often switches. As we found no more transitions from MES to other subtypes than to be expected by chance, we concluded that MES is an end-state. On average, tumor cell percentages decreased from ~67% to ~50% mostly due to an increase in TAM/microglia. Differential expression analysis was performed with correction for tumor cell percentages. While expression of most known oncogenes did not change considerably over time, marker genes of TAM/microglia, neurons and oligodendrocytes were up-regulated whereas endothelial cell markers were down-regulated over time. Furthermore, a cluster of ~30 extracellular matrix-associated genes increase significantly over time. A signature representing the gene-set was significantly associated with poor survival; high signatures were in particular associated to survival in secondary resections (P = 6.613e-06, Kaplan-Meier estimator). This suggests that the increase of extracellular matrix expression fulfils an important role in glioblastoma evolution. Conclusion: Using a large cohort, we interrogated changes in the glioblastoma transcriptome over time and found that in particular the composition of the tumor and its environment changes. The tumor cell percentage drops, suggesting more invasion or recruitment of non-malignant cells or a combination of both. This change is independent of an increase in the prognostic increase in extracellular matrix expression. Citation Format: Youri Hoogstrate, Kaspar Draaisma, Santoesha A. Ghisai, Iris de Heer, Levi van Hijfte, Wouter Coppieters, Melissa Kerkhof, Astrid Weyerbrock, Marc Sanson, Ann Hoeben, Slávka Lukacova, Giuseppe Lombardi, Sieger Leenstra, Monique Hanse, Ruth Fleischeuer, Colin Watts, Joseph McAbee, Nicos Angelopoulos, Thierry Gorlia, Vassilis Golfinopoulos, Johan M. Kros, Vincent Bours, Martin J. van den Bent, Pierre A. Robe, Pim J. French. Transcriptional evolution of glioblastoma reveals changes in bulk composition, mesenchymal sub-type as end-state, and a prognostic association with increased extracellular matrix gene expression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6140.

Background Despite maximal therapy with surgery, chemotherapy, and radiotherapy, glioblastoma (GBM) patients have a median survival of only 15 months. Almost all patients inevitably experience symptomatic tumor recurrence. A hallmark of this tumor type is the large heterogeneity between patients and within tumors itself which relates to the failure of standardized tumor treatment. In this study, tissue samples of paired primary and recurrent GBM tumors were investigated to identify individual factors related to tumor progression. Methods Paired primary and recurrent GBM tumor tissues from 8 patients were investigated with a multiomics approach using transcriptomics, proteomics, and phosphoproteomics. Results In the studied patient cohort, large variations between and within patients are observed for all omics analyses. A few pathways affected at the different omics levels partly overlapped if patients are analyzed at the individual level, such as synaptogenesis (containing the SNARE complex) and cholesterol metabolism. Phosphoproteomics revealed increased STMN1(S38) phosphorylation as part of ERBB4 signaling. A pathway tool has been developed to visualize and compare different omics datasets per patient and showed potential therapeutic drugs, such as abobotulinumtoxinA (synaptogenesis) and afatinib (ERBB4 signaling). Afatinib is currently in clinical trials for GBM. Conclusions A large variation on all omics levels exists between and within GBM patients. Therefore, it will be rather unlikely to find a drug treatment that would fit all patients. Instead, a multiomics approach offers the potential to identify affected pathways on the individual patient level and select treatment options.

Despite clinical intervention, glioblastoma (GBM) remains the deadliest brain tumor in adults. Its incurability is partly related to the establishment of drug resistance, both to standard and novel treatments. In fact, even though small kinase inhibitors have changed the standard clinical practice for several solid cancers, in GBM, they did not fulfill this promise. Drug resistance is thought to arise from the heterogeneity of GBM, which leads the development of several different mechanisms. A better understanding of the evolution and characteristics of drug resistance is of utmost importance to improve the current clinical practice. Therefore, the development of clinically relevant preclinical in vitro models which allow careful dissection of these processes is crucial to gain insights that can be translated to improved therapeutic approaches. In this review, we first discuss the heterogeneity of GBM, which is reflected in the development of several resistance mechanisms. In particular, we address the potential role of drug resistance mechanisms in the failure of small kinase inhibitors in clinical trials. Finally, we discuss strategies to overcome therapy resistance, particularly focusing on the importance of developing in vitro models, and the possible approaches that could be applied to the clinic to manage drug resistance.

INTRODUCTION The search for effective therapies for gliomas is progressively moving towards patient-specific medicine. In order to test patient-tailored therapies, it is vital to develop protocols for reliable establishment of patient-derived glioma cultures. We present a method for reliable culture establishment, with a 95% success rate in 114 consecutive high-grade samples. METHODS Cell cultures were established from either traditionally-resected tumor tissue or ultrasonic surgical aspirator (CUSA) derived tissue fragments, and expanded in serum-free culture, with selection of astrocytic populations if required. Cultures were started from single cells or small tumor fragments of 0.5-3mm (3D). Whole exome and RNA sequencing were carried out with the Illumina Novaseq and HiSeq platforms. Methylation profiling was performed with the Infinium MethylationEPIC array. Cultures and tumors were compared through analysis of single nucleotide polymorphisms and copy number profiles with the Infinium Global Screening Array. Intra-tumoral heterogeneity in cultures was investigated with single-cell transcriptomic sequencing (SORT-seq). We studied tumor-initiating potential by orthotopic injection of cultures in NOD-SCID mice. RESULTS Cultures started from single cells were established from CUSA material more efficiently (92%) than from traditional resection material (70%). 3D-derived cultures had a higher overall efficiency (95% for CUSA, 85% for traditional resection material). We confirmed high concordance in driver mutations, copy number and methylation profiles between tumors and derived cultures. Transcriptomics analysis, comparing tumors and derived cultures, revealed high consistency in gene expression distribution as demonstrated by correlation analysis (r=0.88). Singe-cell RNA-seq shows increased heterogeneity in CUSA derived-cultures, and decreased heterogeneity with passaging over time. Cultures faithfully produce tumors after orthotopic injection in NOD-SCID mice. CONCLUSION We present a highly successful method for the establishment of glioma cultures from patient material, with CUSA-derived cultures revealing greater heterogeneity. Cultures faithfully represent important molecular characteristics of parental tumors and can be used to test potential therapies in vitro.

BACKGROUND Glioblastoma (GBM) is the most aggressive primary brain tumor. The well-known cellular heterogeneity of this cancer, which includes glioma tumor-initiating cells with stem cell characteristics (GSCs), (co)influences therapy resistance and tumor recurrence. Bone Morphogenetic Protein-4 (BMP4) promotes differentiation of GSCs towards astroglial lineage while suppressing oligodendrocyte maturation. Treatment with BMP4 is associated with increased survival in mice. BMPs exert effects in cell-type and context dependent fashion, but also generate subtle heterogeneity in transcriptional response among similar cells. We studied first the cell viability of BMP4-treated primary GBM cultures followed by single-cell RNA-sequencing (scRNA-seq) on two differently responding cultures, and found correlation between their responses and therapy sensitivity. MATERIAL AND METHODS Cell viability, proliferation and apoptosis were assessed in 17 patient-derived BMP4-treated GBM cell cultures. We selected one culture in which this treatment induced high in vitro therapeutic efficacy, and one in which the treatment was ineffective, for analysis by scRNA-seq and then compared the results on the initial panel of 17 cultures. RESULTS After 7 days of treatment with BMP4, cell viability ranged from 28% (referred to as highest in vitro therapeutic efficacy) to 132% compared to untreated cells. scRNA-seq of the previously mentioned cultures in passage 7 showed that all neural cell types that are usually found in freshly resected GBM, were also present in our cultures. In the culture where BMP4 induced high in vitro therapeutic efficacy, BMP4 induces the formation of a large new cell population displaying decreased cell proliferation, increased migration and cell death, while the pro-inflammatory cells were depleted. RNA-velocity analysis revealed that, the cycling of cells was greatly diminished in the culture where therapy with BMP4 was efficacious, whereas this was enhanced in the GBM culture with the lowest cell viability after treatment. Astroglial differentiation was induced in all BMP4-treated cultures, while neuronal differentiation was reduced most in the cultures in which BMP4 induced lower or no in vitro therapeutic efficacy. In the culture without therapeutic efficacy of BMP4 cell cycle arrest was not induced anymore. In addition, OLIG1/2 mRNA and protein levels seemed predictive for BMP4-therapy efficacy, while activation of translation-associated genes (RPL27A, RPS27) was a suitable, immediate post-therapeutic marker for this. CONCLUSION scRNA-seq of in vitro GBM cultures provides advanced insights into the mechanism underlying therapy efficacy of BMP4. Neural differentiation status is distinctive for therapeutic efficacy of BMP4 in vitro before and after therapy.

PURPOSE Precision medicine trials in glioblastoma (GBM) are often conducted at tumor recurrence. However, second surgeries for recurrent GBM are not routinely performed, and therefore, molecular data for trial inclusion are predominantly derived from the primary sample. This study aims to establish whether molecular targets change during tumor progression and, if so, whether this affects precision medicine trial design. MATERIALS AND METHODS We collected 186 pairs of primary-recurrent GBM samples from patients receiving chemoradiotherapy with temozolomide and sequenced approximately 300 cancer genes. MGMT, TERT, and EGFRvIII status was individually determined. RESULTS The molecular profile of our cohort was identical to that of other GBM cohorts ( IDH wild-type [WT], 95%; EGFR amplified, approximately 50%), indicating that patients amenable to second surgery do not represent a specific molecular subtype. Molecular events in IDH WT GBMs were stable in approximately 80% of events, but changes in mutation status were observed for all examined genes (range, approximately 90% and 60% for TERT and EGFR mutations, respectively), and such changes strongly affected targeted trial size and design. A similar pattern of GBM driver instability was observed within MGMT promoter–methylated tumors. MGMT promoter methylation status remained prognostic at tumor recurrence. The observation that hypermutation at GBM recurrence was rare (8%) and not correlated with outcome was relevant for immunotherapy-based treatments. CONCLUSION This large cohort of matched primary and recurrent IDH WT tumors establishes the frequency of GBM driver instability after chemoradiotherapy with temozolomide. This allows per gene or pathway calculation of trial size at tumor recurrence, using molecular data of the primary tumor only. We also identify genes for which repeat surgery is necessary because of low mutation retention rate.

Recent publications on the molecular characterization of malignant glioma have had profound impact on the appreciation of tumoral heterogeneity within and between patients. Both these phenomena are implicated in the variability in clinical outcome between patients, as well as the inevitable recurrence of these tumors after conventional treatment. The advent of selective cell culture protocols for the propagation of patient-derived glioma stem-like cells (GSCs) provides researchers the ability to selectively study the cells that could be at the root of tumor proliferation and resistance to therapy. As these techniques are widely applied in contemporary studies and becoming the preferred in vitro model, molecular characterization of GSCs is considered pivotal for the identification and advancement of novel therapies for this devastating disease. This review aims to provide an overview of canonical molecular alterations defining subtypes of malignant glioma as derived from genotypic, transcriptomic and epigenetic profiling in relation to their representation in GSC models. The distribution of these hallmark alterations as found in characterization studies of GSCs is compared between publications. Finally, conclusions of these studies with respect to coverage of driving alterations and translational relevance are provided. By doing so, we provide a contemporary overview of scientific results derived from GSC models and hopefully create appreciation of the advantages and caveats of utilizing these models for studying malignant glioma.

// Subramanian Venkatesan 1 , Marlous Hoogstraat 2, 3 , Ester Caljouw 4 , Tessa Pierson 1 , Jochem K.H. Spoor 1 , Lona Zeneyedpour 5 , Hendrikus J. Dubbink 6 , Lennard J. Dekker 5 , Marielle van der Kaaij 1 , Jenneke Kloezeman 1 , Lotte M.E. Berghauser Pont 1 , Nicolle J.M. Besselink 2, 3, 7 , Theo M. Luider 5 , Jos Joore 4 , John W. Martens 8, 9 , Martine L.M. Lamfers 1 , Stefan Sleijfer 3, 8, 9 , Sieger Leenstra 1, 10 1 Department of Neurosurgery, Brain Tumor Center Erasmus MC, Rotterdam, The Netherlands 2 Department of Medical Oncology, University Medical Center Utrecht, Utrecht, The Netherlands 3 Center for Personalized Cancer Treatment (CPCT), University Medical Center Utrecht, Utrecht, The Netherlands 4 Pepscope BV, Utrecht, The Netherlands 5 Department of Neurology, Erasmus MC Cancer Institute, Rotterdam, The Netherlands 6 Department of Pathology, Erasmus MC Cancer Institute, Rotterdam, The Netherlands 7 Department of Genetics, University Medical Center Utrecht, Utrecht, The Netherlands 8 Department of Medical Oncology, Erasmus MC Cancer Institute, Rotterdam, The Netherlands 9 Cancer Genomics Netherlands, Erasmus MC Cancer Institute, Rotterdam, The Netherlands 10 Department of Neurosurgery, St. Elisabeth Hospital Tilburg, Tilburg, The Netherlands Correspondence to: Sieger Leenstra, email: s.leenstra@erasmusmc.nl Keywords: brain tumor, personalized medicine, genetic biomarkers, small molecule kinase inhibitors, resistance Received: January 11, 2016 Accepted: July 26, 2016 Published: August 11, 2016 ABSTRACT Background: Glioblastoma is the most malignant tumor of the central nervous system and still lacks effective treatment. This study explores mutational biomarkers of 11 drugs targeting either the RTK/Ras/PI3K, the p53 or the Rb pathway using 25 patient-derived glioblastoma stem-like cell cultures (GSCs). Results: We found that TP53 mutated GSCs were approximately 3.5 fold more sensitive to dual inhibition of mammalian target of rapamycin complex 1 and 2 (mTORC1/2) compared to wild type GSCs. We identified that Bcl-2(Thr56/Ser70) phosphorylation contributed to the resistance of TP53 wild type GSCs against dual mTORC1/2 inhibition. The Bcl-2 inhibitor ABT-263 (navitoclax) increased sensitivity to the mTORC1/2 inhibitor AZD8055 in TP53 wild type GSCs, while sensitivity to AZD8055 in TP53 mutated GSCs remained unchanged. Conclusion: Our data suggest that Bcl-2 confers resistance to mTORC1/2 inhibitors in TP53 wild type GSCs and that combined inhibition of both mTORC1/2 and Bcl-2 is worthwhile to explore further in TP53 wild type glioblastomas, whereas in TP53 mutated glioblastomas dual mTORC1/2 inhibitors should be explored.

INTRODUCTION: In spite of significant attention IDH mutations have attracted in glioma research, in vitro model systems with endogenous IDH mutations remain scarce. Development of these models is crucial not only for unraveling the molecular mechanisms that contribute to this disease entity, but especially for the development of new therapeutic interventions. METHODS: Fresh glioma tissue was obtained directly from the operating theater, dissociated and cultured under serum-free conditions. The presence of IDH mutations was verified at multiple passages using Sanger sequencing. D2-HG levels were measured through mass spectrometry. Cell proliferation was assessed through cell counting, cell viability was measured with an ATP-based viability assay. Drug screens were carried out with an FDA-approved Oncology Drugs Set from the NIH and multiple IDH mutant-specific inhibitors. RESULTS: Over 800 consecutive low grade and high grade glioma samples were processed for cell culture. From seven tumors one or more successful IDH mutant cell cultures were established that maintain the mutation in culture for at least five passages. All cultures were derived from astrocytic tumors. From one patient both the primary grade II astrocytoma and recurrent grade IV glioblastoma formed successful IDH-mutant cell lines. IDH-mutant specific inhibitors showed modest effects on cell viability, indicating limited dependence on D2-HG for growth. Drug screening revealed a subset of compounds that decrease viability at clinically-feasible drug concentrations which warrant further investigation. Whole exome sequencing in parallel with matched fresh-frozen tumor tissue, as well as RNA sequencing analyses are underway. CONCLUSION: We established a set of cell cultures derived from seven IDH mutant gliomas and characterized these on both genetic and transcriptional levels, and investigated drug sensitivity and D2-HG dependence. This unique set can be utilized to investigate novel therapeutic strategies.

BACKGROUND The development of new therapeutic agents generally takes many years to translate to clinically-effective new treatments. Among the strategies to reduce this time frame, efforts are now being undertaken to investigate drug repurposing. With this approach, compounds available for a specific disease are evaluated for their therapeutic efficacy in other diseases. We have set up a patient-derived cell culture model to apply this strategy for glioblastoma (GBM). MATERIAL AND METHODS Fresh patient-derived tumour tissue was dissociated and cultured in serum-free medium supplemented with EGF and bFGF. MGMT status was determined by methylation-specific PCR.Drug screening was performed using the NIH anti-cancer collection containing 114 approved oncology drugs. Compounds include chemotherapeutic agents as well as small molecule targeted agents. Readout for drug effects is based on ATP-based viability assay. Using systems modelling approaches, integrated analysis of both mutational and expression data of each tumour is applied to identify key pathways involved in response to specific compounds. RESULTS Molecular analysis demonstrated that copy number variations are preserved under serum-free culture conditions and that the MGMT methylation status is retained in over 75% of cases. Screening of the NIH anti-cancer collection on 55 GBM cell cultures revealed high intertumoral variation in response to most drugs. This included subsets of GBM revealing exceptionally high sensitivity to specific agents at clinically-feasible concentrations. Further ranking of the compounds was made based on the therapeutic index (IC50 tumour versus normal human astrocytes) and predicted blood-brain-barrier crossing capability. Currently, integrated analysis of molecular profiles of the tumour in relation to the drug response data is ongoing with the aim of identifying response predictors to these clinically-approved anti-cancer agents. CONCLUSION Our patient-derived in vitro drug screening assay may offer a tool to identify available anti-cancer agents that are effective in a subpopulation of GBM patients and that may be implemented in future stratified clinical trials for this patient group.

Objective: In recent years, gamma knife radiosurgery (GKRS) has become increasingly more popular as a salvage treatment modality for patients diagnosed with recurrent gliomas. The goal of GKRS for recurrent glioma patients is to improve survival rates with minimal burden for these patients. The emphasis of this report is on local tumor control (TC), clinical outcome and survival analysis. Methods: We performed a retrospective analysis of prospectively collected data of all patients who underwent GKRS for gliomas at the Gamma Knife Center Tilburg between 23-09-2002 and 21-05-2015. In total, 94 patients with glioma were treated with GKRS. Two patients were excluded because GKRS was used as a first stage treatment. The other 92 patients were included for analysis. Results: TC was 37% for all tumors (TC was 50% in LGGs and 27% in HGGs). Local progression (LP) was 46% for all tumors (LP was 31% in LGGs and 58% in HGGs). New distant lesions were seen in 18% of all patients (in 5% of LGG patients and 31% of HGG patients). Median progression-free and overall survival (PFS and OS) for all patients were 10.5 and 34.4 months, respectively. Median PFS was 50.1 and 5.7 months for low and high grade tumors, respectively. Median OS was 86.6 and 12.8 months for low and high grade tumors, respectively. No serious adverse events were noted post-GKRS. Conclusion: GKRS can safely be used as salvage treatment for recurrent glioma and seems to improve survival rates in (high grade) glioma patients with minimal burden.

BACKGROUND Despite considerable scientific efforts, endogenous in vitro isocitrate dehydrogenase (IDH)-mutated glioma models remain scarce. Availability of these models is key to understanding underlying molecular mechanisms and vital for the development of new therapeutic interventions. We established and characterized a set of seven cell lines derived from IDH1-mutated gliomas and utilized them to investigate IDH-mutant glioma drug-response in vitro. MATERIAL AND METHODS Fresh tumor material was collected directly from the operating room, mechanically and enzymatically dissociated and cultured under serum-free culture conditions. IDH mutation status was verified at several passages with Sanger sequencing, as well as with whole exome sequencing. D-2-hydroxyglutarate (D2-HG) levels were determined with mass-spectrometry. Genome-wide methylation profiling was performed using the Infinium MethylationEpic BeadChip array. Cell viability was measured using the ATP-based CellTiter-Glo assay. Cell proliferation was determined through cell counting. Drug screens were performed with an FDA-approved anti-cancer drug set from the NIH as well as two IDH-mutant specific inhibitors. RESULTS Over the last decade our lab processed over 800 glioma samples from all WHO grades and IDH mutational status. Optimisation of culture conditions led to the establishment of seven IDH1-mutant glioma cell lines. The IDH1-mutation was present during all tested passages in these cell lines. Whole exome sequencing showed a high concordance between tumor and cell lines with regard to driver gene mutations. Similarly, copy-number changes based on genome-wide methylation data also show a close resemblance between the parental tumor and resulting cell lines. The Heidelberg methylation profiler classified each cell line and its parental tumor as IDH mutant glioma. When exposed to an IDH1-mutant specific inhibitor, all cell lines showed a concentration-dependent decrease of D2-HG levels. The inhibitors showed little to no effect on viability up to 10uM during 8 days, however, long term treatment up to 4 weeks revealed a decrease in cell doubling time in 2 of 6 cultures. Drug screen results either alone or in combination with an IDH1-mutant specific inhibitor identified several interesting candidates that are currently followed up in additional experiments. CONCLUSION We established a unique set of patient-derived IDH1-mutant glioma cell lines that closely resemble their respective tumors and can be used to identify new therapeutic strategies.

BACKGROUND Glioblastoma multiforme is the most common and aggressive brain tumor in adults, with an average overall survival of 14 months. Current standard of care consists of tumor resection followed by radiotherapy with concomitant temozolomide and adjuvant temozolomide. However, glioblastoma recurs in all patients. The causes reside in the enhanced invasiveness and resistance to treatment, giving a clear indication that recurrent and resistant glioblastoma biology must be understood better in order to achieve future treatment strategies to benefit the patients. The complex nature of recurrent glioblastoma makes its understanding still a challenging achievement in the field. Nowadays multi-omics approaching is developing further and further and it may be used to unravel, by combining different layers of biological information, a comprehensive view of the changes occurring during the treatment. MATERIAL AND METHODS A discovery set of 13 primary patient-derived glioblastoma stem-like cultures were analysed, comprising selected resistant, induced resistant and with pre-existing resistance conditions. A characterization of transcriptome, proteome and phosphoproteome was performed using RNAseq and liquid chromatography mass spectrometry. Additional 10 paired primary and recurrent tumor tissues were utilized as a validation set. The data obtained was visualised, explored and integrated through TIBCO Spotfire, Ingenuity Pathway Analysis, STRING and COREMINE medical software. RESULTS Genetic regulatory processes such as DNA repair mechanism, mRNA splicing and chromatin assembly were shown to be common over-represented trends in resistant and recurrent glioblastomas as a result of increased genomic instability and stress deriving from acute an repeated temozolomide exposure. Due to the immense heterogeneity of glioblastomas, other proteins and genes here identified as differentially expressed need a further investigation as they also may play an important role in relevant biological processes in a patient-specific way. CONCLUSION This study provides further understanding of glioblastoma biology revealing an association with processes of recurrence and temozolomide resistance, moreover offering potential therapeutic targets for better treatment options for glioblastoma patients.

BACKGROUND Glioblastoma (GBM) is the most prevalent and lethal primary brain tumor. Its recurrence and resistance to current therapies, i.e. temozolomide (TMZ), is thought to result from a subpopulation of cells exhibiting stem cell properties, called glioblastoma stem-like cells (GSCs). Bone Morphogenetic Protein 4 (BMP4) induces GSCs differentiation, leading to a less resistant phenotype. In this study we show that co-treatment of BMP4 with TMZ has therapeutic benefit in a subset of GBM cell lines. Furthermore, we looked for patients’ molecular signatures that could predict sensitivity to this combination treatment. MATERIAL AND METHODS A panel of 17 primary GBM cultures (passages 5–10) were treated with increasing concentrations of TMZ, BMP4 and TMZ + BMP4 for 5 and 7 days and cell viability has been measured. The Combination Index (CI) of the two drugs was calculated to assess the response of each line to TMZ and BMP4 treatment. DNA was used to determine the MGMT promotor methylation status and for targeted exome sequencing. Expression levels of BMP signaling components and differentiation associated genes were determined by qPCR. RESULTS 12 cultures of primary GBMs and 5 cultures of recurrent GBMs were included. Overall, 71% of the tested cell lines was resistant to TMZ, while 41% was resistant to BMP4. Strikingly, 53% of primary cultures show synergy between TMZ and BMP4 (CI < 1 at a Fraction Affected of 50% (Fa50)). There was no significant difference in synergy between five or seven days of treatment. However, combination treatment of BMP4 and TMZ was more effective than sequential treatment. CONCLUSION Co-treatment of BMP4 and TMZ could be of therapeutic benefit in GBM patients, irrespective of their sensitivity to TMZ. Further research regarding the mechanism behind this synergy is necessary as to identify predictive markers for treatment response.

Glial tumors consist of a heterogeneous group of primary CNS neoplasms. While the clinical outcome of these tumors varies substantially between different grades (World Health Organization [WHO] grades I–IV), only grade I tumors can be treated curatively.1 In the last decade, substantial effort has been put into characterizing the pathogenic mechanisms underlying this complex group of glial cell–derived tumors. These efforts have led to the establishment of several molecular subclasses of glioma, which differentiate types of gliomas based on their intrinsic molecular distinctions and similarities, rather than the conventional characteristics used for histology-based grading.2–5 Indeed, gene expression–based clustering of glioma from several grades within the WHO grading system proved more predictive of survival than the histological classification.4, 6 Thus far, however, this gain of insight has not led to improved clinical outcome for patients, since there are no specific treatment strategies designed to target specific subtypes of glioma as of yet. Consequently, the development of preclinical models that accurately reflect the molecular heterogeneity of glioma is imperative for both translationally relevant drug screening programs and the advancement of patient-tailored treatment options. Commercially available cell cultures of glioma have been demonstrated to poorly mimic the molecular aberrations found in patient samples.7,8 Furthermore, the xenograft models derived from these cultures do not sufficiently recapitulate the histological hallmarks of glioma.9 With the advent of the cancer stem cell hypothesis, several groups have implemented serum-free (SF) cell culture regimens, originally developed for neural stem cell propagation, to establish glioma stem-like cell (GSC) cultures from fresh tumor tissue.10,11 By analyzing gene expression profiles of GSCs and serum supplemented (SS) cultures, a distinct separation between the aforementioned and the parental tumors was revealed by unsupervised clustering.7 Several groups have reported GSC cultures to be superior with regard to retaining the original patient gene expression signature and histological phenotype in xenografts.7,11,12 This has led to a wide variety of applications for these cell culture assays, ranging from inquiries into fundamental hypotheses13,14 to preclinical testing of novel agents.15–17 Several contradicting publications have since been published on the optimal cell culture methodology,18,19 mandatory molecular aberrations for successful propagation,20–22 and positive selection for gene expression signatures related to specific molecular subtypes in vitro.14, 23 Most of these previous publications have focused on the characterization of successfully propagated specimens of glioblastoma multiforme (GBM; WHO grade IV) from adults. Reports on the culture success of grade II and grade III gliomas are sparse.20,24 Therefore, we undertook a characterization study of a large cohort (N = 261), which addresses the distribution of glioma from all histological entities for the outcome of GSC culture attempt. Within equal WHO grades, correlations between cell culture outcome and patient overall survival were assessed. Tumor samples of both successful and unsuccessful cultures (n = 46 in total) were also subjected to molecular analysis, and a number of molecular traits that influence cell culture success rate were identified, as well as genes that may play a role in this process. These results emphasize the need for, and provide leads to, the development of improved culture protocols supporting growth of all subtypes of glioma. This is essential for implementation of this model in drug screening programs for personalized treatment strategies.

The conditionally replicating oncolytic adenovirus Delta24-RGD (Ad) is currently under investigation in clinical trials for glioblastoma, including in combination with temozolomide (TMZ), the standard chemotherapy for this tumor. Previously, we showed that the efficacy of Delta24-RGD in a murine model is primarily dependent on the virus-induced anti-tumor immune response. As observed with most chemotherapies, TMZ has pronounced immune-modulating effects. Here, we studied the combined effects of these treatments in a murine glioma model. In vitro, we observed a synergistic activity between Delta24-RGD and TMZ. In vivo, C57BL/6 mice bearing intracranial GL261 tumors were treated with TMZ for 5 days either prior to intratumoral Delta24-RGD injection (TMZ/Ad) or post virus injection (Ad/TMZ). Notably, the Ad/TMZ regimen led to similar tumoral CD8+ T cell influx as the virus-only treatment, but increased the ability of CD8+ T cells to specifically recognize the tumor cells. This was accompanied by improved survival. The TMZ/Ad regimen also improved survival significantly compared to controls, but not compared to virus alone. In this group, the influx of dendritic cells is impaired, followed by a significantly lower number of tumor-infiltrating CD8+ T cells and no recognition of tumor cells. Depletion of either CD4+ T cells or CD8+ T cells impaired the efficacy of Delta24-RGD, underscoring the role of these cells in therapeutic activity of the virus. Overall, we show that the addition of TMZ to Delta24-RGD treatment leads to a significant increase in survival and that the order of sequence of these treatments affects the CD8+T cell anti-tumor activity.

Early resection is standard of care for presumed low-grade gliomas. This is based on studies including only tumors that were post-surgically confirmed as low-grade glioma. Unfortunately this does not represent the clinicians’ situation wherein he/she has to deal with a lesion on MRI that is suspect for low-grade glioma (i.e. without prior knowledge on the histological diagnosis). We therefore aimed to determine the optimal initial strategy for patients with a lesion suspect for low-grade glioma, but not histologically proven yet. We retrospectively identified 150 patients with a resectable presumed low-grade-glioma and who were otherwise in good clinical condition. In this cohort we compared overall survival between three types of initital treatment strategy: a wait-and-scan approach (n = 38), early resection (n = 83), or biopsy for histopathological verification (n = 29). In multivariate analysis, no difference was observed in overall survival for early resection compared to wait-and-scan: hazard ratio of 0.92 (95% CI 0.43–2.01; p = 0.85). However, biopsy strategy showed a shorter overall survival compared to wait-and-scan: hazard ratio of 2.69 (95% CI 1.19–6.06; p = 0.02). In this cohort we failed to confirm superiority of early resection over a wait-and-scan approach in terms of overall survival, though longer follow-up is required for final conclusion. Biopsy was associated with shorter overall survival.

INTRODUCTION: Current efforts to improve patient survival in recurrent glioblastomas (GBMs) are often based on targeting the tumors acquired genetic changes. However, most molecular data is derived from the initial tumor: resections of recurrent GBMs are seldom performed. This study aims to assess whether genetic traits of initial GBMs are still present at recurrence. METHODS: DNA/RNA was isolated from pairs of initial and recurrent Stupp-treated GBM tumor samples (FFPE) and sequenced on a panel of 365 cancer genes. MGMT promotor methylation was determined by MS-PCR, EGFRvIII by RT-PCR and TERT-promotor mutations by SNaPshot. RESULTS: 276 patients from ten medical centers in six countries were identified with median age of 54.1 years. Median survival was 23.7 months, and median time to second surgery 13.0 months. Only 10 of 186 sequenced matched tumor pairs were IDH-mutated (5.4%). Overall, genetic traits of initial GBMs were stable with a median retention rate of coding mutations of 81.8%. Similarly, copy number changes (CNVkit/GISTIC) generally were retained at tumor recurrence. However, the retention rate was also ~80% in all of the major GBM driver pathways (TP53 signaling, RAS-RAF-MEK-ERK, RTK signaling). This is important as decreases 20% of loss of a marker requires a doubling of the number of included patients to achieve a similar power (assuming an objective response rate of 40% as a positive outcome). One noticeable change was a loss of TERT-promoter mutations in 7.5% of recurrent samples. Only four tumors were hypermutated (> 100 coding mutations) at recurrence, though more samples (n=12) acquired mutations in MSH2 and MSH6. Only one EGFR-amplified tumor was hypermutated. CONCLUSION: EGFR-amplified GBMs seldom transform into hypermutated tumors which suggests immune-checkpoint inhibitors have limited clinical use in recurrent GBMs. Re-sampling should be considered prior to inclusion in targeted therapy trials to ensure proper patient selection.

Introduction Treatment of gliomas is complicated by variable response rates of individual patients’ tumours to therapies. The Department of Neurosurgery of the Erasmus MC has developed a 2D culture platform (GLIOscreen) for screening patient-derived glioma tissues with potential therapeutic compounds. Unfortunately, not all patient-derived glioma tissues are amenable to 2D culture, probably caused by tumour heterogeneity, raising the necessity for additional, complementing culture models. Material and methods Here we show the development of an organotypic glioma model in the OrganoPlate to establish screenable cellular models for all glioma patients. The OrganoPlate is an easy to use, high throughput microfluidic platform enabling 3D cell culture and co-culture options, creating physiologically relevant models with a minimal requirement of cell material. Results and discussions The 3D glioma model will be used to culture individual patient’s cancer cells for the screening of potential effective (combinatorial) treatments, such as Temozolomide, a first line therapy in glioma treatment. GLIOscreen-derived glioma cell line GS261 was seeded in BME2 (reduced growth factor ECM), in the OrganoPlate and cultured for 8 days. On day 8 cells were analysed by phase contrast imaging and the live/dead cell viability assay (Life Technologies). The cell viability can also be studied in time with RealTime-Glo (Promega), a non-toxic cell viability assay. Conclusion Glioma cells can be cultured in the OrganoPlate for up to 2 months and are suitable for high-throughput chemotherapeutic drug screening.

BACKGROUND: Potential treatment-related neurotoxicity and the indolent course of the disease mainly feed the controversy concerning the optimal timing of surgery and radiotherapy in meningioma patients. OBJECT: To quantify the additional negative effects of conventional radiotherapy compared to surgery alone on neurocognitive functioning and health-related quality of life (HRQOL) in patients with WHO grade I meningiomas. METHODS: Neurocognitive functioning and HRQOL (SF36, EORTC-BCM20) were assessed in consecutive patients (1999-2005) with WHO grade I meningiomas at least 1 year after surgical treatment in two centers for brain tumor patients. Subsequently, we selected all patients who underwent surgery and conformal external beam fractioned radiotherapy (n=18) and matched these patients for age, sex, and educational level with the same number of patients who had had surgery only (n=18), as well as with the same number of healthy controls. RESULTS: No significant differences in neurocognitive functioning were found between the two meningioma patient groups; however, even meningioma patients who were treated with surgery only had a significantly lower neurocognitive functioning than healthy controls. Meningioma patients who were treated with surgery and radiotherapy had significantly lower HRQOL scores than meningioma patients who were treated with surgery only, who had HRQOL ratings comparable with healthy controls; these differences, however, disappeared after correction for the duration of disease. CONCLUSIONS: In contrast with conventional thinking, long-term neurocognitive functioning was significantly impaired in our meningioma patients. Additional radiotherapy following surgery, however, does not have additional deleterious effects on neurocognitive outcome in these patients.

Supratentorial primitive neuro-ectodermal tumors (PNET) in adults are very rare. Extraneural metastasis are unusual and the optimal palliative chemotherapy regimen is not established. We present a 26-year-old patient with local recurrence and distant metastasis of supratentorial PNET successfully treated with intensive induction chemotherapy and maintenance temozolomide.

Object In nearly all patients with glioblastoma multiforme (GBM) a local recurrence develops within a short period of time. In this paper the authors describe two patients in whom a second GBM developed after a relatively long time interval at a site remote from the primary tumor. The genetic profiles of the tumors were compared to discriminate between distant recurrence and a second primary tumor. Methods Both patients harboring a supratentorial GBM were treated with surgery and local high-dose radiotherapy. Local control of the disease at the primary tumor site was achieved. Within 2 years, a second GBM developed in both patients, not only outside the previously irradiated target areas but infratentorially in one patient and in the opposite hemisphere in the other. The tumors were examined for the presence of several genetic alterations that are frequently found in GBMs—a loss of heterozygosity at chromosome regions 1p36, 10p15, 19q13, and 22q13, and at the CDKN2A, PTEN, DMBT1, and TP53 gene regions; a TP53 mutation; and EGFR amplification. In the first patient, genetic profiling revealed that the primary tumor had an allelic imbalance for markers in several chromosome regions for which the second tumor displayed a complete loss. In the second patient, genetic profiling demonstrated the presence of genetic changes in the second tumor that were identical with and additional to those found in the primary tumor. Conclusions Based on the similarities between the genetic profiles of the primary and the second tumors in these patients, the authors decided that in each case the second distant GBM was a distant recurrence rather than a second independent primary tumor.

PURPOSE: Investigation of cell migration and proliferation of human glioma cell line spheroids (CLS) and evaluation of morphology, apoptosis, and immunohistochemical expression of MIB-1, p53, and p21 of organotypic muticellular spheroids (OMS) following cisplatin (CDDP) and irradiation (RT).MATERIAL AND METHODS: Spheroids of the GaMg glioma cell line and OMS prepared from biopsy tissue of six glioblastoma patients were used. Radiochemosensitvity (5 microg/ml CDDP followed by RT) was determined using migration and proliferation assays on CLS. In OMS, histology and immunohistochemical studies of MIB-1, p53, and p21 expression were examined 24 and 48 h following treatment.RESULTS: Combination treatment led to a migration inhibition of 38% (CDDP 13%; RT 27%) and specific growth delay of 2.6 (CDDP 1.3; RT 2.1) in CLS. Cell cycle analysis after combination treatment showed an accumulation of cells in the G2/M phase. In OMS, apoptosis increased, cell proliferation decreased, and p53/p21 expression increased more pronounced following CDDP+RT. No morphological damage was observed.CONCLUSION: CDDP can lead to enhancement of the RT effect in spheroids of both human glioma cell line spheroids and biopsy spheroids from glioblastoma specimens. The exerted effect is additive rather than synergistic.

Glioblastoma multiforme (GBM) is the most prevalent and aggressive primary brain tumor. Its recurrence and rapidly acquired resistance to current therapies, including treatment with temozolomide (TMZ), results from the rapid expansion of re-emerging Glioblastoma Tumor Initiating stem cell-like Cells (GTICs). Targeted Exome Sequencing (TES) revealed that in such recurrent human GBM missense mutations arise in genes of the Bone Morphogenetic Protein (BMP) system in addition to those of other molecular systems found in primary GBM. To determine in which subpopulation BMP system mutations accumulate, we subjected two GBM primary cultures with a known mutations in ZFYVE16 (A290S) or BMPR1B (K222E) to targeted sequencing after FAC-sorting these cells based on the acknowledged stem cell markers CD44 and CD133. We found that ZFYVE16 (A290S) and BMPR1B (K222E) mutations accumulate within the CD133+/CD44- GTICs. Treatment of the primary culture with the latter mutation, with BMP4 suggests that the response to BMP signals is markedly reduced. Cells from these cultured were also cultured again after FAC-sorting for CD133 and CD44. Surprisingly, after three passages all subpopulations acquired a variation in expression of these markers specific for each patient. This shows that a 2D culture is able to loose and acquire stem cell markers and might resemble the original heterogeneity of the parental tumor more closely than we originally expected. In a separate set of experiments, RNA-sequencing of a TMZ-resistant primary GBM culture (GS295), created during the course of this work, compared to the TMZ-sensitive GS295, shows that several genes of the BMP system (NOGGIN, BMP7, ID1 and GATA3) are differentially expressed. Our results suggest that BMP system mutations tend to accumulate in CD133+ GTICs, that BMP signaling is altered within GBM primary cultures and that several mutated genes of the BMP system may correlate with acquisition of TMZ resistance.

As the value of grading of ependymomas is currently debated we studied the expression of proliferation- and apoptosis-related proteins in these tumors as these mechanisms both are suggested to be important in tumor growth. We characterized the immunohistochemical expression of p53, Mdm2, Bcl-2, and Bax in 51 intracranial ependymomas. We also assessed the apoptosis- and proliferation-index, measured by MIB-1, PCNA-immunohistochemistry, and analyzed the clinical parameters. Of all used antibodies, the correlation with survival and the correlation among ordered categories was assessed. None of the analyzed immunohistochemical variables were significantly correlated with tumor grade. On the other hand, PCNA, MIB-1, and p53 were significantly related to the survival of the patient. In multivariate analysis, p53 was the only independent predictive variable (p = 0.0132).

AIM: Determination of in-vitro radiosensitivity and genetic alterations of cell cultures derived from human glioma biopsy tissue and established glioma cell lines.MATERIAL AND METHODS: Fresh brain tumor specimens of six patients were processed to early passage cell cultures. In addition the cell lines D 384 and Gli 6 were used. Cell cultures were irradiated with doses from 2 to 10 Gy. Following irradiation, cell survival was determined by clonogenic assay and survival curves were generated. The surviving fractions after 2 Gy (SF2) and 4 Gy (SF4) were used as radiosensitivity parameters. Genetic analysis included determination of the mutational and loss of heterozygosity (LOH) status of TP 53 (exons 5-8), the LOH 10- and epidermal growth factor receptor gene (EGFR) amplification status.RESULTS: The SF2 and SF4 values ranged from 0.54 to 0.88 (mean: 0.70) and from 0.13 to 0.52 (mean: 0.32), respectively. Genetic alterations were found in the Gli 6 cell line and in two primary cell cultures. The genetic profile of Gli 6 showed LOH but no TP 53 mutation, complete LOH 10 and no EGFR amplification. The VU 15 cell culture showed TP 53 mutation but no LOH 10 or EGFR amplification, while VU 24 showed incomplete LOH 10, EGFR amplification and no TP 53 mutation. In the other four cell cultures and D 384 cell line no genetic alterations were diagnosed. Histopathological classification of glioblastoma multiforme and/or genetic alterations resulted in lower radiosensitivity.CONCLUSION: In this small series of early passage glioma cell cultures low radiosensitivity and alterations in cell regulatory genes were seen. Further testing of biological behavior in larger series of patient-derived material is ongoing.

HDAC inhibitors have radiosensitizing effects in established cancer cell lines. This study was conducted to compare the efficacy of SAHA, LBH589, Valproic Acid (VPA), MS275 and Scriptaid in the patient-derived glioblastoma model. In more detail, SAHA and LBH589 were evaluated to determine predictors of response. Acetylated-histone-H3, gamma H2AX/53BP1, (p)Chek2/ATM, Bcl-2/Bcl-XL, p21(CIP1/WAF1) and caspase-3/7 were studied in relation to response. SAHA sensitized 50% of cultures, LBH589 45%, VPA and Scriptaid 40% and MS275 60%. Differences after treatment with SAHA/RTx or LBH589/RTx in a sensitive and resistant culture were increased acetylated-H3, caspase-3/7 and prolonged DNA damage repair gamma H2AX/53BP1 foci. pChek2 was found to be associated with both SAHA/RTx and LBH589/RTx response with a positive predictive value (PPV) of 90%. Bcl-XL had a PPV of 100% for LBH589/RTx response. Incubation with HDACi 24 and 48 hours pre-RTx resulted in the best efficacy of combination treatment. In conclusion a subset of patient-derived glioblastoma cultures were sensitive to HDACi/RTx. For SAHA and LBH589 responses were strongly associated with pChek2 and Bcl-XL, which warrant further clinical exploration. Additional information on responsiveness was obtained by DNA damage response markers and apoptosis related proteins. (C) 2014 Elsevier Ireland Ltd. All rights reserved.

The oncolytic adenovirus Delta24-RGD represents a new promising therapeutic agent for patients with a malignant glioma and is currently under investigation in clinical phase I/II trials. Earlier preclinical studies showed that Delta24-RGD is able to effectively lyse tumor cells, yielding promising results in various immune-deficient glioma models. However, the role of the immune response in oncolytic adenovirus therapy for glioma has never been explored. To this end, we assessed Delta24-RGD treatment in an immune-competent orthotopic mouse model for glioma and evaluated immune responses against tumor and virus. Delta24-RGD treatment led to long-term survival in 50% of mice and this effect was completely lost upon administration of the immunosuppressive agent dexamethasone. Delta24-RGD enhanced intra-tumoral infiltration of F4/80+ macrophages, CD4+ and CD8+ T-cells, and increased the local production of pro-inflammatory cytokines and chemokines. In treated mice, T cell responses were directed to the virus as well as to the tumor cells, which was reflected in the presence of protective immunological memory in mice that underwent tumor rechallenge. Together, these data provide evidence that the immune system plays a vital role in the therapeutic efficacy of oncolytic adenovirus therapy of glioma, and may provide angles to future improvements on Delta24-RGD therapy.

PURPOSE: To evaluate the health-related quality of life (HRQOL) and cognitive functioning of high-grade glioma patients in the postneurosurgical period. PATIENTS AND METHODS: The HRQOL, as assessed by the Short-Form Health Survey-36, tumor-specific symptoms, and objective and subjective neuropsychologic functioning, of 68 newly diagnosed glioma patients were compared with that of 50 patients with non–small-cell lung cancer (NSCLC) and to age- and sex-matched healthy controls. The association between tumor lateralization, extent of resection, and use of medication, and the HRQOL outcomes was also investigated. RESULTS: The HRQOL of the two patient groups was similar but significantly lower than that of the healthy controls. Glioma patients reported significantly more neurologic symptoms and poorer objective and subjective neuropsychologic functioning than the NSCLC patients. Using healthy controls as the reference group, cognitive impairment assessed at the individual patient level was observed in all glioma patients and 52% of the NSCLC patients. Poor performance on timed tasks in the glioma group could be attributed, in large part, to visual and motor deficits. Tumor lateralization was found to affect neuropsychologic functioning in a predictable manner. The extent of resection was not related significantly to neuropsychologic functioning. Corticosteroid use was associated with better recognition memory, whereas antiepileptic drug use was correlated negatively with working memory capacity. CONCLUSION: The general HRQOL of glioma patients is similar to that of patients with NSCLC. However, they suffer from a number of condition-specific neurologic and neuropsychologic problems that have a significant impact on their daily lives in the postsurgical period, before treatment with radiotherapy.

The value of chemotherapy in patients with malignant astrocytoma remains controversial. In our laboratories in vitro experiments with organotypic spheroid cultures showed superior effectiveness of anthracyclines. Systemic administration did not provide in therapeutic concentrations so far. Because recent studies on Daunorubicin in liposomes in the treatment of Kaposi sarcoma have shown effectiveness with diminished systemic toxicity, we administered intravenously a single dose of Daunorubicin in liposomes in eight patients at different intervals prior to surgery (12-50 h). In samples taken from tumor, tumor-edge and where possible from adjacent brain, the levels of Daunorubicin and its active metabolite Daunorubicinol were assessed with high performance liquid chromatography. Here we report that high concentrations of Daunorubicin and Daunorubicinol were found in malignant gliomas after systemic administration of liposomal Daunorubicin.

PURPOSE: Investigation of the in vitro cytotoxic effect of X-rays, either alone or combined with cisplatin on early passage cell cultures derived from human glioblastoma multiforme biopsy tissue.MATERIALS AND METHODS: Fresh tumour specimens from four patients were processed to cell cultures. The U373 glioma cell line was used as a reference. Early passage cell cultures were X-irradiated (0-8 Gy) either alone or in combination with cisplatin (0.5-1 microgram/ml). Cell survival was determined by either clonogenic assay or the colorimetric MTT assay. Survival curves were generated and mathematically analysed using the linear quadratic model, to obtain the radiosensitivity parameters alpha, beta, and SF2, i.e., the Surviving Fraction after 2 Gy.RESULTS: Two patient-derived glioma cell cultures and the U373 cell line showed rather high SF2 values of 0.61-0.72 in the clonogenic assay, indicating relative high radiation resistance. Cisplatin alone (1 microgram/ml) reduced cell survival by 10-30% (n = 4). When combined with irradiation, a clear additive cytotoxic effect of cisplatin was demonstrated by the unaltered value of the alpha-parameter for reproductive cell death.CONCLUSION: Cisplatin exerted an additive rather than radiosensitising cytotoxic effect in uncharacterised patient derived glioma cell cultures.

Background Glioblastoma is a highly malignant brain tumor for which no cure is available. To identify new therapeutic targets, we performed a mutation analysis of kinase genes in glioblastoma. Methods Database mining and a literature search identified 76 kinases that have been found to be mutated at least twice in multiple cancer types before. Among those we selected 34 kinase genes for mutation analysis. We also included IDH1, IDH2, PTEN, TP53 and NRAS, genes that are known to be mutated at considerable frequencies in glioblastoma. In total, 174 exons of 39 genes in 113 glioblastoma samples from 109 patients and 16 high-grade glioma (HGG) cell lines were sequenced. Results Our mutation analysis led to the identification of 148 non-synonymous somatic mutations, of which 25 have not been reported before in glioblastoma. Somatic mutations were found in TP53, PTEN, IDH1, PIK3CA, EGFR, BRAF, EPHA3, NRAS, TGFBR2, FLT3 and RPS6KC1. Mapping the mutated genes into known signaling pathways revealed that the large majority of them plays a central role in the PI3K-AKT pathway. Conclusions The knowledge that at least 50% of glioblastoma tumors display mutational activation of the PI3K-AKT pathway should offer new opportunities for the rational development of therapeutic approaches for glioblastomas. However, due to the development of resistance mechanisms, kinase inhibition studies targeting the PI3K-AKT pathway for relapsing glioblastoma have mostly failed thus far. Other therapies should be investigated, targeting early events in gliomagenesis that involve both kinases and non-kinases. Electronic supplementary material The online version of this article (doi:10.1186/1471-2407-14-718) contains supplementary material, which is available to authorized users.

Item does not contain fulltext BACKGROUND: Genetic and epigenetic profiling of glioblastomas has provided a comprehensive list of altered cancer genes of which only O(6)-methylguanine-methyltransferase (MGMT) methylation is used thus far as a predictive marker in a clinical setting. We investigated the prognostic significance of genetic and epigenetic alterations in glioblastoma patients. METHODS: We screened 98 human glioblastoma samples for genetic and epigenetic alterations in 10 genes and chromosomal loci by PCR and multiplex ligation-dependent probe amplification (MLPA). We tested the association between these genetic and epigenetic alterations and glioblastoma patient survival. Subsequently, we developed a 2-gene survival predictor. RESULTS: Multivariate analyses revealed that mutations in isocitrate dehydrogenase 1 (IDH1), promoter methylation of MGMT, irradiation dosage, and Karnofsky Performance Status (KFS) were independent prognostic factors. A 2-gene predictor for glioblastoma survival was generated. Based on the genetic and epigenetic status of IDH1 and MGMT, glioblastoma patients were stratified into 3 clinically different genotypes: glioblastoma patients with IDH1mt/MGMTmet had the longest survival, followed by patients with IDH1mt/MGMTunmet or IDH1wt/MGMTmet, and patients with IDH1wt/MGMTunmet had the shortest survival. This 2-gene predictor was an independent prognostic factor and performed significantly better in predicting survival than either IDH1 mutations or MGMT methylation alone. The predictor was validated in 3 external datasets. DISCUSSION: The combination of IDH1 mutations and MGMT methylation outperforms either IDH1 mutations or MGMT methylation alone in predicting survival of glioblastoma patients. This information will help to increase our understanding of glioblastoma biology, and it may be helpful for baseline comparisons in future clinical trials.

In the present study organotypic multicellular spheroids (OMS) were used to study the effects of chemotherapeutic agents on malignant gliomas. Compared with the frequently used cell line models, OMS have several advantages with respect to the preservation of the cellular heterogeneity and the structure of the original tumour. OMS prepared from seven glioma specimens were treated with 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), daunorubicin or doxorubicin. After exposure to these drugs, the histology and cell proliferation of the OMS were analysed by immunohistochemistry and image analysis. Furthermore, the expression of P-glycoprotein (P-gp) and multidrug resistance-related protein (MRP), which both can contribute to resistance to daunorubicin and doxorubicin, were immunohistochemically investigated. We found that OMS from gliomas are sensitive for daunorubicin and doxorubicin but not for BCNU in terms of tissue destruction and decrease in cell proliferation. In addition, all gliomas were P-gp and MRP negative, which is in accordance with the sensitivity for daunorubicin and doxorubicin. Considering the potential use of several new alternative drug delivery methods, such as intratumoural implantation of drug-impregnated polymers or liposomal encapsulation of cytostatic drugs, daunorubicin and doxorubicin might be effective in the treatment of malignant gliomas. Images Figure 1 Figure 2 Figure 3

Expression of CD44, particularly of certain splice variants, has been linked to tumor progression and metastatic potential in a number of different animal and human cancers. Although differential expression of CD44 standard epitopes (CD44s) in human brain tumors has been reported, the expression of CD44 variant exon encoded sequences (CD44v) in primary brain tumors in situ has not been studied in detail. In the present study, the expression of CD44s and CD44v epitopes was analyzed immunohistochemically on frozen sections of primary brain tumors. In addition, the expression of CD44 on cultured glioma cells was investigated by immunofluorescence flow cytometry. The results demonstrate the presence of CD44s epitopes and of CD44 splice variants containing CD44v4, v5 and v10 sequences in various types of brain tumors. A subgroup of highly malignant gliomas showed a strong (focal) expression of CD44v5. CD44v6 was absent in all brain tumors examined. CD44s appeared to be the dominant form of CD44 expressed in primary brain tumors, its expression was not correlated with tumor grade. We envisage that CD44 isoforms, in particular CD44s, may contribute to the invasive character of primary tumors by interacting with hyaluronate, one of the most abundant molecules in the extracellular matrix of the brain.