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Background: Immune checkpoint inhibitors (ICIs) have efficacy in several solid tumors but limited efficacy in glioblastoma (GBM). This study evaluated the safety of anti-CTLA-4 and anti-PD-1 ICIs alone or in combination in newly diagnosed GBM after completion of standard radiochemotherapy with the subsequent intent to test combinatorial ICIs in this setting., Methods: The primary endpoint was dose-limiting toxicity (DLT) for adults with unifocal, supratentorial newly diagnosed GBM after resection and chemoradiation. Ipilimumab and nivolumab were tested separately and in combination with a planned expansion cohort dependent upon DLT results., Results: Thirty-two patients were enrolled at 9 institutions: 6 to each DLT assessment cohort and 14 to the expansion cohort. Median age: 55 years, 67.7% male, 83.9% White. Treatment was well tolerated with 16% Grade 4 events; the combination did not have unexpectedly increased toxicity, with no Grade 5 events. One DLT was seen in each single-agent treatment; none were observed in the combination, leading to expanded accrual of the combined treatment. The median follow-up was 19.6 months. For all patients receiving combination treatment, median overall survival (OS) and progression-free survival (PFS) were 20.7 and 16.1 months, respectively., Conclusions: IPI and NIVO are safe and tolerable with toxicities similar to those noted with other cancers when given in combination with adjuvant temozolomide for newly diagnosed GBM. Combination IPI + NIVO is not substantially more toxic than single agents. These results support a subsequent efficacy trial to test the combination of ICIs in Phase II/III for patients with newly diagnosed GBM., Clinicaltrials.gov Registration: NCT02311920., (© 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.)

Purpose: Patients with glioblastoma (GBM) have a dismal prognosis. Although the DNA alkylating agent temozolomide (TMZ) is the mainstay of chemotherapy, therapeutic resistance rapidly develops in patients. Base excision repair inhibitor TRC102 (methoxyamine) reverses TMZ resistance in preclinical glioma models. We aimed to investigate the efficacy and safety of oral TRC102+TMZ in recurrent GBM (rGBM)., Patients and Methods: A preregistered (NCT02395692), nonrandomized, multicenter, phase 2 clinical trial (BERT) was planned and conducted through the Adult Brain Tumor Consortium (ABTC-1402). Arm 1 included patients with bevacizumab-naïve GBM at the first recurrence, with the primary endpoint of response rates. If sufficient activity was identified, a second arm was planned for the bevacizumab-refractory patients. The secondary endpoints were overall survival (OS), progression-free survival (PFS), PFS at 6 months (PFS6), and toxicity., Results: Arm 1 enrolled 19 patients with a median of two treatment cycles. Objective responses were not observed; hence, arm 2 did not open. The median OS was 11.1 months [95% confidence interval (CI), 8.2-17.9]. The median PFS was 1.9 months (95% CI, 1.8-3.7). The PFS6 was 10.5% (95% CI, 1.3%-33.1%). Most toxicities were grades 1 and 2, with two grade 3 lymphopenias and one grade 4 thrombocytopenia. Two patients with PFS ≥ 17 months and OS > 32 months were deemed "extended survivors." RNA sequencing of tumor tissue, obtained at diagnosis, demonstrated significantly enriched signatures of DNA damage response (DDR), chromosomal instability (CIN70, CIN25), and cellular proliferation (PCNA25) in "extended survivors.", Conclusions: These findings confirm the safety and feasibility of TRC102+TMZ in patients with rGBM. They also warrant further evaluation of combination therapy in biomarker-enriched trials enrolling GBM patients with baseline hyperactivated DDR pathways., (©2024 American Association for Cancer Research.)

Purpose: Glioblastoma (GB) is the most common primary malignant brain tumor with the highest incidence occurring in older adults with a median age at diagnosis of 64 years old. While treatment often improves survival it brings toxicities and adverse events (AE). Here we identify sex differences in treatment patterns and AE in individuals ≥ 66 years at diagnosis with GB., Methods: Using the SEER-Medicare dataset sex differences in adverse events were assessed using multivariable logistic regression performed to calculate the male/female odds ratio (M/F OR) and 95% confidence intervals [95% CI] of experiencing an AE adjusted for demographic variables and Elixhauser comorbidity score., Results: Males with GB were more likely to receive standard of care (SOC; Surgery with concurrent radio-chemotherapy) [20%] compared to females [17%], whereas females were more likely to receive no treatment [26%] compared to males [21%]. Females with GB receiving SOC were more likely to develop gastrointestinal disorders (M/F OR = 0.76; 95% CI,0.64-0.91, p = 0.002) or blood and lymphatic system disorders (M/F OR = 0.79; 95% CI,0.66-0.95, p = 0.012). Males with GB receiving SOC were more likely to develop cardiac disorders (M/F OR = 1.21; 95% CI,1.02-1.44, p = 0.029) and renal disorders (M/F OR = 1.65; 95% CI,1.37-2.01, p < 0.001)., Conclusions: Sex differences for individuals, 66 years and older, diagnosed with GB exist in treatment received and adverse events developed across different treatment modalities., (© 2024. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Glioblastoma is the most lethal primary brain tumor with glioblastoma stem cells (GSCs) atop a cellular hierarchy. GSCs often reside in a perivascular niche, where they receive maintenance cues from endothelial cells, but the role of heterogeneous endothelial cell populations remains unresolved. Here, we show that lymphatic endothelial-like cells (LECs), while previously unrecognized in brain parenchyma, are present in glioblastomas and promote growth of CCR7-positive GSCs through CCL21 secretion. Disruption of CCL21-CCR7 paracrine communication between LECs and GSCs inhibited GSC proliferation and growth. LEC-derived CCL21 induced KAT5-mediated acetylation of HMGCS1 on K273 in GSCs to enhance HMGCS1 protein stability. HMGCS1 promoted cholesterol synthesis in GSCs, favorable for tumor growth. Expression of the CCL21-CCR7 axis correlated with KAT5 expression and HMGCS1 K273 acetylation in glioblastoma specimens, informing patient outcome. Collectively, glioblastomas contain previously unrecognized LECs that promote the molecular crosstalk between endothelial and tumor cells, offering potentially alternative therapeutic strategies., (© 2024. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Objectives: To test the feasibility of using 3D MRF maps with radiomics analysis and machine learning in the characterization of adult brain intra-axial neoplasms., Methods: 3D MRF acquisition was performed on 78 patients with newly diagnosed brain tumors including 33 glioblastomas (grade IV), 6 grade III gliomas, 12 grade II gliomas, and 27 patients with brain metastases. Regions of enhancing tumor, non-enhancing tumor, and peritumoral edema were segmented and radiomics analysis with gray-level co-occurrence matrices and gray-level run-length matrices was performed. Statistical analysis was performed to identify features capable of differentiating tumors based on type, grade, and isocitrate dehydrogenase (IDH1) status. Receiver operating curve analysis was performed and the area under the curve (AUC) was calculated for tumor classification and grading. For gliomas, Kaplan-Meier analysis for overall survival was performed using MRF T1 features from enhancing tumor region., Results: Multiple MRF T1 and T2 features from enhancing tumor region were capable of differentiating glioblastomas from brain metastases. Although no differences were identified between grade 2 and grade 3 gliomas, differentiation between grade 2 and grade 4 gliomas as well as between grade 3 and grade 4 gliomas was achieved. MRF radiomics features were also able to differentiate IDH1 mutant from the wild-type gliomas. Radiomics T1 features for enhancing tumor region in gliomas correlated to overall survival (p < 0.05)., Conclusion: Radiomics analysis of 3D MRF maps allows differentiating glioblastomas from metastases and is capable of differentiating glioblastomas from metastases and characterizing gliomas based on grade, IDH1 status, and survival., Key Points: • 3D MRF data analysis using radiomics offers novel tissue characterization of brain tumors. • 3D MRF with radiomics offers glioma characterization based on grade, IDH1 status, and overall patient survival., (© 2022. The Author(s), under exclusive licence to European Society of Radiology.)

Purpose: Gliomagenesis and resistance of glioblastoma (GBM) are believed to be mediated by glioma stem cells (GSC). Evidence suggests that SHH signaling promotes GSC proliferation and self-renewal., Methods: ABTC-0904 was a two-arm, multicenter phase 0/II study of GDC-0449, an oral inhibitor of Smoothened (SMO) in patients undergoing resection for recurrent GBM. All patients (Arms I and II) had surgery and received drug post-operatively. Only patients in Arm I received drug prior to surgery. The primary objective was to determine 6-month progression free survival (PFS-6). Secondary endpoints include median PFS (mPFS) and overall survival (mOS), response rate, and toxicity. Correlative studies included bioanalysis of GDC-0449, and inhibition of SHH signaling, GSC proliferation and self-renewal., Results: Forty-one patients were enrolled. Pharmacokinetics of GDC-0449 in plasma demonstrated levels within expected therapeutic range in 75% of patients. The proportion of tumorcells producing CD133 + neurospheres, neurosphere proliferation, self-renewal, and expression of the SHh downstream signaling was significantly decreased in Arm I following GDC-0449 treatment (p < 0.005; p < 0.001 respectively) compared to Arm II (no drug pre-op). Treatment was well tolerated. There were no objective responders in either arm. Overall PFS-6 was 2.4% (95% CI 0.9-11.1%). Median PFS was 2.3 months (95% CI 1.9-2.6) and mOS was 7.8 months (95% CI 5.4-10.1)., Conclusions: GDC-0449 was well tolerated, reached tumor, and inhibited CD133 + neurosphere formation, but had little clinical efficacy as a single agent in rGBM. This suggests growth and maintenance of rGBM is not solely dependent on the SHH pathway thus targeting SMO may require combined approaches., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Although machine learning (ML) has shown promise across disciplines, out-of-sample generalizability is concerning. This is currently addressed by sharing multi-site data, but such centralization is challenging/infeasible to scale due to various limitations. Federated ML (FL) provides an alternative paradigm for accurate and generalizable ML, by only sharing numerical model updates. Here we present the largest FL study to-date, involving data from 71 sites across 6 continents, to generate an automatic tumor boundary detector for the rare disease of glioblastoma, reporting the largest such dataset in the literature (n = 6, 314). We demonstrate a 33% delineation improvement for the surgically targetable tumor, and 23% for the complete tumor extent, over a publicly trained model. We anticipate our study to: 1) enable more healthcare studies informed by large diverse data, ensuring meaningful results for rare diseases and underrepresented populations, 2) facilitate further analyses for glioblastoma by releasing our consensus model, and 3) demonstrate the FL effectiveness at such scale and task-complexity as a paradigm shift for multi-site collaborations, alleviating the need for data-sharing., (© 2022. The Author(s).)

Introduction: Glioblastoma multiforme (GBM) constitutes one of the deadliest tumors to afflict humans, although it is still considered an orphan disease. Despite testing multiple new and innovative therapies in ongoing clinical trials, the median survival for this type of malignancy is less than two years after initial diagnosis, regardless of therapy. One class of promising new therapies are chimeric antigen receptor T cells or CAR-T which have been shown to be very effective at treating refractory liquid tumors such as B-cell malignancies. However, CAR-T effectivity against solid tumors such as GBM has been limited thus far., Methods: A Pubmed, Google Scholar, Directory of Open Access Journals, and Web of Science literature search using the terms chimeric antigen receptor or CAR-T, GBM, solid tumor immunotherapy, immunotherapy, and CAR-T combination was performed for publication dates between January 1987 and November 2021., Results: In the current review, we present a comprehensive list of CAR-T cells developed to treat GBM, we describe new possible T-cell engineering strategies against GBM while presenting a short introductory history to the reader regarding the origin(s) of this cutting-edge therapy. We have also compiled a unique list of anti-GBM CAR-Ts with their specific protein sequences and their functions as well as an inventory of clinical trials involving CAR-T and GBM., Conclusions: The aim of this review is to introduce the reader to the field of T-cell engineering using CAR-Ts to treat GBM and describe the obstacles that may need to be addressed in order to significantly delay the relentless growth of GBM., (© 2021. The Author(s).)

Background/purpose: Glioblastoma (GBM) is the most common primary malignant brain tumor. Sex has been shown to be an important prognostic factor for GBM. The purpose of this study was to develop and independently validate sex-specific nomograms for estimation of individualized GBM survival probabilities using data from 2 independent NRG Oncology clinical trials., Methods: This analysis included information on 752 (NRG/RTOG 0525) and 599 (NRG/RTOG 0825) patients with newly diagnosed GBM. The Cox proportional hazard models by sex were developed using NRG/RTOG 0525 and significant variables were identified using a backward selection procedure. The final selected models by sex were then independently validated using NRG/RTOG 0825., Results: Final nomograms were built by sex. Age at diagnosis, KPS, MGMT promoter methylation and location of tumor were common significant predictors of survival for both sexes. For both sexes, tumors in the frontal lobes had significantly better survival than tumors of multiple sites. Extent of resection, and use of corticosteroids were significant predictors of survival for males., Conclusions: A sex specific nomogram that assesses individualized survival probabilities (6-, 12- and 24-months) for patients with GBM could be more useful than estimation of overall survival as there are factors that differ between males and females. A user friendly online application can be found here- https://npatilshinyappcalculator.shinyapps.io/SexDifferencesInGBM/ ., (© 2021. This is a U.S. government work and not under copyright protection in the U.S.; foreign copyright protection may apply.)

Purpose: This is a radiomics study investigating the ability of texture analysis of MRF maps to improve differentiation between intra-axial adult brain tumors and to predict survival in the glioblastoma cohort., Methods: Magnetic resonance fingerprinting (MRF) acquisition was performed on 31 patients across 3 groups: 17 glioblastomas, 6 low-grade gliomas, and 8 metastases. Using regions of interest for the solid tumor and peritumoral white matter on T1 and T2 maps, second-order texture features were calculated from gray-level co-occurrence matrices and gray-level run length matrices. Selected features were compared across the three tumor groups using Wilcoxon rank-sum test. Receiver operating characteristic curve analysis was performed for each feature. Kaplan-Meier method was used for survival analysis with log rank tests., Results: Low-grade gliomas and glioblastomas had significantly higher run percentage, run entropy, and information measure of correlation 1 on T1 than metastases (p < 0.017). The best separation of all three tumor types was seen utilizing inverse difference normalized and homogeneity values for peritumoral white matter in both T1 and T2 maps (p < 0.017). In solid tumor T2 maps, lower values in entropy and higher values of maximum probability and high-gray run emphasis were associated with longer survival in glioblastoma patients (p < 0.05). Several texture features were associated with longer survival in glioblastoma patients on peritumoral white matter T1 maps (p < 0.05)., Conclusion: Texture analysis of MRF-derived maps can improve our ability to differentiate common adult brain tumors by characterizing tumor heterogeneity, and may have a role in predicting outcomes in patients with glioblastoma.

Background: Cancer stem-like cells are a major cause of resistance to therapy in patients with glioblastoma (GBM) as well as other cancers. Tumor cells are maintained in a stem-like proliferative state in large part through the Notch signaling pathway. The function of this pathway in turn depends on gamma secretase activity. Inhibition of this enzyme therefore inhibits the Notch pathway and tumor growth as measured by a reduction in the formation of brain tumor neurospheres in murine models. RO4929097 is an oral gamma secretase inhibitor., Objective: To estimate the 6-mo progression-free survival rate (PFS6) in patients with progressive GBM and to inhibit by 50% the generation of neurospheres in fresh tissue resected from patients treated with RO4929097., Methods: In this phase II and pharmacodynamic study, patients with recurrent GBM received RO4929097 in a study of 2 groups. Group A patients had unresectable disease and received drug in a standard phase II design. Group B patients had resectable disease and received drug before and after surgical resection. Endpoints included PFS6 and the inhibition of neurosphere formation in the resected tumor samples., Results: A total of 47 patients received treatment, 7 of whom had tumor resection. The PFS6 was 4%, and the inhibition of neurosphere formation occurred in 1 of 7 patient samples., Conclusion: RO4929097 was inactive in recurrent GBM patients and demonstrated minimal inhibition of neurosphere formation in fresh tissue samples., (Copyright © 2020 by the Congress of Neurological Surgeons.)

Background: The tumorigenic potential of glioma stem cells (GSCs) is associated with multiple reversible molecular alternations, but the role of posttranslational protein sumoylation in GSCs has not been elucidated. The development of GSC-targeting drugs relies on the discovery of GSC-preferential molecular modifications and the relevant signaling pathways. In this work, we investigated the protein sumoylation status, the major sumoylated substrate, and the key regulatory enzyme in GSCs to explore the therapeutic potential of disrupting protein sumoylation for glioblastoma (GBM) treatment., Methods: Patient-derived GSCs, primary GBM sections, and intracranial GBM xenografts were used to determine protein sumoylation and the related molecular mechanisms by immunoblot, quantitative PCR, immunoprecipitation, immunofluorescence, and immunohistochemistry. Orthotopic GBM xenograft models were applied to investigate the inhibition of tumor growth by disrupting protein sumoylation with short hairpin (sh)RNAs or molecular inhibitors., Results: We show that high levels of small ubiquitin-related modifier 1 (SUMO1)-but not SUMO2/3-modified sumoylation are preferentially present in GSCs. The promyelocytic leukemia (PML) protein is a major SUMO1-sumoylated substrate in GSCs, whose sumoylation facilitates its interaction with c-Myc to stabilize c-Myc proteins. The prolyl-isomerase Pin1 is preferentially expressed in GSCs and functions as the key enzyme to promote SUMO1 sumoylation. Disruption of SUMO1 sumoylation by Pin1 silencing with shRNAs or inhibition with its inhibitor Juglone markedly abrogated GSC maintenance and mitigated GSC-driven tumor growth., Conclusions: Our findings indicate that high SUMO1-modified protein sumoylation as a feature of GSCs is critical for GSC maintenance, suggesting that targeting SUMO1 sumoylation may effectively improve GBM treatment., (© The Author(s) 2020. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Nuclear matrix-associated proteins (NMPs) play critical roles in regulating chromatin organization and gene transcription by binding to the matrix attachment regions (MARs) of DNA. However, the functional significance of NMPs in glioblastoma (GBM) progression remains unclear. Here, we show that the Special AT-rich Binding Protein-2 (SATB2), one of crucial NMPs, recruits histone acetyltransferase CBP to promote the FOXM1-mediated cell proliferation and tumor growth of GBM. SATB2 is preferentially expressed by glioma stem cells (GSCs) in GBM. Disrupting SATB2 markedly inhibited GSC proliferation and GBM malignant growth by down-regulating expression of key genes involved in cell proliferation program. SATB2 activates FOXM1 expression to promote GSC proliferation through binding to the MAR sequence of FOXM1 gene locus and recruiting CBP to the MAR. Importantly, pharmacological inhibition of SATB2/CBP transcriptional activity by the CBP inhibitor C646 suppressed GSC proliferation in vitro and GBM growth in vivo. Our study uncovers a crucial role of the SATB2/CBP-mediated transcriptional regulation in GBM growth, indicating that targeting SATB2/CBP may effectively improve GBM treatment., (© 2020 The Authors. Published under the terms of the CC BY 4.0 license.)

Glioblastoma multiforme (GBM) is the most malignant primary brain cancer affecting adults. Therapeutic options for GBM have remained the same for over a decade with no significant improvement. Many therapies that are successful in culture have failed in patients, likely due to the complex microenvironment in the brain, which has yet to be reproduced in any culture model. Furthermore, the high passage number of cultured cells and clonal selection fail to recapitulate the molecular and genomic signatures of GBM. We have established orthotopic patient-derived xenografts (PDX) from 37 GBM patients with human GBM. Of the 69 patient samples analyzed, we were successful in passaging 37 lines three or more generations (53.6%). After phenotypic characterization of the xenografted tumor tissue, two different growth patterns emerged highly invasive or localized. The phenotype was dependent on malignancy and previous treatment of the patient from which the xenograft was derived. Physiologically, mice exhibited symptoms more quickly with each subsequent passage, particularly in the localized tumors. Study of these physiologically relevant human xenografts in mice will enable therapeutic screenings in a microenvironment that more closely resembles GBM and may allow development of individualized patient models which may eventually be used for simulating treatment.

Importance: Per the World Health Organization 2016 integrative classification, newly diagnosed glioblastomas are separated into isocitrate dehydrogenase gene 1 or 2 (IDH)-wild-type and IDH-mutant subtypes, with median patient survival of 1.2 and 3.6 years, respectively. Although maximal resection of contrast-enhanced (CE) tumor is associated with longer survival, the prognostic importance of maximal resection within molecular subgroups and the potential importance of resection of non-contrast-enhanced (NCE) disease is poorly understood., Objective: To assess the association of resection of CE and NCE tumors in conjunction with molecular and clinical information to develop a new road map for cytoreductive surgery., Design, Setting, and Participants: This retrospective, multicenter cohort study included a development cohort from the University of California, San Francisco (761 patients diagnosed from January 1, 1997, through December 31, 2017, with 9.6 years of follow-up) and validation cohorts from the Mayo Clinic (107 patients diagnosed from January 1, 2004, through December 31, 2014, with 5.7 years of follow-up) and the Ohio Brain Tumor Study (99 patients with data collected from January 1, 2008, through December 31, 2011, with a median follow-up of 10.9 months). Image accessors were blinded to patient groupings. Eligible patients underwent surgical resection for newly diagnosed glioblastoma and had available survival, molecular, and clinical data and preoperative and postoperative magnetic resonance images. Data were analyzed from November 15, 2018, to March 15, 2019., Main Outcomes and Measures: Overall survival., Results: Among the 761 patients included in the development cohort (468 [61.5%] men; median age, 60 [interquartile range, 51.6-67.7] years), younger patients with IDH-wild-type tumors and aggressive resection of CE and NCE tumors had survival similar to that of patients with IDH-mutant tumors (median overall survival [OS], 37.3 [95% CI, 31.6-70.7] months). Younger patients with IDH-wild-type tumors and reduction of CE tumor but residual NCE tumors fared worse (median OS, 16.5 [95% CI, 14.7-18.3] months). Older patients with IDH-wild-type tumors benefited from reduction of CE tumor (median OS, 12.4 [95% CI, 11.4-14.0] months). The results were validated in the 2 external cohorts. The association between aggressive CE and NCE in patients with IDH-wild-type tumors was not attenuated by the methylation status of the promoter region of the DNA repair enzyme O6-methylguanine-DNA methyltransferase., Conclusions and Relevance: This study confirms an association between maximal resection of CE tumor and OS in patients with glioblastoma across all subgroups. In addition, maximal resection of NCE tumor was associated with longer OS in younger patients, regardless of IDH status, and among patients with IDH-wild-type glioblastoma regardless of the methylation status of the promoter region of the DNA repair enzyme O6-methylguanine-DNA methyltransferase. These conclusions may help reassess surgical strategies for individual patients with newly diagnosed glioblastoma.

Purpose: To construct a multi-institutional radiomic model that supports upfront prediction of progression-free survival (PFS) and recurrence pattern (RP) in patients diagnosed with glioblastoma multiforme (GBM) at the time of initial diagnosis., Patients and Methods: We retrospectively identified data for patients with newly diagnosed GBM from two institutions (institution 1, n = 65; institution 2, n = 15) who underwent gross total resection followed by standard adjuvant chemoradiation therapy, with pathologically confirmed recurrence, sufficient follow-up magnetic resonance imaging (MRI) scans to reliably determine PFS, and available presurgical multiparametric MRI (MP-MRI). The advanced software suite Cancer Imaging Phenomics Toolkit (CaPTk) was leveraged to analyze standard clinical brain MP-MRI scans. A rich set of imaging features was extracted from the MP-MRI scans acquired before the initial resection and was integrated into two distinct imaging signatures for predicting mean shorter or longer PFS and near or distant RP. The predictive signatures for PFS and RP were evaluated on the basis of different classification schemes: single-institutional analysis, multi-institutional analysis with random partitioning of the data into discovery and replication cohorts, and multi-institutional assessment with data from institution 1 as the discovery cohort and data from institution 2 as the replication cohort., Results: These predictors achieved cross-validated classification performance (ie, area under the receiver operating characteristic curve) of 0.88 (single-institution analysis) and 0.82 to 0.83 (multi-institution analysis) for prediction of PFS and 0.88 (single-institution analysis) and 0.56 to 0.71 (multi-institution analysis) for prediction of RP., Conclusion: Imaging signatures of presurgical MP-MRI scans reveal relatively high predictability of time and location of GBM recurrence, subject to the patients receiving standard first-line chemoradiation therapy. Through its graphical user interface, CaPTk offers easy accessibility to advanced computational algorithms for deriving imaging signatures predictive of clinical outcome and could similarly be used for a variety of radiomic and radiogenomic analyses.

Glioblastoma ranks among the most aggressive and lethal of all human cancers. Functionally defined glioma stem cells (GSC) contribute to this poor prognosis by driving therapeutic resistance and maintaining cellular heterogeneity. To understand the molecular processes essential for GSC maintenance and tumorigenicity, we interrogated the superenhancer landscapes of primary glioblastoma specimens and in vitro GSCs. GSCs epigenetically upregulated ELOVL2, a key polyunsaturated fatty-acid synthesis enzyme. Targeting ELOVL2 inhibited glioblastoma cell growth and tumor initiation. ELOVL2 depletion altered cellular membrane phospholipid composition, disrupted membrane structural properties, and diminished EGFR signaling through control of fatty-acid elongation. In support of the translational potential of these findings, dual targeting of polyunsaturated fatty-acid synthesis and EGFR signaling had a combinatorial cytotoxic effect on GSCs. SIGNIFICANCE: Glioblastoma remains a devastating disease despite extensive characterization. We profiled epigenomic landscapes of glioblastoma to pinpoint cell state-specific dependencies and therapeutic vulnerabilities. GSCs utilize polyunsaturated fatty-acid synthesis to support membrane architecture, inhibition of which impairs EGFR signaling and GSC proliferation. Combinatorial targeting of these networks represents a promising therapeutic strategy. See related commentary by Affronti and Wellen, p. 1161 . This article is highlighted in the In This Issue feature, p. 1143 ., (©2019 American Association for Cancer Research.)

Glioblastoma is an incurable brain cancer characterized by high genetic and pathological heterogeneity. Here, we mapped active chromatin landscapes with gene expression, whole exomes, copy number profiles, and DNA methylomes across 44 patient-derived glioblastoma stem cells (GSCs), 50 primary tumors, and 10 neural stem cells (NSCs) to identify essential super-enhancer (SE)-associated genes and the core transcription factors that establish SEs and maintain GSC identity. GSCs segregate into two groups dominated by distinct enhancer profiles and unique developmental core transcription factor regulatory programs. Group-specific transcription factors enforce GSC identity; they exhibit higher activity in glioblastomas versus NSCs, are associated with poor clinical outcomes, and are required for glioblastoma growth in vivo. Although transcription factors are commonly considered undruggable, group-specific enhancer regulation of the MAPK/ERK pathway predicts sensitivity to MEK inhibition. These data demonstrate that transcriptional identity can be leveraged to identify novel dependencies and therapeutic approaches., (© 2019 Mack et al.)

Genetic drivers of cancer can be dysregulated through epigenetic modifications of DNA. Although the critical role of DNA 5-methylcytosine (5mC) in the regulation of transcription is recognized, the functions of other non-canonical DNA modifications remain obscure. Here, we report the identification of novel N 6 -methyladenine (N 6 -mA) DNA modifications in human tissues and implicate this epigenetic mark in human disease, specifically the highly malignant brain cancer glioblastoma. Glioblastoma markedly upregulated N 6 -mA levels, which co-localized with heterochromatic histone modifications, predominantly H3K9me3. N 6 -mA levels were dynamically regulated by the DNA demethylase ALKBH1, depletion of which led to transcriptional silencing of oncogenic pathways through decreasing chromatin accessibility. Targeting the N 6 -mA regulator ALKBH1 in patient-derived human glioblastoma models inhibited tumor cell proliferation and extended the survival of tumor-bearing mice, supporting this novel DNA modification as a potential therapeutic target for glioblastoma. Collectively, our results uncover a novel epigenetic node in cancer through the DNA modification N 6 -mA., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Background: Standard therapy for glioblastoma includes surgery, radiotherapy, and temozolomide. This Phase 3 trial evaluates the addition of an autologous tumor lysate-pulsed dendritic cell vaccine (DCVax ® -L) to standard therapy for newly diagnosed glioblastoma., Methods: After surgery and chemoradiotherapy, patients were randomized (2:1) to receive temozolomide plus DCVax-L (n = 232) or temozolomide and placebo (n = 99). Following recurrence, all patients were allowed to receive DCVax-L, without unblinding. The primary endpoint was progression free survival (PFS); the secondary endpoint was overall survival (OS)., Results: For the intent-to-treat (ITT) population (n = 331), median OS (mOS) was 23.1 months from surgery. Because of the cross-over trial design, nearly 90% of the ITT population received DCVax-L. For patients with methylated MGMT (n = 131), mOS was 34.7 months from surgery, with a 3-year survival of 46.4%. As of this analysis, 223 patients are ≥ 30 months past their surgery date; 67 of these (30.0%) have lived ≥ 30 months and have a Kaplan-Meier (KM)-derived mOS of 46.5 months. 182 patients are ≥ 36 months past surgery; 44 of these (24.2%) have lived ≥ 36 months and have a KM-derived mOS of 88.2 months. A population of extended survivors (n = 100) with mOS of 40.5 months, not explained by known prognostic factors, will be analyzed further. Only 2.1% of ITT patients (n = 7) had a grade 3 or 4 adverse event that was deemed at least possibly related to the vaccine. Overall adverse events with DCVax were comparable to standard therapy alone., Conclusions: Addition of DCVax-L to standard therapy is feasible and safe in glioblastoma patients, and may extend survival. Trial registration Funded by Northwest Biotherapeutics; Clinicaltrials.gov number: NCT00045968; https://clinicaltrials.gov/ct2/show/NCT00045968?term=NCT00045968&rank=1 ; initially registered 19 September 2002.

Glioblastomas are lethal cancers defined by angiogenesis and pseudopalisading necrosis. Here, we demonstrate that these histological features are associated with distinct transcriptional programs, with vascular regions showing a proneural profile, and hypoxic regions showing a mesenchymal pattern. As these regions harbor glioma stem cells (GSCs), we investigated the epigenetic regulation of these two niches. Proneural, perivascular GSCs activated EZH2, whereas mesenchymal GSCs in hypoxic regions expressed BMI1 protein, which promoted cellular survival under stress due to downregulation of the E3 ligase RNF144A. Using both genetic and pharmacologic inhibition, we found that proneural GSCs are preferentially sensitive to EZH2 disruption, whereas mesenchymal GSCs are more sensitive to BMI1 inhibition. Given that glioblastomas contain both proneural and mesenchymal GSCs, combined EZH2 and BMI1 targeting proved more effective than either agent alone both in culture and in vivo, suggesting that strategies that simultaneously target multiple epigenetic regulators within glioblastomas may be effective in overcoming therapy resistance caused by intratumoral heterogeneity.

Glioblastoma (GBM) is the most aggressive and lethal type of brain cancer with a median survival of less than two years even following aggressive treatment (Stupp et al., N Engl J Med 352:987-996, 2005). Among the many challenges in treating patients with this devastating disease is the ability to differentiate Magnetic Resonance Imaging (MRI) images that appear following radiation therapy, often termed "radiation necrosis" from true GBM recurrence. Radiation necrosis (RN) and GBM are very difficult to distinguish and currently only a brain biopsy can conclusively differentiate these pathologies. In the present study, we introduce a differential diagnostic approach using a newly identified Myeloid-Derived Suppressor Cell (MDSC) biomarker, vascular non-inflammatory molecule 2 (VNN2 + ), in combination with expression of traditional HLA-DR on peripheral blood CD14 + monocytes isolated from GBM and/or RN patients. We performed proof-of-principle experiments confirming the sensitivity and specificity of this approach based upon the combined expression levels of HLA-DR and VNN2 among CD14 + Mo-MDSC, which we called the DR-Vanin Index or DVI. The DVI was able to distinguish GBM from RN patients with a high degree of certainty (n = 18 and n = 6 respectively; p = 0.0004). This novel, quick and inexpensive blood-based liquid biopsy could potentially replace invasive brain biopsies in differentiating GBM from RN patients using a minimally-invasive technique.

Background: To elucidate molecular features associated with disproportionate survival of glioblastoma (GB) patients, we conducted deep genomic comparative analysis of a cohort of patients receiving standard therapy (surgery plus concurrent radiation and temozolomide); "GB outliers" were identified: long-term survivor of 33 months (LTS; n = 8) versus short-term survivor of 7 months (STS; n = 10)., Methods: We implemented exome, RNA, whole genome sequencing, and DNA methylation for collection of deep genomic data from STS and LTS GB patients., Results: LTS GB showed frequent chromosomal gains in 4q12 (platelet derived growth factor receptor alpha and KIT) and 12q14.1 (cyclin-dependent kinase 4), and deletion in 19q13.33 (BAX, branched chain amino-acid transaminase 2, and cluster of differentiation 33). STS GB showed frequent deletion in 9p11.2 (forkhead box D4-like 2 and aquaporin 7 pseudogene 3) and 22q11.21 (Hypermethylated In Cancer 2). LTS GB showed 2-fold more frequent copy number deletions compared with STS GB. Gene expression differences showed the STS cohort with altered transcriptional regulators: activation of signal transducer and activator of transcription (STAT)5a/b, nuclear factor-kappaB (NF-κB), and interferon-gamma (IFNG), and inhibition of mitogen-activated protein kinase (MAPK1), extracellular signal-regulated kinase (ERK)1/2, and estrogen receptor (ESR)1. Expression-based biological concepts prominent in the STS cohort include metabolic processes, anaphase-promoting complex degradation, and immune processes associated with major histocompatibility complex class I antigen presentation; the LTS cohort features genes related to development, morphogenesis, and the mammalian target of rapamycin signaling pathway. Whole genome methylation analyses showed that a methylation signature of 89 probes distinctly separates LTS from STS GB tumors., Conclusion: We posit that genomic instability is associated with longer survival of GB (possibly with vulnerability to standard therapy); conversely, genomic and epigenetic signatures may identify patients where up-front entry into alternative, targeted regimens would be a preferred, more efficacious management., (© The Author(s) 2016. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background: Glioblastoma (GBM) is the most common primary malignant brain tumor. Nomograms are often used for individualized estimation of prognosis. This study aimed to build and independently validate a nomogram to estimate individualized survival probabilities for patients with newly diagnosed GBM, using data from 2 independent NRG Oncology Radiation Therapy Oncology Group (RTOG) clinical trials., Methods: This analysis included information on 799 (RTOG 0525) and 555 (RTOG 0825) eligible and randomized patients with newly diagnosed GBM and contained the following variables: age at diagnosis, race, gender, Karnofsky performance status (KPS), extent of resection, O6-methylguanine-DNA methyltransferase (MGMT) methylation status, and survival (in months). Survival was assessed using Cox proportional hazards regression, random survival forests, and recursive partitioning analysis, with adjustment for known prognostic factors. The models were developed using the 0525 data and were independently validated using the 0825 data. Models were internally validated using 10-fold cross-validation, and individually predicted 6-, 12-, and 24-month survival probabilities were generated to measure the predictive accuracy and calibration against the actual survival status., Results: A final nomogram was built using the Cox proportional hazards model. Factors that increased the probability of shorter survival included greater age at diagnosis, male gender, lower KPS, not having total resection, and unmethylated MGMT status., Conclusions: A nomogram that assesses individualized survival probabilities (6-, 12-, and 24-mo) for patients with newly diagnosed GBM could be useful to health care providers for counseling patients regarding treatment decisions and optimizing therapeutic approaches. Free software for implementing this nomogram is provided: http://cancer4.case.edu/rCalculator/rCalculator.html., (© The Author(s) 2016. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com)

Glioblastoma is the most lethal brain tumor and harbors glioma stem cells (GSCs) with potent tumorigenic capacity. The function of GSCs in tumor propagation is maintained by several core transcriptional regulators including c-Myc. c-Myc protein is tightly regulated by posttranslational modification. However, the posttranslational regulatory mechanisms for c-Myc in GSCs have not been defined. In this study, we demonstrate that the deubiquitinase USP13 stabilizes c-Myc by antagonizing FBXL14-mediated ubiquitination to maintain GSC self-renewal and tumorigenic potential. USP13 was preferentially expressed in GSCs, and its depletion potently inhibited GSC proliferation and tumor growth by promoting c-Myc ubiquitination and degradation. In contrast, overexpression of the ubiquitin E3 ligase FBXL14 induced c-Myc degradation, promoted GSC differentiation, and inhibited tumor growth. Ectopic expression of the ubiquitin-insensitive mutant T58A-c-Myc rescued the effects caused by FBXL14 overexpression or USP13 disruption. These data suggest that USP13 and FBXL14 play opposing roles in the regulation of GSCs through reversible ubiquitination of c-Myc., (© 2017 Fang et al.)

OBJECTIVE Laser interstitial thermal therapy (LITT), sometimes referred to as "stereotactic laser ablation," has demonstrated utility in a subset of high-risk surgical patients with difficult to access (DTA) intracranial neoplasms. However, the treatment of tumors larger than 10 cm 3 is associated with suboptimal outcomes and morbidity. This may limit the utility of LITT in dealing with precisely those large or deep tumors that are most difficult to treat with conventional approaches. Recently, several groups have reported on minimally invasive transsulcal approaches utilizing tubular retracting systems. However, these approaches have been primarily used for intraventricular or paraventricular lesions, and subtotal resections have been reported for intraparenchymal lesions. Here, the authors describe a combined approach of LITT followed by minimally invasive transsulcal resection for large and DTA tumors. METHODS The authors retrospectively reviewed the results of LITT immediately followed by minimally invasive, transsulcal, transportal resection in 10 consecutive patients with unilateral, DTA malignant tumors > 10 cm 3 . The patients, 5 males and 5 females, had a median age of 65 years. Eight patients had glioblastoma multiforme (GBM), 1 had a previously treated GBM with radiation necrosis, and 1 had a melanoma brain metastasis. The median tumor volume treated was 38.0 cm 3 . RESULTS The median tumor volume treated to the yellow thermal dose threshold (TDT) line was 83% (range 76%-92%), the median tumor volume treated to the blue TDT line was 73% (range 60%-87%), and the median extent of resection was 93% (range 84%-100%). Two patients suffered mild postoperative neurological deficits, one transiently. Four patients have died since this analysis and 6 remain alive. Median progression-free survival was 280 days, and median overall survival was 482 days. CONCLUSIONS Laser interstitial thermal therapy followed by minimally invasive transsulcal resection, reported here for the first time, is a novel option for patients with large, DTA, malignant brain neoplasms. There were no unexpected neurological complications in this series, and operative characteristics improved as surgeon experience increased. Further studies are needed to elucidate any differences in survival or quality of life metrics.

The nearly universal recurrence of glioblastoma (GBM) is driven in part by a treatment-resistant subpopulation of GBM stem cells (GSC). To identify improved therapeutic possibilities, we combined the EGFR/HER2 inhibitor lapatinib with a novel small molecule, CBL0137, which inhibits FACT (facilitates chromatin transcription), a histone chaperone complex predominantly expressed in undifferentiated cells. Lapatinib and CBL0137 synergistically inhibited the proliferation of patient-derived GBM cells. Compared with non-stem tumor cells (NSTC) enriched from the same specimens, the GSCs were extremely sensitive to CBL0137 monotherapy or FACT knockdown. FACT expression was elevated in GSCs compared with matched NSTCs and decreased in GSCs upon differentiation. Acute exposure of GSCs to CBL0137 increased asymmetric cell division, decreased GSC marker expression, and decreased the capacity of GSCs to form tumor spheres in vitro and to initiate tumors in vivo Oral administration of CBL0137 to mice bearing orthotopic GBM prolonged their survival. Knockdown of FACT reduced the expression of genes encoding several core stem cell transcription factors (SOX2, OCT4, NANOG, and OLIG2), and FACT occupied the promoters of these genes. FACT expression was elevated in GBM tumors compared with non-neoplastic brain tissues, portended a worse prognosis, and positively correlated with GSC markers and stem cell gene expression signatures. Preferential targeting of GSCs by CBL0137 and synergy with EGFR inhibitors support the development of clinical trials combining these two agents in GBM. Cancer Res; 76(8); 2432-42. ©2016 AACR., (©2016 American Association for Cancer Research.)

Many cancers feature cellular hierarchies that are driven by tumor-initiating cancer stem cells (CSC) and rely on complex interactions with the tumor microenvironment. Standard cell culture conditions fail to recapitulate the original tumor architecture or microenvironmental gradients and are not designed to retain the cellular heterogeneity of parental tumors. Here, we describe a three-dimensional culture system that supports the long-term growth and expansion of tumor organoids derived directly from glioblastoma specimens, including patient-derived primary cultures, xenografts, genetically engineered glioma models, or patient samples. Organoids derived from multiple regions of patient tumors retain selective tumorigenic potential. Furthermore, organoids could be established directly from brain metastases not typically amenable to in vitro culture. Once formed, tumor organoids grew for months and displayed regional heterogeneity with a rapidly dividing outer region of SOX2(+), OLIG2(+), and TLX(+) cells surrounding a hypoxic core of primarily non-stem senescent cells and diffuse, quiescent CSCs. Notably, non-stem cells within organoids were sensitive to radiotherapy, whereas adjacent CSCs were radioresistant. Orthotopic transplantation of patient-derived organoids resulted in tumors displaying histologic features, including single-cell invasiveness, that were more representative of the parental tumor compared with those formed from patient-derived sphere cultures. In conclusion, we present a new ex vivo model in which phenotypically diverse stem and non-stem glioblastoma cell populations can be simultaneously cultured to explore new facets of microenvironmental influences and CSC biology. Cancer Res; 76(8); 2465-77. ©2016 AACR., (©2016 American Association for Cancer Research.)

Aims: To determine the efficacy of methylguanine methyltransferase (MGMT) depletion + BCNU [1,3-bis(2-chloroethyl)-1- nitrosourea: carmustine] therapy and the impact of methylation status in adults with glioblastoma multiforme (GBM) and gliosarcoma., Methods: Methylation analysis was performed on GBM patients with adequate tissue samples. Patients with newly diagnosed GBM or gliosarcoma were eligible for this Phase III open-label clinical trial. At registration, patients were randomized to Arm 1, which consisted of therapy with O(6)-benzylguanine (O(6)-BG) + BCNU 40 mg/m(2) (reduced dose) + radiation therapy (RT) (O6BG + BCNU arm), or Arm 2, which consisted of therapy with BCNU 200 mg/m(2) + RT (BCNU arm)., Results: A total of 183 patients with newly diagnosed GBM or gliosarcoma from 42 U.S. institutions were enrolled in this study. Of these, 90 eligible patients received O(6)-BG + BCNU + RT and 89 received BCNU + RT. The trial was halted at the first interim analysis in accordance with the guidelines for stopping the study due to futility (<40 % improvement among patients on the O6BG + BCNU arm). Following adjustment for stratification factors, there was no significant difference in overall survival (OS) or progression-free survival (PFS) between the two groups (one sided p = 0.94 and p = 0.88, respectively). Median OS was 11 [95 % confidence interval (CI) 8-13] months for patients in the O6BG + BCNU arm and 10 (95 % CI 8-12) months for those in the BCNU arm. PFS was 4 months for patients in each arm. Adverse events were reported in both arms, with significantly more grade 4 and 5 events in the experimental arm., Conclusions: The addition of O(6)-BG to the standard regimen of radiation and BCNU for the treatment patients with newly diagnosed GBM and gliosarcoma did not provide added benefit and in fact caused additional toxicity.

Extra-CNS metastasis from glioblastoma (ECMGBM) is an emerging but little known clinical entity. We review pre-clinical and translational publications assessing the ability of GBM to spread locally and outside the CNS. Reported cases demonstrating ECMGBM are reviewed providing a summary of presentations for the entity. Special attention is placed on transmission of GBM through organ transplantation. Finally, predictions are made as to the future significance of ECMGBM, especially in the context of better outcomes in CNS GBM.

Glioblastoma (GBM) is a prototypical heterogeneous brain tumor refractory to conventional therapy. A small residual population of cells escapes surgery and chemoradiation, resulting in a typically fatal tumor recurrence ∼ 7 mo after diagnosis. Understanding the molecular architecture of this residual population is critical for the development of successful therapies. We used whole-genome sequencing and whole-exome sequencing of multiple sectors from primary and paired recurrent GBM tumors to reconstruct the genomic profile of residual, therapy resistant tumor initiating cells. We found that genetic alteration of the p53 pathway is a primary molecular event predictive of a high number of subclonal mutations in glioblastoma. The genomic road leading to recurrence is highly idiosyncratic but can be broadly classified into linear recurrences that share extensive genetic similarity with the primary tumor and can be directly traced to one of its specific sectors, and divergent recurrences that share few genetic alterations with the primary tumor and originate from cells that branched off early during tumorigenesis. Our study provides mechanistic insights into how genetic alterations in primary tumors impact the ensuing evolution of tumor cells and the emergence of subclonal heterogeneity., (© 2015 Kim et al.; Published by Cold Spring Harbor Laboratory Press.)

Tumour-associated macrophages (TAMs) are enriched in glioblastoma multiformes (GBMs) that contain glioma stem cells (GSCs) at the apex of their cellular hierarchy. The correlation between TAM density and glioma grade suggests a supportive role for TAMs in tumour progression. Here we interrogated the molecular link between GSCs and TAM recruitment in GBMs and demonstrated that GSCs secrete periostin (POSTN) to recruit TAMs. TAM density correlates with POSTN levels in human GBMs. Silencing POSTN in GSCs markedly reduced TAM density, inhibited tumour growth, and increased survival of mice bearing GSC-derived xenografts. We found that TAMs in GBMs are not brain-resident microglia, but mainly monocyte-derived macrophages from peripheral blood. Disrupting POSTN specifically attenuated the tumour-supportive M2 type of TAMs in xenografts. POSTN recruits TAMs through the integrin αvβ₃ as blocking this signalling by an RGD peptide inhibited TAM recruitment. Our findings highlight the possibility of improving GBM treatment by targeting POSTN-mediated TAM recruitment.

Glioblastomas are highly lethal brain tumors containing tumor-propagating glioma stem cells (GSCs). The molecular mechanisms underlying the maintenance of the GSC phenotype are not fully defined. Here we demonstrate that the zinc finger and X-linked transcription factor (ZFX) maintains GSC self-renewal and tumorigenic potential by upregulating c-Myc expression. ZFX is differentially expressed in GSCs relative to non-stem glioma cells and neural progenitor cells. Disrupting ZFX by shRNA reduced c-Myc expression and potently inhibited GSC self-renewal and tumor growth. Ectopic expression of c-Myc to its endogenous level rescued the effects caused by ZFX disruption, supporting that ZFX controls GSC properties through c-Myc. Furthermore, ZFX binds to a specific sequence (GGGCCCCG) on the human c-Myc promoter to upregulate c-Myc expression. These data demonstrate that ZFX functions as a critical upstream regulator of c-Myc and plays essential roles in the maintenance of the GSC phenotype. This study also supports that c-Myc is a dominant driver linking self-renewal to malignancy., (© 2014 AlphaMed Press.)

Glioblastoma (GBM) contains a self-renewing, tumorigenic cancer stem cell (CSC) population which contributes to tumor propagation and therapeutic resistance. While the tumor microenvironment is essential to CSC self-renewal, the mechanisms by which CSCs sense and respond to microenvironmental conditions are poorly understood. Scavenger receptors are a broad class of membrane receptors well characterized on immune cells and instrumental in sensing apoptotic cellular debris and modified lipids. Here, we provide evidence that CSCs selectively use the scavenger receptor CD36 to promote their maintenance using patient-derived CSCs and in vivo xenograft models. CD36 expression was observed in GBM cells in addition to previously described cell types including endothelial cells, macrophages, and microglia. CD36 was enriched in CSCs and was able to functionally distinguish self-renewing cells. CD36 was coexpressed with integrin alpha 6 and CD133, previously described CSC markers, and CD36 reduction resulted in concomitant loss of integrin alpha 6 expression, self-renewal, and tumor initiation capacity. We confirmed oxidized phospholipids, ligands of CD36, were present in GBM and found that the proliferation of CSCs, but not non-CSCs, increased with exposure to oxidized low-density lipoprotein. CD36 was an informative biomarker of malignancy and negatively correlated to patient prognosis. These results provide a paradigm for CSCs to thrive by the selective enhanced expression of scavenger receptors, providing survival, and metabolic advantages., (© 2014 AlphaMed Press.)

Background: Gliomas are the most common primary malignant brain tumors in adults with great heterogeneity in histopathology and clinical course. The intent was to evaluate the relevance of known glioblastoma (GBM) expression and methylation based subtypes to grade II and III gliomas (ie. lower grade gliomas)., Methods: Gene expression array, single nucleotide polymorphism (SNP) array and clinical data were obtained for 228 GBMs and 176 grade II/II gliomas (GII/III) from the publically available Rembrandt dataset. Two additional datasets with IDH1 mutation status were utilized as validation datasets (one publicly available dataset and one newly generated dataset from MD Anderson). Unsupervised clustering was performed and compared to gene expression subtypes assigned using the Verhaak et al 840-gene classifier. The glioma-CpG Island Methylator Phenotype (G-CIMP) was assigned using prediction models by Fine et al., Results: Unsupervised clustering by gene expression aligned with the Verhaak 840-gene subtype group assignments. GII/IIIs were preferentially assigned to the proneural subtype with IDH1 mutation and G-CIMP. GBMs were evenly distributed among the four subtypes. Proneural, IDH1 mutant, G-CIMP GII/III s had significantly better survival than other molecular subtypes. Only 6% of GBMs were proneural and had either IDH1 mutation or G-CIMP but these tumors had significantly better survival than other GBMs. Copy number changes in chromosomes 1p and 19q were associated with GII/IIIs, while these changes in CDKN2A, PTEN and EGFR were more commonly associated with GBMs., Conclusions: GBM gene-expression and methylation based subtypes are relevant for GII/III s and associate with overall survival differences. A better understanding of the association between these subtypes and GII/IIIs could further knowledge regarding prognosis and mechanisms of glioma progression.

Few studies had investigated genome-wide methylation in glioblastoma multiforme (GBM). Our goals were to study differential methylation across the genome in gene promoters using an array-based method, as well as repetitive elements using surrogate global methylation markers. The discovery sample set for this study consisted of 54 GBM from Columbia University and Case Western Reserve University, and 24 brain controls from the New York Brain Bank. We assembled a validation dataset using methylation data of 162 TCGA GBM and 140 brain controls from dbGAP. HumanMethylation27 Analysis Bead-Chips (Illumina) were used to interrogate 26,486 informative CpG sites in both the discovery and validation datasets. Global methylation levels were assessed by analysis of L1 retrotransposon (LINE1), 5 methyl-deoxycytidine (5m-dC) and 5 hydroxylmethyl-deoxycytidine (5hm-dC) in the discovery dataset. We validated a total of 1548 CpG sites (1307 genes) that were differentially methylated in GBM compared to controls. There were more than twice as many hypomethylated genes as hypermethylated ones. Both the discovery and validation datasets found 5 tumor methylation classes. Pathway analyses showed that the top ten pathways in hypomethylated genes were all related to functions of innate and acquired immunities. Among hypermethylated pathways, transcriptional regulatory network in embryonic stem cells was the most significant. In the study of global methylation markers, 5m-dC level was the best discriminant among methylation classes, whereas in survival analyses, high level of LINE1 methylation was an independent, favorable prognostic factor in the discovery dataset. Based on a pathway approach, hypermethylation in genes that control stem cell differentiation were significant, poor prognostic factors of overall survival in both the discovery and validation datasets. Approaches that targeted these methylated genes may be a future therapeutic goal.

Background: Outcomes for patients with recurrent glioblastoma multiforme (GBM) are poor and may be improved by immunotherapy. We investigated the safety and efficacy of an autologous heat-shock protein peptide complex-96 (HSPPC-96) vaccine for patients with recurrent GBM., Methods: In this open-label, single-arm, phase II study, adult patients with surgically resectable recurrent GBM were given vaccine after gross total resection. The primary endpoint was overall survival at 6 months. Secondary endpoints included overall survival, progression-free survival, safety, and immune profiling. Outcome analyses were performed in the intention-to-treat and efficacy populations., Results: Between October 3, 2007 and October 24, 2011, 41 patients underwent gross total resection of recurrent GBM and received a median of 6 doses of HSPPC-96 vaccine. Following treatment, 90.2% of patients were alive at 6 months (95% confidence interval [CI]: 75.9-96.8) and 29.3% were alive at 12 months (95% CI: 16.6-45.7). Median overall survival was 42.6 weeks (95% CI: 34.7-50.5). Twenty-seven (66%) patients were lymphopenic prior to therapy, and patients with lymphocyte counts below the cohort median demonstrated decreased overall survival (hazard ratio: 4.0; 95% CI: 1.4-11.8; P = .012). There were no treatment-related deaths. There were 37 serious (grades 3-5) adverse events reported, with 17 attributable to surgical resection and a single grade 3 constitutional event related to the vaccine., Conclusion: The HSPPC-96 vaccine is safe and warrants further study of efficacy for the treatment of recurrent GBM. Significant pretreatment lymphopenia may impact the outcomes of immunotherapy and deserves additional investigation.

Glioblastoma is one of the most common primary brain tumors in adults and the prognosis is very poor. The standard treatment is gross total resection and postoperative radiotherapy with concurrent and adjuvant chemotherapy with temozolomide. Given its propensity to spread to areas of brain parenchyma surrounding the gross tumor volume (GTV), a generous margin is typically created around the GTV. Peritumoral edema is frequently included in the GTV, especially in the USA. The margin of expansion from GTV to clinical target volume also varies widely among different institutions. There is a lack of consensus as to what constitutes the most appropriate clinical target volume for glioblastoma.

Glioblastomas display cellular hierarchies with self-renewing tumor-initiating cells (TIC), also known as cancer stem cells, at the apex. Although the TIC hypothesis remains controversial and the functional assays to define the TIC phenotype are evolving, we and others have shown that TICs may contribute to therapeutic resistance, tumor spread, and angiogenesis. The identification of TICs has been informed by the use of markers characterized in normal stem cells, but this approach has an inherent limitation to selectively identify TICs. To develop reagents that enrich TICs but not matched non-TICs or tissue-specific stem cells, we adopted Cell-Systematic Evolution of Ligands by Exponential Enrichment (Cell-SELEX) to identify glioblastoma TIC-specific nucleic acid probes-aptamers-that specifically bind TICs. In this study, using Cell-SELEX with positive selection for TICs and negative selection for non-TICs and human neural progenitor cells, we identified TIC aptamers that specifically bind to TICs with excellent dissociation constants (Kd). These aptamers select and internalize into glioblastoma cells that self-renew, proliferate, and initiate tumors. As aptamers can be modified to deliver payloads, aptamers may represent novel agents that could selectively target or facilitate imaging of TICs., (©2013 AACR.)

Object: Laser interstitial thermal therapy has been used as an ablative treatment for glioma; however, its development was limited due to technical issues. The NeuroBlate System incorporates several technological advances to overcome these drawbacks. The authors report a Phase I, thermal dose-escalation trial assessing the safety and efficacy of NeuroBlate in recurrent glioblastoma multiforme (rGBM)., Methods: Adults with suspected supratentorial rGBM of 15- to 40-mm dimension and a Karnofsky Performance Status score of ≥ 60 were eligible. After confirmatory biopsy, treatment was delivered using a rigid, gas-cooled, side-firing laser probe. Treatment was monitored using real-time MRI thermometry, and proprietary software providing predictive thermal damage feedback was used by the surgeon, along with control of probe rotation and depth, to tailor tissue coagulation. An external data safety monitoring board determined if toxicity at lower levels justified dose escalation., Results: Ten patients were treated at the Case Comprehensive Cancer Center (Cleveland Clinic and University Hospitals-Case Medical Center). Their average age was 55 years (range 34-69 years) and the median preoperative Karnofsky Performance Status score was 80 (range 70-90). The mean tumor volume was 6.8 ± 5 cm(3) (range 2.6-19 cm(3)), the percentage of tumor treated was 78% ± 12% (range 57%-90%), and the conformality index was 1.21 ± 0.33 (range 1.00-2.04). Treatment-related necrosis was evident on MRI studies at 24 and 48 hours. The median survival was 316 days (range 62-767 days). Three patients improved neurologically, 6 remained stable, and 1 worsened. Steroid-responsive treatment-related edema occurred in all patients but one. Three had Grade 3 adverse events at the highest dose., Conclusions: NeuroBlate represents new technology for delivering laser interstitial thermal therapy, allowing controlled thermal ablation of deep hemispheric rGBM. CLINICAL TRIAL REGISTRATION NO.: NCT00747253 ( ClinicalTrials.gov ).

Glioblastomas (GBMs) are highly vascular and lethal brain tumors that display cellular hierarchies containing self-renewing tumorigenic glioma stem cells (GSCs). Because GSCs often reside in perivascular niches and may undergo mesenchymal differentiation, we interrogated GSC potential to generate vascular pericytes. Here, we show that GSCs give rise to pericytes to support vessel function and tumor growth. In vivo cell lineage tracing with constitutive and lineage-specific fluorescent reporters demonstrated that GSCs generate the majority of vascular pericytes. Selective elimination of GSC-derived pericytes disrupts the neovasculature and potently inhibits tumor growth. Analysis of human GBM specimens showed that most pericytes are derived from neoplastic cells. GSCs are recruited toward endothelial cells via the SDF-1/CXCR4 axis and are induced to become pericytes predominantly by transforming growth factor β. Thus, GSCs contribute to vascular pericytes that may actively remodel perivascular niches. Therapeutic targeting of GSC-derived pericytes may effectively block tumor progression and improve antiangiogenic therapy., (Copyright © 2013 Elsevier Inc. All rights reserved.)

To determine a molecular basis for prognostic differences in glioblastoma multiforme (GBM), we employed a combinatorial network analysis framework to exhaustively search for molecular patterns in protein-protein interaction (PPI) networks. We identified a dysregulated molecular signature distinguishing short-term (survival<225 days) from long-term (survival>635 days) survivors of GBM using whole genome expression data from The Cancer Genome Atlas (TCGA). A 50-gene subnetwork signature achieved 80% prediction accuracy when tested against an independent gene expression dataset. Functional annotations for the subnetwork signature included "protein kinase cascade," "IκB kinase/NFκB cascade," and "regulation of programmed cell death" - all of which were not significant in signatures of existing subtypes. Finally, we used label-free proteomics to examine how our subnetwork signature predicted protein level expression differences in an independent GBM cohort of 16 patients. We found that the genes discovered using network biology had a higher probability of dysregulated protein expression than either genes exhibiting individual differential expression or genes derived from known GBM subtypes. In particular, the long-term survivor subtype was characterized by increased protein expression of DNM1 and MAPK1 and decreased expression of HSPA9, PSMD3, and CANX. Overall, we demonstrate that the combinatorial analysis of gene expression data constrained by PPIs outlines an approach for the discovery of robust and translatable molecular signatures in GBM.

Objective: Glioblastomas (GBMs) are lethal cancers that display cellular hierarchies parallel to normal brain. At the apex are GBM stem cells (GSCs), which are relatively resistant to conventional therapy. Interactions with the adjacent perivascular niche are an important driver of malignancy and self-renewal in GSCs. Extracellular matrix (ECM) cues instruct neural stem/progenitor cell-niche interactions, and the objective of our study was to elucidate its composition and contribution to GSC maintenance in the perivascular niche., Methods: We interrogated human tumor tissue for immunofluorescence analysis and derived GSCs from tumor tissues for functional studies. Bioinformatics analyses were conducted by mining publicly available databases., Results: We find that laminin ECM proteins are localized to the perivascular GBM niche and inform negative patient prognosis. To identify the source of laminins, we characterized cellular elements within the niche and found that laminin α chains were expressed by nonstem tumor cells and tumor-associated endothelial cells (ECs). RNA interference targeting laminin α2 inhibited GSC growth and self-renewal. In co-culture studies of GSCs and ECs, laminin α2 knockdown in ECs resulted in decreased tumor growth., Interpretation: Our studies highlight the contribution of nonstem tumor cell-derived laminin juxtracrine signaling. As laminin α2 has recently been identified as a molecular marker of aggressive ependymoma, we propose that the brain vascular ECM promotes tumor malignancy through maintenance of the GSC compartment, providing not only a molecular fingerprint but also a possible therapeutic target., (Copyright © 2012 American Neurological Association.)

Growth factor-mediated proliferation and self-renewal maintain tissue-specific stem cells and are frequently dysregulated in cancers. Platelet-derived growth factor (PDGF) ligands and receptors (PDGFRs) are commonly overexpressed in gliomas and initiate tumors, as proven in genetically engineered models. While PDGFRα alterations inform intertumoral heterogeneity toward a proneural glioblastoma (GBM) subtype, we interrogated the role of PDGFRs in intratumoral GBM heterogeneity. We found that PDGFRα is expressed only in a subset of GBMs, while PDGFRβ is more commonly expressed in tumors but is preferentially expressed by self-renewing tumorigenic GBM stem cells (GSCs). Genetic or pharmacological targeting of PDGFRβ (but not PDGFRα) attenuated GSC self-renewal, survival, tumor growth, and invasion. PDGFRβ inhibition decreased activation of the cancer stem cell signaling node STAT3, while constitutively active STAT3 rescued the loss of GSC self-renewal caused by PDGFRβ targeting. In silico survival analysis demonstrated that PDGFRB informed poor prognosis, while PDGFRA was a positive prognostic factor. Our results may explain mixed clinical responses of anti-PDGFR-based approaches and suggest the need for integration of models of cancer as an organ system into development of cancer therapies.

Glioblastoma multiforme (GBM) is the most lethal primary brain tumor. Extensive proliferation and dispersal of GBM tumor cells within the brain limits patient survival to approximately 1 year. Hence, there is a great need for the development of better means to treat GBM. Receptor protein tyrosine phosphatase (PTP)µ is proteolytically cleaved in GBM to yield fragments that promote dispersal of GBM cells. While normal brain tissue retains expression of full-length PTPµ, low-grade human astrocytoma samples have varying amounts of full-length PTPµ and cleaved PTPµ. In the highest-grade astrocytomas (i.e., GBM), PTPµ is completely proteolyzed into fragments. We demonstrate that short hairpin RNA mediated knockdown of full-length PTPµ and PTPµ fragments reduces glioma cell growth and survival in vitro. The reduction in growth and survival following PTPµ knockdown is enhanced when cells are grown in the absence of serum, suggesting that PTPµ may regulate autocrine signaling. Furthermore, we show for the first time that reduction of PTPµ protein expression decreases the growth and survival of glioma cells in vivo using mouse xenograft flank and i.c. tumor models. Inhibitors of PTPµ could be used to reduce the growth and survival of GBM cells in the brain, representing a promising therapeutic target for GBM.

Activation of TNF receptor 1 (TNF-R1) can generate signals that promote either apoptosis or survival. In this study, we show that these signals can be determined by the character of the extracellular matrix in the tumor microenvironment. Specifically, through studies of glioblastoma, we showed that TNFα stimulation induced apoptosis of primary brain endothelial cells (EC) attached to collagen or fibronectin (which engage integrins α2β1/α3β1 and α5β1, respectively), but did not induce apoptosis of ECs attached to laminin (which engages integrins α6β1 and α3β1). TNF-R1 expression was significantly higher in ECs in glioblastoma (GBM) tumors compared with ECs in normal brain specimens. TNFα was also expressed in GBM tumor-associated ECs, which was associated with longer patient survival. ECs plated on anti-integrin α2 or α3 antibody were susceptible to TNFα-induced apoptosis, whereas those plated on anti-integrin α6 antibody were not. Moreover, the ECs plated on laminin, but not collagen, expressed cellular FLICE inhibitory protein (cFLIP) and TNFα stimulation of laminin-attached cells in which cFLIP had been downregulated resulted in the induction of apoptosis. In contrast, attachment to laminin did not induce cFLIP expression in GBM tumor stem cells. Together, our findings indicate that the laminin receptor integrin α6β1 promotes the survival of brain ECs by inhibiting prodeath signaling by TNF-R1, in part by inducing cFLIP expression.

Glioblastoma multiforme (GBM) is the most malignant and lethal form of astrocytoma. The GBM patient survival time of approximately 1 year necessitates the identification of novel molecular targets and more effective therapeutics. Cadherin-11, a calcium-dependent cell-cell adhesion molecule and mesenchymal marker, plays a role in both normal tissue development and in cancer cell migration. The functional significance of cadherin-11 in GBM has not been investigated. Here, we show that cadherin-11 is expressed in human GBM tumors and human glioma stem-like cells by immunohistochemical labeling. In addition, we show that cadherin-11 is expressed in human glioma cell lines by immunoblotting. Short hairpin RNA-mediated knockdown of cadherin-11 expression in human glioma cell lines results in decreased migration and growth factor-independent cell survival in vitro. More importantly, knockdown of cadherin-11 inhibits glioma cell survival in heterotopic and orthotopic mouse xenograft models. Together, our results show the functional significance of cadherin-11 expression in GBM and provide evidence for a novel role of cadherin-11 in promoting glioma cell survival in an in vivo environment. Thus, our studies suggest cadherin-11 is a viable molecular target for therapeutic intervention in GBM.

Human cancers are driven by the acquisition of somatic mutations. Separating the driving mutations from those that are random consequences of general genomic instability remains a challenge. New sequencing technology makes it possible to detect mutations that are present in only a minority of cells in a heterogeneous tumor population. We sought to leverage the power of ultra-deep sequencing to study various levels of tumor heterogeneity in the serial recurrences of a single glioblastoma multiforme patient. Our goal was to gain insight into the temporal succession of DNA base-level lesions by querying intra- and inter-tumoral cell populations in the same patient over time. We performed targeted "next-generation" sequencing on seven samples from the same patient: two foci within the primary tumor, two foci within an initial recurrence, two foci within a second recurrence, and normal blood. Our study reveals multiple levels of mutational heterogeneity. We found variable frequencies of specific EGFR, PIK3CA, PTEN, and TP53 base substitutions within individual tumor regions and across distinct regions within the same tumor. In addition, specific mutations emerge and disappear along the temporal spectrum from tumor at the time of diagnosis to second recurrence, demonstrating evolution during tumor progression. Our results shed light on the spatial and temporal complexity of brain tumors. As sequencing costs continue to decline and deep sequencing technology eventually moves into the clinic, this approach may provide guidance for treatment choices as we embark on the path to personalized cancer medicine.