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Background: Maximum tumor resection improves overall survival (OS) in patients with glioblastoma. The extent of resection (EOR) is historically dichotomized. The RANO resect group recently proposed criteria for volumetry-based EOR assessment in patients that were treated according to Stupp´s protocol. The purpose of this study was (1) to investigate the prognostic value of EOR in patients receiving combined chemotherapy with lomustine (CCNU)/temozolomide (TMZ), and (2) to analyse the prognostic performance of binary EOR assessment compared to volumetric assessment., Methods: 78 patients with newly diagnosed MGMT-methylated GBM undergoing tumor resection followed by radiochemotherapy with CCNU/TMZ were included in this study. Residual contrast-enhancing (CE) tumor volume after the first resection was measured and its influence on OS and PFS was analysed using uni- and multivariable Cox regression analysis as well as two-sided log rank test. Patients were divided into RTV ≤1 cm³, >1 cm³ - ≤5 cm³ and >5 cm³ following the proposed criteria of the RANO resect group., Results: Prolonged OS was associated with age <60 years, low RTV, and gross total resection (GTR). Residual tumor volume (RTV) had a superior prognostic value compared to binary EOR assessment. Patients with total or near total resection of CE tumor (≤1 cm³ RTV) showed prolonged OS (median 54.4 months, 95% CI 46.94-not reached), with a 5-year survival rate of 49%., Conclusion: Low RTV is associated with increased survival in glioblastoma patients undergoing radiochemotherapy with CCNU/TMZ. This study demonstrates the applicability of the recently proposed RANO resect criteria in this subgroup of patients., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Diffuse Midline Gliomas (DMGs) are universally fatal, primarily pediatric malignancies affecting the midline structures of the central nervous system. Despite decades of clinical trials, treatment remains limited to palliative radiation therapy. A major challenge is the coexistence of molecularly distinct malignant cell states with potentially orthogonal drug sensitivities. To address this challenge, we leveraged established network-based methodologies to elucidate Master Regulator (MR) proteins representing mechanistic, non-oncogene dependencies of seven coexisting subpopulations identified by single-cell analysis-whose enrichment in essential genes was validated by pooled CRISPR/Cas9 screens. Perturbational profiles of 372 clinically relevant drugs helped identify those able to invert the activity of subpopulation-specific MRs for follow-up in vivo validation. While individual drugs predicted to target individual subpopulations-including avapritinib, larotrectinib, and ruxolitinib-produced only modest tumor growth reduction in orthotopic models, systemic co-administration induced significant survival extension, making this approach a valuable contribution to the rational design of combination therapy.

Diffuse intrinsic pontine glioma (DIPG) is a devastating brain tumor with a need for novel therapies. So far, monotherapies have failed to prolong survival for these patients, and combinatorial strategies have often shown severe, dose-limiting toxicities. In this issue of the JCI, Duchatel, Jackson, and colleagues address this challenge by introducing a drug combination that mitigates side effects and overcomes resistance. After identifying the PI3K/mTOR pathway as a therapeutic vulnerability, they treated DIPG-bearing mice with paxalisib and saw responses but also observed hyperglycemia as a severe side effect. Combining paxalisib with metformin mitigated this toxicity, but also upregulated protein kinase C (PKC) signaling. To tackle this mechanism of resistance, the authors added the PKC inhibitor enzastaurin to their drug combination and showed that this triple therapy led to improved survival. This approach paves the way for improved outcomes for patients with DIPG and other brain tumors.

Recent clinical trials for H3K27-altered diffuse midline gliomas (DMGs) have shown much promise. We present a consensus roadmap and identify three major barriers: (1) refinement of experimental models to include immune and brain-specific components; (2) collaboration among researchers, clinicians, and industry to integrate patient-derived data through sharing, transparency, and regulatory considerations; and (3) streamlining clinical efforts including biopsy, CNS-drug delivery, endpoint determination, and response monitoring. We highlight the importance of comprehensive collaboration to advance the understanding, diagnostics, and therapeutics for DMGs., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Background: Extracellular vesicles (EVs) originating from the central nervous system (CNS) can enter the blood stream and carry molecules characteristic of disease states. Therefore, circulating CNS-derived EVs have the potential to serve as liquid-biopsy markers for early diagnosis and follow-up of neurodegenerative diseases and brain tumors. Monitoring and profiling of CNS-derived EVs using multiparametric analysis would be a major advance for biomarker as well as basic research. Here, we explored the performance of a multiplex bead-based flow-cytometry assay (EV Neuro) for semi-quantitative detection of CNS-derived EVs in body fluids., Methods: EVs were separated from culture of glioblastoma cell lines (LN18, LN229, NCH82) and primary human astrocytes and measured at different input amounts in the MACSPlex EV Kit Neuro, human. In addition, EVs were separated from blood samples of small cohorts of glioblastoma (GB), multiple sclerosis (MS) and Alzheimer's disease patients as well as healthy controls (HC) and subjected to the EV Neuro assay. To determine statistically significant differences between relative marker signal intensities, an unpaired samples t-test or Wilcoxon rank sum test were computed. Data were subjected to tSNE, heatmap clustering, and correlation analysis to further explore the relationships between disease state and EV Neuro data., Results: Glioblastoma cell lines and primary human astrocytes showed distinct EV profiles. Signal intensities were increasing with higher EV input. Data normalization improved identification of markers that deviate from a common profile. Overall, patient blood-derived EV marker profiles were constant, but individual EV populations were significantly increased in disease compared to healthy controls, e.g. CD36 + EVs in glioblastoma and GALC + EVs in multiple sclerosis. tSNE and heatmap clustering analysis separated GB patients from HC, but not MS patients from HC. Correlation analysis revealed a potential association of CD107a + EVs with neurofilament levels in blood of MS patients and HC., Conclusions: The semi-quantitative EV Neuro assay demonstrated its utility for EV profiling in complex samples. However, reliable statistical results in biomarker studies require large sample cohorts and high effect sizes. Nonetheless, this exploratory trial confirmed the feasibility of discovering EV-associated biomarkers and monitoring circulating EV profiles in CNS diseases using the EV Neuro assay. Video Abstract., (© 2023. BioMed Central Ltd., part of Springer Nature.)

Purpose: The AVAglio trial reported a significant survival benefit for first line bevacizumab treatment in patients with IDH wildtype glioblastoma of the proneural gene expression subtype. We here aim to replicate these findings in an independent trial cohort., Methods: We evaluate the treatment benefit of bevacizumab according to gene expression subtypes of pretreatment tumor samples (n = 123) in the GLARIUS trial (NCT00967330) for MGMT unmethylated glioblastoma patients with Kaplan-Meier analyses, log-rank tests and Cox regression models., Results: Employing the Phillips classifier, bevacizumab conferred a significant PFS advantage in patients with proneural IDH wild-type tumors (10.4 vs. 6.0 months, p = 0.002), but no OS advantage (16.4 vs. 17.4 months, p = 0.6). Multivariable analysis adjusting for prognostic covariates confirmed the absence of a significant OS advantage from bevacizumab (hazard ratio, 1.05, 95% CI, 0.42 to 2.64; p = 0.14). Further, there was no interaction between the proneural subtype and treatment arm (p = 0.15). These results were confirmed in analyses of tumor subgroups according to the Verhaak classifier., Conclusion: In contrast to AVAglio, glioblastoma gene expression subgroups were not associated with a differential OS benefit from first-line bevacizumab in the GLARIUS trial., (© 2023. The Author(s).)

Purpose: In the randomized CeTeG/NOA-09 trial, lomustine/temozolomide (CCNU/TMZ) was superior to TMZ therapy regarding overall survival (OS) in MGMT promotor-methylated glioblastoma. Progression-free survival (PFS) and pseudoprogression rates (about 10%) were similar in both arms. Further evaluating this discrepancy, we analyzed patterns of postprogression survival (PPS) and MRI features at first progression according to modified RANO criteria (mRANO)., Methods: We classified the patients of the CeTeG/NOA-09 trial according to long vs. short PPS employing a cut-off of 18 months and compared baseline characteristics and survival times. In patients with available MRIs and confirmed progression, the increase in T 1 -enhancing, FLAIR hyperintense lesion volume and the change in ADC mean value of contrast-enhancing tumor upon progression were determined., Results: Patients with long PPS in the CCNU/TMZ arm had a particularly short PFS (5.6 months). PFS in this subgroup was shorter than in the long PPS subgroup of the TMZ arm (11.1 months, p = 0.01). At mRANO-defined progression, patients of the CCNU/TMZ long PPS subgroup had a significantly higher increase of mean ADC values (p = 0.015) and a tendency to a stronger volumetric increase in T 1 -enhancement (p = 0.22) as compared to long PPS patients of the TMZ arm., Conclusion: The combination of survival and MRI analyses identified a subgroup of CCNU/TMZ-treated patients with features that sets them apart from other patients in the trial: short first PFS despite long PPS and significant increase in mean ADC values upon mRANO-defined progression. The observed pattern is compatible with the features commonly observed in pseudoprogression suggesting mRANO-undetected pseudoprogressions in the CCNU/TMZ arm of CeTeG/NOA-09., (© 2023. The Author(s).)

Psychoemotional distress affects patients with cancer, including patients with a diagnosis of a malignant brain tumor. Empathy, professional expertise, and conversational skills are required to ensure successful communication with patients. The purpose of this study was to assess whether knowing the communication needs of patients would be helpful to neuro-oncologists before meeting with them. Patients in our neuro-oncology center were asked to complete the National Comprehensive Cancer Network Distress Thermometer (DT) and a study-specific questionnaire on patients' expectations for communication with the treating physician. The questions targeted issues such as attention/caring and awareness of their disease and prognosis. Importance ratings were compared between patients, with high vs. low distress scores to analyze the impact of distress on the patient's needs in physician-patient communication. A total of 81 patients completed the DT and questionnaire. One third ( n = 27) had IDH wild-type astrocytoma, and 42 patients (51.9%) were undergoing therapy for primary or recurrent disease. Mean distress was 4.88 (standard deviation ± 2.64) in the whole cohort, and 56.8% of patients had a high distress score (≥ 5 on a 10-point scale). All issues were assessed as important or very important for communication by the majority of patients, and importance ratings increased in patients with high distress levels for most items. Mean importance ratings correlated significantly with distress scores ( p < .001). Distress was increased in neuro-oncology patients. Patients with higher distress levels considered issues of both attention/caring and medical information about the disease as more important than patients with lower distress levels. Using distress assessment may help physicians and advanced practitioners to tailor the contents of their discussion for successful communication with patients., Competing Interests: The authors have no conflicts of interest to disclose., (© 2023 Harborside™.)

Detection of tumor progression in patients with glioblastoma remains a major challenge. Extracellular vesicles (EVs) are potential biomarkers and can be detected in the blood of patients with glioblastoma. In our study, we evaluated the potential of serum-derived EVs from glioblastoma patients to serve as biomarker for tumor progression. EVs from serum of glioblastoma patients and healthy volunteers were separated by size exclusion chromatography and ultracentrifugation. EV markers were defined by using a proximity-extension assay and bead-based flow cytometry. Tumor progression was defined according to modified RANO criteria. EVs from the serum of glioblastoma patients (n = 67) showed an upregulation of CD29, CD44, CD81, CD146, C1QA and histone H3 as compared to serum EVs from healthy volunteers (P value range: <.0001 to .08). For two independent cohorts of glioblastoma patients, we noted upregulation of C1QA, CD44 and histone H3 upon tumor progression, but not in patients with stable disease. In a multivariable logistic regression analysis, a combination of CD29, CD44, CD81, C1QA and histone H3 correlated with RANO-defined tumor progression with an AUC of 0.76. Measurement of CD29, CD44, CD81, C1QA and histone H3 in serum-derived EVs of glioblastoma patients, along with standard MRI assessment, has the potential to improve detection of true tumor progression and thus could be a useful biomarker for clinical decision making., (© 2022 The Authors. International Journal of Cancer published by John Wiley & Sons Ltd on behalf of UICC.)

Purpose: In the randomized phase III trial CeTeG/NOA-09, temozolomide (TMZ)/lomustine (CCNU) combination therapy was superior to TMZ in newly diagnosed MGMT methylated glioblastoma, albeit reporting more frequent hematotoxicity. Here, we analyze high grade hematotoxicity and its prognostic relevance in the trial population., Methods: Descriptive and comparative analysis of hematotoxicity adverse events ≥ grade 3 (HAE) according to the Common Terminology of Clinical Adverse Events, version 4.0 was performed. The association of HAE with survival was assessed in a landmark analysis. Logistic regression analysis was performed to predict HAE during the concomitant phase of chemotherapy., Results: HAE occurred in 36.4% and 28.6% of patients under CCNU/TMZ and TMZ treatment, respectively. The median onset of the first HAE was during concomitant chemotherapy (i.e. first CCNU/TMZ course or daily TMZ therapy), and 42.9% of patients with HAE receiving further courses experienced repeat HAE. Median HAE duration was similar between treatment arms (CCNU/TMZ 11.5; TMZ 13 days). Chemotherapy was more often discontinued due to HAE in CCNU/TMZ than in TMZ (19.7 vs. 6.3%, p = 0.036). The occurrence of HAE was not associated with survival differences (p = 0.76). Regression analysis confirmed older age (OR 1.08) and female sex (OR 2.47), but not treatment arm, as predictors of HAE., Conclusion: Older age and female sex are associated with higher incidence of HAE. Although occurrence of HAE was not associated with shorter survival, reliable prediction of patients at risk might be beneficial to allow optimal management of therapy and allocation of supportive measures., Trial Registration: NCT01149109., (© 2023. The Author(s).)

Purpose: The role of obesity in glioblastoma remains unclear, as previous analyses have reported contradicting results. Here, we evaluate the prognostic impact of obesity in two trial populations; CeTeG/NOA-09 (n = 129) for MGMT methylated glioblastoma patients comparing temozolomide (TMZ) to lomustine/TMZ, and GLARIUS (n = 170) for MGMT unmethylated glioblastoma patients comparing TMZ to bevacizumab/irinotecan, both in addition to surgery and radiotherapy., Methods: The impact of obesity (BMI ≥ 30 kg/m 2 ) on overall survival (OS) and progression-free survival (PFS) was investigated with Kaplan-Meier analysis and log-rank tests. A multivariable Cox regression analysis was performed including known prognostic factors as covariables., Results: Overall, 22.6% of patients (67 of 297) were obese. Obesity was associated with shorter survival in patients with MGMT methylated glioblastoma (median OS 22.9 (95% CI 17.7-30.8) vs. 43.2 (32.5-54.4) months for obese and non-obese patients respectively, p = 0.001), but not in MGMT unmethylated glioblastoma (median OS 17.1 (15.8-18.9) vs 17.6 (14.7-20.8) months, p = 0.26). The prognostic impact of obesity in MGMT methylated glioblastoma was confirmed in a multivariable Cox regression (adjusted odds ratio: 2.57 (95% CI 1.53-4.31), p < 0.001) adjusted for age, sex, extent of resection, baseline steroids, Karnofsky performance score, and treatment arm., Conclusion: Obesity was associated with shorter survival in MGMT methylated, but not in MGMT unmethylated glioblastoma patients., (© 2022. The Author(s).)

Extracellular vesicles (EVs) are reminiscent of their cell of origin and thus represent a valuable source of biomarkers. However, for EVs to be used as biomarkers in clinical practice, simple, comparable, and reproducible analytical methods must be applied. Although progress is being made in EV separation methods for human biofluids, the implementation of EV assays for clinical diagnosis and common guidelines are still lacking. We conducted a comprehensive analysis of established EV separation techniques from human serum and plasma, including ultracentrifugation and size exclusion chromatography (SEC), followed by concentration using (a) ultracentrifugation, (b) ultrafiltration, or (c) precipitation, and immunoaffinity isolation. We analyzed the size, number, protein, and miRNA content of the obtained EVs and assessed the functional delivery of EV cargo. Our results demonstrate that all methods led to an adequate yield of small EVs. While no significant difference in miRNA content was observed for the different separation methods, ultracentrifugation was best for subsequent flow cytometry analysis. Immunoaffinity isolation is not suitable for subsequent protein analyses. SEC + ultracentrifugation showed the best functional delivery of EV cargo. In summary, combining SEC with ultracentrifugation gives the highest yield of pure and functional EVs and allows reliable analysis of both protein and miRNA contents. We propose this combination as the preferred EV isolation method for biomarker studies from human serum or plasma.

The CeTeG/NOA-09 trial showed a survival benefit for combined CCNU/TMZ therapy in MGMT-promoter-methylated glioblastoma patients (quantitative methylation-specific PCR [qMSP] ratio > 2). Here, we report on the prognostic value of the MGMT promoter methylation ratio determined by qMSP and evaluate the concordance of MGMT methylation results obtained by qMSP, pyrosequencing (PSQ) or DNA methylation arrays (MGMT-STP27). A potential association of qMSP ratio with survival was analyzed in the CeTeG/NOA-09 trial population (n = 129; log-rank tests, Cox regression analyses). The concordance of MGMT methylation assays (qMSP, PSQ and MGMT-STP27) was evaluated in 76 screened patients. Patients with tumors of qMSP ratio > 4 showed superior survival compared to those with ratios 2-4 (P = .0251, log-rank test). In multivariate analysis, the qMSP ratio was not prognostic across the study cohort (hazard ratio [HR] = 0.88; 95% CI: 0.72-1.08). With different cutoffs for qMSP ratio (4, 9, 12 or 25), the CCNU/TMZ benefit tended to be larger in subgroups with lower ratios (eg, for cutoff 9: HR 0.32 for lower subgroup, 0.73 for higher subgroup). The concordance rates with qMSP were 94.4% (PSQ) and 90.2% (MGMT-STP27). Discordant results were restricted to tumors with qMSP ratios ≤4 and PSQ mean methylation rate ≤25%. Despite a shorter survival in MGMT-promoter-methylated patients with lower methylation according to qMSP, these patients had a benefit from combined CCNU/TMZ therapy, which even tended to be stronger than in patients with higher methylation rates. With acceptable concordance rates, decisions on CCNU/TMZ therapy may also be based on PSQ or MGMT-STP27., (© 2020 The Authors. International Journal of Cancer published by John Wiley & Sons Ltd on behalf of Union for International Cancer Control.)

Background: The metabolic enzyme carbonic anhydrase 12 (CA12/CAXII) emerges as a promising cancer therapeutic target with drug development projects underway. Previous reports proposed the relevance of CA12 in the context of glioma but are limited in patient data quantity, ignore ethnic diversity of patients or rely on semi-quantitative, thereby out of date, methodology. Moreover, little is known on the association of CA12 to brain tumor stemness or on the effect of anti-CAXII-directed monotherapies on glioma stem cells (GSCs), in particular their response regarding mesenchymal differentiation status., Methods: We performed in silico analysis on three independent, large-scale patient datasets interrogating state of the art molecular diagnostics alongside clinical outcomes. We analyzed CAXII abundance on a collection of GSCs and functionally tested their response to exposure to CAXII blocking antibody 6A10., Results: CA12 is highly expressed in glial tumors compared with normal tissue and predicts for poor clinical course of tumor patients. CA12 expression in glioblastoma significantly correlates with clinically established, molecular markers of IDH1 WT DNA, WHO grade IV or absence of 1p/19q chromosome arm co-deletion. Furthermore, tumors with elevated CA12 cluster into the mesenchymal transcription subclass of the disease. CAXII abundance in different GSCs ranges from almost absent to high levels and does not correlate to stem cell marker CD133/AC133 cell surface expression. Moreover, aiming to pharmacologically block CAXII in our cells with antibody 6A10 caused significant functional response only in one of the tested GSCs models, featuring suppression of cell invasion accompanied by reduction of ZEB1 protein and other stem cell markers., Conclusion: CA12 represents a clinically relevant and molecular brain tumor-subtype specific therapeutic target. Our correlative data from experimental and clinical samples does not support CA12/CAXII to be GSC specific. 6A10 possesses promising potential to impede the invasive capacity of glioma cells and supports the emerging concept that CAXII interacts with cancer EMT programs. However, further mechanistic studies are required to comprehensively assess the therapeutic potential of 6A10 and to identify different resistance mechanisms of GSCs., Competing Interests: Prof. Reinhard Zeidler reports a patent, EP10004646.5, issued. The authors report no other potential conflicts of interest in this work., (© 2021 Li et al.)

Glioblastoma is a devastating disease, for which biomarkers allowing a prediction of prognosis are urgently needed. microRNAs have been described as potentially valuable biomarkers in cancer. Here, we studied a panel of microRNAs in extracellular vesicles (EVs) from the serum of glioblastoma patients and evaluated their correlation with the prognosis of these patients. The levels of 15 microRNAs in EVs that were separated by size-exclusion chromatography were studied by quantitative real-time PCR, followed by CD44 immunoprecipitation (SEC + CD44), and compared with those from the total serum of glioblastoma patients ( n = 55) and healthy volunteers ( n = 10). Compared to total serum, we found evidence for the enrichment of miR-21-3p and miR-106a-5p and, conversely, lower levels of miR-15b-3p, in SEC + CD44 EVs. miR-15b-3p and miR-21-3p were upregulated in glioblastoma patients compared to healthy subjects. A significant correlation with survival of the patients was found for levels of miR-15b-3p in total serum and miR-15b-3p, miR-21-3p, miR-106a-5p, and miR-328-3p in SEC + CD44 EVs. Combining miR-15b-3p in serum or miR-106a-5p in SEC + CD44 EVs with any one of the other three microRNAs in SEC + CD44 EVs allowed for a prognostic stratification of glioblastoma patients. We have thus identified four microRNAs in glioblastoma patients whose levels, in combination, can predict the prognosis for these patients., Competing Interests: U.H. reports grants and personal fees from Roche; personal fees and non-financial support from Medac and Bristol-Myers Squibb; and personal fees from Novocure, Novartis, Daichii-Sankyo, Riemser, and Noxxon. N.S. reports personal fees and other support from Roche and received honoraria for advisory board from Bayer. M.G. reports grants, personal fees, and other support from Novocure and Medac, and personal fees from Merck. C.S. reports personal fees from Roche. S.W. and C.C. are employed by Miltenyi Biomedicine. All other authors declare no conflicts of interest.

Purpose: In the EF-14 trial for newly diagnosed glioblastoma (ndGBM) patients addition of Tumour Treating Fields (TTFields) to temozolomide treatment resulted in a significantly improved overall survival (OS). In the NOA-09/CeTeG trial, combination of lomustine and temozolomide was superior to temozolomide monotherapy in patients with O6-methylguanine DNA methyltransferase (MGMT) promoter methylated (MGMTm) ndGBM. We evaluated combination of these two treatment modalities in patients with MGMTm ndGBM. There have been so far no data on the combination of these two efficient regimens., Methods: This bicentric retrospective analysis investigated 16 patients. Parameters evaluated included safety outcome as measured by Common Toxicity Criteria for Adverse Events (CTCAE), clinical outcomes, and compliance to treatment., Results: Hematologic adverse events CTCAE ≥ 3 were observed in seven, hepatotoxic adverse events of CTCAE ≥ 3 in four patients. Mild to moderate skin toxicity was detected in six patients. At data cutoff, patients demonstrated a median progression-free survival (PFS) of 20 months. The usage rate of TTFields showed a high median adherence (83%) to the therapy., Conclusions: This analysis provides first indication that the combination of TTFields/lomustine/temozolomide is safe and feasible. The observed survival outcomes might suggest potential beneficial effects.

Background: The CeTeG/NOA-09 trial showed significantly longer overall survival with combined lomustine-temozolomide therapy compared with standard temozolomide for patients with glioblastoma with methylated MGMT promoter. The trial also aimed to investigate the effect of lomustine-temozolomide therapy on health-related quality of life (HRQOL) and neurocognitive function, which we report here., Methods: In this randomised, multicentre, open-label, phase 3 trial, newly diagnosed, chemoradiotherapy-naive patients with MGMT-methylated glioblastoma, aged 18-70 years, with a Karnofsky performance score of 70% or higher, were recruited and enrolled at 17 university hospitals in Germany. Patients received standard radiotherapy (60 Gy) and were randomly assigned (1:1, stratified by centre by allocating complete blocks of six to a centre, without masking) to either six 6-week courses of oral combined lomustine (100 mg/m 2 on day 1) plus temozolomide (100-200 mg/m 2 on days 2-6) or standard oral temozolomide (75 mg/m 2 daily during radiotherapy plus six 4-week courses of temozolomide [150-200 mg/m 2 ] on days 1-5, every 4 weeks). The primary endpoint was overall survival. HRQOL, assessed using the European Organisation for Research and Treatment of Cancer (EORTC) quality of life questionnaire core-30 and the EORTC brain cancer module (BN20); and neurocognitive function, assessed using the Mini Mental State Examination (MMSE), plus a neurocognitive test battery (NOA-07), including Trail Making Test A and B (TMT-A and B), working memory tests, and tests for lexical (Controlled Oral Word Association [COWA]) and semantic verbal fluency, were secondary endpoints analysed in the modified intention-to-treat population (mITT; all randomly assigned patients who received at least one dose of study chemotherapy). We used linear mixed-model analyses to investigate differences between treatment groups regarding HRQOL (clinically relevant ≥10 points) and MMSE scores (clinically relevant ≥3 points). The trial is registered with ClinicalTrials.gov, NCT01149109., Findings: Between June 17, 2011 and April 8, 2014, 141 patients were randomly assigned and 129 patients began treatment and were included in the mITT population (63 in the temozolomide and 66 in the lomustine-temozolomide group). Median follow-up for HRQOL (the item global health) was 19·4 months (IQR 7·8-38·6), for MMSE was 15·3 months (4·1-29·6), and for COWA was 11·0 months (0-27·5). We found no significant impairment regarding any item of HRQOL in the lomustine-temozolomide group (difference between the groups for global health 0·30 [95% CI -0·23 to 0·83]; p=0·26). Differences in MMSE were in favour of the temozolomide group (difference -0·11 [95% CI -0·19 to -0·03]; p=0·0058) but were not clinically relevant (1·76/30 points over 4 years). We found no significant difference between the groups in any subtest of the neurocognitive test battery (difference for COWA 0·04 [95% CI -0·01 to 0·09]; p=0·14)., Interpretation: The absence of systematic and clinically relevant changes in HRQOL and neurocognitive function combined with the survival benefit of lomustine-temozolomide versus temozolomide alone suggests that a long-term net clinical benefit exists for patients with newly diagnosed glioblastoma with methylation of the MGMT promoter and supports the use of lomustine-temozolomide as a treatment option for these patients., Funding: German Federal Ministry of Education and Research., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Purpose: The phase II GLARIUS trial assigned patients with newly diagnosed, O-6-methylguanine-DNA methyltransferase promoter non-methylated glioblastoma to experimental bevacizumab/irinotecan (BEV/IRI) or standard temozolomide (TMZ). To identify subpopulations with a particularly favorable course, we assessed the prognostic potential of magnetic resonance imaging (MRI) markers before treatment onset., Methods: MRIs at baseline (before treatment onset) were analyzed for T1-hyperintense and diffusion-restricted lesions; as well as the presence of both hyperintense and diffusion-restricted (double positive) lesions. The MRI findings were correlated with overall and progression-free survival., Results: MRI scans were evaluable in 71% of the GLARIUS modified intention-to-treat population (n = 121 of 170; 88 patients in the BEV/IRI arm, and 33 patients in the TMZ control arm). Diffusion-restricted and T1 hyperintense lesions were present in 60% and 65% of patients in BEV/IRI arm, while 57% and 63% were found in the TMZ arm, respectively. Double positive lesions were found in 37% of BEV/IRI patients and in 39% of TMZ patients. Neither the presence of T1-hyperintense, diffusion-restricted lesions, nor double positive lesions were associated with improved survival., Conclusions: Baseline T1-hyperintense and diffusion-restricted lesions are not suitable to predict progression-free or overall survival of patients treated with bevacizumab/irinotecan or temozolomide.

Background: There is an urgent need for more effective therapies for glioblastoma. Data from a previous unrandomised phase 2 trial suggested that lomustine-temozolomide plus radiotherapy might be superior to temozolomide chemoradiotherapy in newly diagnosed glioblastoma with methylation of the MGMT promoter. In the CeTeG/NOA-09 trial, we aimed to further investigate the effect of lomustine-temozolomide therapy in the setting of a randomised phase 3 trial., Methods: In this open-label, randomised, phase 3 trial, we enrolled patients from 17 German university hospitals who were aged 18-70 years, with newly diagnosed glioblastoma with methylated MGMT promoter, and a Karnofsky Performance Score of 70% and higher. Patients were randomly assigned (1:1) with a predefined SAS-generated randomisation list to standard temozolomide chemoradiotherapy (75 mg/m 2 per day concomitant to radiotherapy [59-60 Gy] followed by six courses of temozolomide 150-200 mg/m 2 per day on the first 5 days of the 4-week course) or to up to six courses of lomustine (100 mg/m 2 on day 1) plus temozolomide (100-200 mg/m 2 per day on days 2-6 of the 6-week course) in addition to radiotherapy (59-60 Gy). Because of the different schedules, patients and physicians were not masked to treatment groups. The primary endpoint was overall survival in the modified intention-to-treat population, comprising all randomly assigned patients who started their allocated chemotherapy. The prespecified test for overall survival differences was a log-rank test stratified for centre and recursive partitioning analysis class. The trial is registered with ClinicalTrials.gov, number NCT01149109., Findings: Between June 17, 2011, and April 8, 2014, 141 patients were randomly assigned to the treatment groups; 129 patients (63 in the temozolomide and 66 in the lomustine-temozolomide group) constituted the modified intention-to-treat population. Median overall survival was improved from 31·4 months (95% CI 27·7-47·1) with temozolomide to 48·1 months (32·6 months-not assessable) with lomustine-temozolomide (hazard ratio [HR] 0·60, 95% CI 0·35-1·03; p=0·0492 for log-rank analysis). A significant overall survival difference between groups was also found in a secondary analysis of the intention-to-treat population (n=141, HR 0·60, 95% CI 0·35-1·03; p=0·0432 for log-rank analysis). Adverse events of grade 3 or higher were observed in 32 (51%) of 63 patients in the temozolomide group and 39 (59%) of 66 patients in the lomustine-temozolomide group. There were no treatment-related deaths., Interpretation: Our results suggest that lomustine-temozolomide chemotherapy might improve survival compared with temozolomide standard therapy in patients with newly diagnosed glioblastoma with methylated MGMT promoter. The findings should be interpreted with caution, owing to the small size of the trial., Funding: German Federal Ministry of Education and Research., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Background: We evaluated patterns of tumor growth in patients with newly diagnosed MGMT-non-methylated glioblastoma who were assigned to undergo radiotherapy in conjunction with bevacizumab/irinotecan (BEV/IRI) or standard temozolomide (TMZ) within the randomized phase II GLARIUS trial., Methods: In 142 patients (94 BEV/IRI, 48 TMZ), we reviewed magnetic resonance imaging scans at baseline and first tumor recurrence. Based on contrast-enhanced T1-weighted and fluid-attenuated inversion recovery images, we assessed tumor growth patterns and tumor invasiveness. Tumor growth patterns were classified as either multifocal or local at baseline and recurrence; at first recurrence, we additionally assessed whether distant lesions appeared. Invasiveness was determined as either diffuse or non-diffuse. Associations with treatment arms were calculated using Fisher's exact test., Results: At baseline, 115 of 142 evaluable patients (81%) had a locally confined tumor. Between treatment arms, there was no significant difference in the fraction of tumors that changed from an initially local tumor growth pattern to a multifocal pattern (12 and 13%, p = 0.55). Distant lesions appeared in 17% (BEV/IRI) and 13% (TMZ) of patients (p = 0.69). 15% of patients in the BEV/IRI arm and 8% in the TMZ arm developed a diffuse growth pattern from an initially non-diffuse pattern (p = 0.42)., Conclusions: The tumor growth and invasiveness patterns do not differ between BEV/IRI and TMZ-treated MGMT-non-methylated glioblastoma patients in the GLARIUS trial. BEV/IRI was not associated with an increased rate of multifocal, distant, or highly invasive tumors at the time of recurrence.

In a subset of glioblastoma (GBM) patients, the differentiation between tumor progression and tumor pseudoprogression (PsP) is challenging. This case describes a male patient suffering from isocitrate dehydrogenase 1 (IDH1) mutant GBM who demonstrated an increasing contrast-enhancing (CE) lesion on a cranial magnetic resonance imaging (cMRI) scan 8 months after radiochemotherapy. In accordance with the response assessment in neuro-oncology (RANO) criteria, the cMRI lesion was classified as recurrent tumor, although 18 F-fluoroethyl-L-tyrosine positron emission tomography ( 18 F-FET-PET) did not indicate vital tumor tissue. The patient underwent re-surgery but histopathology only revealed reactive and necrotic tissue, consistent with PsP. Nine weeks after complete resection of the CE lesion, a new lesion emerged that later regressed in the follow-up cMRI scans, thereby retrospectively establishing the diagnosis of recurrent PsP., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Background: The GLARIUS trial, which investigated the efficacy of bevacizumab (BEV)/irinotecan (IRI) compared with standard temozolomide in the first-line therapy of O6-methylguanine-DNA methyltransferase (MGMT)-nonmethylated glioblastoma, showed that progression-free survival was significantly prolonged by BEV/IRI, while overall survival was similar in both arms. The present report focuses on quality of life (QoL) and Karnofsky performance score (KPS) during the whole course of the disease., Methods: Patients (n = 170) received standard radiotherapy and were randomized (2:1) for BEV/IRI or standard temozolomide. At least every 3 months KPS was determined and QoL was measured using the European Organisation for Research and Treatment of Cancer 30-item Core Quality of Life and 20-item Brain Neoplasm questionnaires. A generalized estimating equation (GEE) model evaluated differences in the course of QoL and KPS over time. Also, the time to first deterioration and the time to postprogression deterioration were analyzed separately., Results: In all dimensions of QoL and KPS, GEE analyses and time to first deterioration analyses did not detect significant differences between the treatment arms. At progression, 82% of patients receiving second-line therapy in the standard arm received BEV second-line therapy. For the dimensions of motor dysfunction and headaches, time to postprogression deterioration was prolonged in the standard arm receiving crossover second-line BEV in the vast majority of patients at the time of evaluation., Conclusions: GLARIUS did not find indications for a BEV-induced detrimental effect on QoL in first-line therapy of MGMT-nonmethylated GBM patients. Moreover, GLARIUS provided some indirect corroborative data supporting the notion that BEV may have beneficial effects upon QoL in relapsed GBM.

BRAF V600E mutations occur frequently in malignant melanoma, but are rare in most malignant glioma subtypes. Besides, more benign brain tumors such as ganglioglioma, dysembryoblastic neuroepithelial tumours and supratentorial pilocytic astrocytomas, only pleomorphic xanthoastrocytomas (50-78%) and epitheloid glioblastoma (50%) regularly exhibit BRAF mutations. In the present study, we report on three patients with recurrent malignant gliomas harbouring a BRAF V600E mutation. All patients presented with markedly disseminated leptomeningeal disease at recurrence and had progressed after radiotherapy and alkylating chemotherapy. Therefore, estimated life expectancy at recurrence was a few weeks. All three patients received dabrafenib as a single agent and all showed a complete or nearly complete response. Treatment is ongoing and patients are stable for 27 months, 7 months and 3 months, respectively. One patient showed a dramatic radiologic and clinical response after one week of treatment. We were able to generate an ex vivo tumor cell culture from CSF in one patient. Treatment of this cell culture with dabrafenib resulted in reduced cell density and inhibition of ERK phosphorylation in vitro. To date, this is the first series on adult patients with BRAF-mutated malignant glioma and leptomeningeal dissemination treated with dabrafenib monotherapy. All patients showed a dramatic response with one patient showing an ongoing response for more than two years.

Ependymoma is a malignant pediatric brain tumor, often incurable under the current treatment regimen. We aimed to evaluate the expression of microRNAs (miRs) in pediatric ependymoma tumors in an attempt to identify prognostic molecular markers which would lead to potential therapeutic targets. Following miR-array expression analysis, we focused on 9 miRs that correlated with relapse which were further validated by quantitative real-time PCR (qRT-PCR) in a cohort of 67 patients. Western blotting and immunohistochemistry were used to measure target protein expression in 20 and 34 tumor samples, respectively. High expression of miR-124-3p significantly correlated with the lower progression-free survival (PFS) of 16% compared to 67% in those expressing low levels (P = .002). Interestingly, in the group of patients with local disease (n = 56) expression levels of this miR distinguished 2 subgroups with a significantly different outcome (P = .001). miR-124-3p was identified as an independent prognostic factor of relapse in the multivariate analysis performed in the whole cohort and in the group with localized disease. In the localized group, a patient expressing high levels of miR-124-3p had a 4.1-fold increased risk for relapse (P = .005). We demonstrated the direct binding of miR-124-3p to its target TP53INP1. Negative TP53INP1 protein levels correlated with a poor outcome (P = .034). We propose miR-124-3p and TP53INP1 as new biomarkers for prognostic stratification that may be possible therapeutic targets for ependymoma., (© 2017 Wiley Periodicals, Inc.)

Contrast enhancement of glioblastomas (GBM) is caused by the decrease in relaxation time, T1. Here, we demonstrate that the quantitative measurement of T1 (qT1) discovers a subtle enhancement in GBM patients that is invisible in standard MRI. We assessed the volume change of this "cloudy" enhancement during radio-chemotherapy and its impact on patients' progression-free survival (PFS). We enrolled 18 GBM patients in this observational, prospective cohort study and measured 3T-MRI pre- and post contrast agent with standard T1-weighted (T1w) and with sequences to quantify T1 before radiation, and at 6-week intervals during radio-chemotherapy. We measured contrast enhancement by subtracting pre from post contrast contrast images, yielding relative signal increase ∆T1w and relative T1 shortening ∆qT1. On ∆qT1, we identified a solid and a cloudy-enhancing compartment and evaluated the impact of their therapy-related volume change upon PFS. In ∆qT1 maps cloudy-enhancing compartments were found in all but two patients at baseline and in all patients during therapy. The qT1 decrease in the cloudy-enhancing compartment post contrast was 21.64% versus 1.96% in the contralateral control tissue (P < 0.001). It was located at the margin of solid enhancement which was also seen on T1w. In contrast, the cloudy-enhancing compartment was visually undetectable on ∆T1w. A volume decrease of more than 21.4% of the cloudy-enhancing compartment at first follow-up predicted longer PFS (P = 0.038). Cloudy-enhancing compartment outside the solid contrast-enhancing area of GBM is a new observation which is only visually detectable with qT1-mapping and may represent tumor infiltration. Its early volume decrease predicts a longer PFS in GBM patients during standard radio-chemotherapy., (© 2016 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.)

Ependymomas in children can arise throughout all compartments of the central nervous system (CNS). Highly malignant paediatric ependymoma subtypes are Group A tumours of the posterior fossa (PF-EPN-A) and RELA-fusion positive (ST-EPN-RELA) tumours in the supratentorial compartment. It was repeatedly reported in smaller series that accumulation of p53 is frequently observed in ependymomas and that immunohistochemical staining correlates with poor clinical outcome, while TP53 mutations are rare. Our TP53 mutation analysis of 130 primary ependymomas identified a mutation rate of only 3%. Immunohistochemical analysis of 398 ependymomas confirmed previous results correlating the accumulation of p53 with inferior outcome. Among the p53-positive ependymomas, the vast majority exhibited a RELA fusion leading to the hypothesis that p53 inactivation might be linked to RELA positivity.In order to assess the potential of p53 reactivation through MDM2 inhibition in ependymoma, we evaluated the effects of Actinomycin-D and Nutlin-3 treatment in two preclinical ependymoma models representing the high-risk subtypes PF-EPN-A and ST-EPN-RELA. The IC-50 of the agent as determined by metabolic activity assays was in the lower nano-molar range (0.2-0.7 nM). Transcriptome analyses of high-dose (100 nM), low-dose (5 nM) and non-treated cells revealed re-expression of p53 dependent genes including p53 upregulated modulator of apoptosis (PUMA) after low-dose treatment. At the protein level, we validated the Actinomycin-D induced upregulation of PUMA, and of p53 interaction partners MDM2 and p21. Proapoptotic effects of low-dose application of the agent were confirmed by flow cytometry. Thus, Actinomycin-D could constitute a promising therapeutic option for ST-EPN-RELA ependymoma patients, whose tumours frequently exhibit p53 inactivation., Competing Interests: The authors of this study declare no conflicts of interest.

Purpose: The adequate second-line therapy of patients with glioblastoma (GBM) is a matter of ongoing debate. This particularly applies to patients with a non-methylated MGMT promotor who are known to have a poor response to alkylating chemotherapy. In some countries, antiangiogenic therapy with BEV is applied as second-line therapy, and in others nitrosourea therapy is second-line choice. It is an open question whether the delay of BEV to third-line therapy has a negative impact on survival., Methods: A total of 61 adult patients (median age 56.9 years) with MGMT-non-methylated relapsed GBM treated with BEV (n = 45) or nitrosourea (n = 16) as second-line therapy were analyzed retrospectively and compared regarding progression-free survival (PFS) and overall survival (OS)., Results: Patients treated with second-line BEV had longer median PFS (107 days, 95 % CI 80.7-133.2 days) than patients with second-line nitrosourea (52 days, 95 % CI 36.3-67.7 days, P = 0.011, logrank test). However, there was no significant difference in overall survival (BEV median 170 days, 95 % CI 87.2-252.8 days; nitrosourea median 256 days, 95 % CI 159.9-352.0 days, P = 0.468). PFS was similar after BEV third-line therapy (median 117 days, 95 % CI 23.6-210.4 days) as compared to second-line BEV therapy (median 107 days, 95 % CI 80.7-133.3 days, P = 0.584)., Conclusion: Our findings suggest that early treatment with BEV in patients with MGMT-non-methylated relapsed GBM is associated with a better PFS, but not with superior OS, possibly implicating that the early, i.e., second-line, use of BEV is not mandatory and BEV treatment may safely be delayed to third-line therapy in this subgroup of patients.

Purpose: Dovitinib (TKI258) is an oral multi-tyrosine kinase inhibitor of FGFR, VEGFR, PDGFR β, and c-Kit. Since dovitinib is able to cross the blood-brain barrier and targets brain tumor-relevant pathways, we conducted a phase I trial to demonstrate its safety in recurrent glioblastoma (GBM)., Patients and Methods: Patients with first or second GBM recurrence started treatment with the maximal tolerated dose (MTD) previously established in systemic cancer patients (500 mg/d, 5 days on/2 days off). A modified 3 + 3 design in three cohorts (500, 400, 300 mg) was used., Results: Twelve patients were enrolled. Seventy-two adverse events (AEs) occurred and 16.7 % of AEs were classified as ≥CTC grade 3 toxicity, mainly including hepatotoxicity and hematotoxicity. Only one out of six patients of the 300-mg cohort showed grade 3 toxicity. The PFS-6 rate was 16.7 %, and it was not associated with detection of the FGFR-TACC gene fusion in the tumor., Conclusion: Dovitinib is safe in patients with recurrent GBM and showed efficacy in only some patients unselected for target expression. The recommended phase II dose of 300 mg would be substantially lower than the recently established MTD in systemic cancer patients. Further personalized trials are recommended.

Purpose: Pseudoprogression (PsP) is characterized by therapy-associated but not tumor growth-associated increases of contrast-enhancing glioblastoma lesions on MRI. Although typically occurring during the first 3 months after radiochemotherapy, PsP may occur later in the course of the disease and may then be particularly difficult to distinguish from true tumor progression. We explored PET using O-(2-[(18)F]fluoroethyl)-L-tyrosine ((18)F-FET-PET) to approach the diagnostic dilemma., Experimental Design: Twenty-six patients with glioblastoma that presented with increasing contrast-enhancing lesions later than 3 months after completion of radiochemotherapy underwent (18)F-FET-PET. Maximum and mean tumor/brain ratios (TBRmax and TBRmean) of (18)F-FET uptake as well as time-to-peak (TTP) and patterns of the time-activity curves were determined. The final diagnosis of true progression versus late PsP was based on follow-up MRI using RANO criteria., Results: Late PsP occurred in 7 patients with a median time from radiochemotherapy completion of 24 weeks while the remaining patients showed true tumor progression. TBRmax and TBRmean were significantly higher in patients with true progression than in patients with late PsP (TBRmax 2.4 ± 0.1 vs. 1.5 ± 0.2, P = 0.003; TBRmean 2.1 ± 0.1 vs. 1.5 ± 0.2, P = 0.012) whereas TTP was significantly shorter (mean TTP 25 ± 2 vs. 40 ± 2 min, P < 0.001). ROC analysis yielded an optimal cutoff value of 1.9 for TBRmax to differentiate between true progression and late PsP (sensitivity 84%, specificity 86%, accuracy 85%, P = 0.015)., Conclusions: O-(2-[(18)F]fluoroethyl)-L-tyrosine PET provides valuable information in assessing the elusive phenomenon of late PsP. Clin Cancer Res; 22(9); 2190-6. ©2015 AACR., (©2015 American Association for Cancer Research.)

Objective: To investigate whether the occurrence or clearance of microhemorrhages in cerebral amyloid angiopathy (CAA)-related vascular inflammation can be modified by immunosuppressive treatment., Methods: Clinical and radiologic follow-up for more than 5 years of a patient with histopathologically confirmed CAA-related vascular inflammation treated with a prolonged and tapered regimen of IV cyclophosphamide and oral steroids., Results: Under long-term immunosuppressive treatment, a reduced number of cortical micobleeds was observed on repeat MRIs because of both the prevention of new microbleeds and the clearance of those existing at baseline., Conclusions: Sustained immunosuppression should be considered and systematically investigated as a treatment option for cortical microbleeds in CAA and related inflammatory phenotypes., Classification of Evidence: This study provides Class IV evidence. This is a single observational study without controls.

Ependymomas are common childhood brain tumours that occur throughout the nervous system, but are most common in the paediatric hindbrain. Current standard therapy comprises surgery and radiation, but not cytotoxic chemotherapy as it does not further increase survival. Whole-genome and whole-exome sequencing of 47 hindbrain ependymomas reveals an extremely low mutation rate, and zero significant recurrent somatic single nucleotide variants. Although devoid of recurrent single nucleotide variants and focal copy number aberrations, poor-prognosis hindbrain ependymomas exhibit a CpG island methylator phenotype. Transcriptional silencing driven by CpG methylation converges exclusively on targets of the Polycomb repressive complex 2 which represses expression of differentiation genes through trimethylation of H3K27. CpG island methylator phenotype-positive hindbrain ependymomas are responsive to clinical drugs that target either DNA or H3K27 methylation both in vitro and in vivo. We conclude that epigenetic modifiers are the first rational therapeutic candidates for this deadly malignancy, which is epigenetically deregulated but genetically bland.

Medulloblastoma is an aggressively growing tumour, arising in the cerebellum or medulla/brain stem. It is the most common malignant brain tumour in children, and shows tremendous biological and clinical heterogeneity. Despite recent treatment advances, approximately 40% of children experience tumour recurrence, and 30% will die from their disease. Those who survive often have a significantly reduced quality of life. Four tumour subgroups with distinct clinical, biological and genetic profiles are currently identified. WNT tumours, showing activated wingless pathway signalling, carry a favourable prognosis under current treatment regimens. SHH tumours show hedgehog pathway activation, and have an intermediate prognosis. Group 3 and 4 tumours are molecularly less well characterized, and also present the greatest clinical challenges. The full repertoire of genetic events driving this distinction, however, remains unclear. Here we describe an integrative deep-sequencing analysis of 125 tumour-normal pairs, conducted as part of the International Cancer Genome Consortium (ICGC) PedBrain Tumor Project. Tetraploidy was identified as a frequent early event in Group 3 and 4 tumours, and a positive correlation between patient age and mutation rate was observed. Several recurrent mutations were identified, both in known medulloblastoma-related genes (CTNNB1, PTCH1, MLL2, SMARCA4) and in genes not previously linked to this tumour (DDX3X, CTDNEP1, KDM6A, TBR1), often in subgroup-specific patterns. RNA sequencing confirmed these alterations, and revealed the expression of what are, to our knowledge, the first medulloblastoma fusion genes identified. Chromatin modifiers were frequently altered across all subgroups. These findings enhance our understanding of the genomic complexity and heterogeneity underlying medulloblastoma, and provide several potential targets for new therapeutics, especially for Group 3 and 4 patients.