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Purpose: PTEN mutation status is a pivotal biomarker for glioblastoma. This study aimed to establish a radiomic signature to predict PTEN mutation status in patients with glioblastoma, and to investigate the genetic background behind this radiomic signature., Methods: In this study, a total of 862 radiomic features were extracted from each patient. The training (n = 69) and validation (n = 40) sets were retrospectively collected from the Cancer Genome Atlas and the Chinese Glioma Genome Atlas, respectively. The minimum redundancy maximum relevance (mRMR) algorithm was used to select the best predictive features of PTEN status. A machine learning model was then built with the selected features using a support vector machine classifier. The predictive performance of each selected feature and the complete model were evaluated via the area under the curve from receiver operating characteristic analysis in both the training and validation sets. The genetic background underlying the radiomic signature was determined using radiogenomic analysis., Results: Six features were selected using the mRMR algorithm, including two features derived from contrast-enhanced images and four features derived from T2-weighted images. The predictive performance of the machine learning model for the training and validation sets were 0.925 and 0.787, respectively, which were better than the individual features. Radiogenomics analysis revealed that the PTEN-associated biological processes could be described using the radiomic signature., Conclusion: These results show that radiomic features derived from preoperative MRI can predict PTEN mutation status in glioblastoma patients, thus providing a novel noninvasive imaging biomarker.

This study aimed to identify the optimal radiomic machine-learning classifier for differentiating glioblastoma (GBM) from solitary brain metastases (MET) preoperatively. Four hundred and twelve patients with solitary brain tumors (242 GBM and 170 solitary brain MET) were divided into training (n = 227) and test (n = 185) cohorts. Radiomic features extraction was performed with PyRadiomics software. In the training cohort, twelve feature selection methods and seven classification methods were evaluated to construct favorable radiomic machine-learning classifiers. The performance of the classifiers was evaluated using the mean area under the curve (AUC) and relative standard deviation in percentile (RSD). In the training cohort, thirteen classifiers had favorable predictive performances (AUC≥0.95 and RSD ≤6). In the test cohort, receiver operating characteristic (ROC) curve analysis revealed that support vector machines (SVM) + least absolute shrinkage and selection operator (LASSO) (AUC, 0.90) classifiers had the highest prediction efficacy. Furthermore, the clinical performance of the best classifier was superior to neuroradiologists in accuracy, sensitivity, and specificity. In conclusion, employing radiomic machine-learning technology could help neuroradiologist in differentiating GBM from solitary brain MET preoperatively., (Copyright © 2019. Published by Elsevier B.V.)

Objectives: The aim of this study was to differentiate primary central nervous system lymphoma (PCNSL) from glioblastomas (GBM) using the fractal analysis of conventional MRI data., Materials and Methods: Sixty patients with PCNSL and 107 patients with GBM with MRI data available were enrolled. Fractal dimension (FD) and lacunarity values of the tumour region were calculated using fractal analysis. A predictive model combining fractal parameters and anatomical characteristics was built using logistic regression. The role of FD, lacunarity and the predictive model in differential diagnosis was evaluated using receiver-operating characteristic (ROC) curve analysis. The association between fractal parameters and anatomical characteristics of tumours was also investigated., Results: PCNSL had lower FD values (p < 0.001) and higher lacunarity values (p < 0.001) than GBM. ROC curve analysis revealed that FD, lacunarity, and the predictive model could distinguish PCNSL from GBM (area under the curve: 0.895, 0.776, and 0.969, respectively). The following associations were observed between fractal parameters and anatomical characteristics: multiple lesions were significantly associated with higher lacunarity (p = 0.024), necrosis with higher FD (p = 0.027), corpus callosum involvement with higher lacunarity (p < 0.001) in PCNSL and subventricular zone involvement with higher FD (p < 0.001) in GBM., Conclusions: The findings of the study indicate that fractal analysis on conventional MRI performs well in distinguishing PCNSL from GBM., Key Points: • Fractal dimension and lacunarity were capable of differentiating PCNSL from GBM. • PCNSL and GBM exhibited different anatomical characteristics. • Fractal parameters were associated with some of these anatomical characteristics.

Necrosis is a hallmark feature of glioblastoma (GBM). This study investigated the prognostic role of necrotic patterns in GBM using fractal dimension (FD) and lacunarity analyses of magnetic resonance imaging (MRI) data and evaluated the role of lacunarity in the biological processes leading to necrosis. We retrospectively reviewed clinical and MRI data of 95 patients with GBM. FD and lacunarity of the necrosis on MRI were calculated by fractal analysis and subjected to survival analysis. We also performed gene ontology analysis in 32 patients with available RNA-seq data. Univariate analysis revealed that FD < 1.56 and lacunarity > 0.46 significantly correlated with poor progression-free survival (p = 0.006 and p = 0.012, respectively) and overall survival (p = 0.008 and p = 0.005, respectively). Multivariate analysis revealed that both parameters were independent factors for unfavorable progression-free survival (p = 0.001 and p = 0.015, respectively) and overall survival (p = 0.002 and p = 0.007, respectively). Gene ontology analysis revealed that genes positively correlated with lacunarity were involved in the suppression of apoptosis and necrosis-associated biological processes. We demonstrate that the fractal parameters of necrosis in GBM can predict patient survival and are associated with the biological processes of tumor necrosis.

Targeting glioblastoma stem cells with γ-secretase inhibitors (GSIs) disrupts the Notch pathway and has shown some benefit in both pre-clinical models and in patients during phase I/II clinical trials. However, it is largely unknown why some glioblastoma (GBM) does not respond to GSI treatment. In this issue of the JCI, Xie et al. determined that GSI-resistant brain tumor-initiating cells (BTICs) from GBM express a higher level of the gene RBPJ, which encodes a mediator of canonical Notch signaling, compared to non-BTICs. Knockdown of RBPJ in BTICs decreased propagation in vitro and in vivo by inducing apoptosis. Interestingly, RBPJ was shown to regulate a different transcription program than Notch in BTICs by binding CDK9, thereby affecting Pol II-regulated transcript elongation. Targeting CDK9 or c-MYC, an upstream regulator of RBPJ, with small molecules also decreased BTIC propagation, and prolonged survival in mice bearing orthotopic GBM xenografts. This study not only provides a mechanism for GSI treatment resistance, but also identifies two potential therapeutic strategies to target GSI-resistant BTICs.

Human telomerase reverse transcriptase (TERT) promoter mutations are important genetic alterations in many kinds of human malignancies, including glioma. The current study aimed to investigate the anatomical specificity of TERT promoter mutations in glioblastomas (GBMs). Clinical information and preoperative magnetic resonance images of 203 patients with GBMs were reviewed. TERT promoter mutation status was assessed by Sanger sequencing in all cases. Tumor lesions were manually segmented and then registered to a standard brain atlas. Then the specific brain regions associated with TERT promoter mutation status were subsequently identified by voxel-based regression analysis. TERT promoter mutations were detected in 94 (46.3 %) of the 203 patients. Voxel-based statistical analysis demonstrated that GBMs with TERT promoter mutations were much more likely to locate in the right temporal lobe, while those with wild-type TERT promoters were more likely to occur in the anterior region of the right lateral ventricle. No significant difference was found in the lesion volumes of the T2-identified tumor or in the contrast enhancement areas between the two groups. The current study demonstrated the anatomic specificity of TERT promoter mutation status in GBM. These findings may provide new insight into the molecular classification of GBM and further our understanding of the associations between tumor-specific molecular alterations and tumor location.

Background: Radiological characteristics may reflect the biological features of brain tumors and may be associated with genetic alterations that occur in tumorigenesis. This study aimed to investigate the relationship between radiological features and IDH1 status as well as their predictive value for survival of glioblastoma patients., Methods: The clinical information and MR images of 280 patients with histologically confirmed glioblastoma were retrospectively reviewed. The radiological characteristics of tumors were examined on MR images, and the IDH1 status was determined using DNA sequencing for all cases. The Kaplan-Meier method and Cox regression model were used to identify prognostic factors for progression-free and overall survival., Results: The IDH1 mutation was associated with longer progression-free survival (P = .022; hazard ratio, 0.602) and overall survival (P = .018; hazard ratio, 0.554). In patients with the IDH1 mutation, tumor contrast enhancement and peritumoral edema indicated worse progression-free survival (P = .015 and P = .024, respectively) and worse overall survival (P = .024 and P = .032, respectively). For tumors with contrast enhancement, multifocal contrast enhancement of the tumor lesion was associated with poor progression-free survival (P = .002) and poor overall survival (P = .010) in patients with wild-type IDH1 tumors., Conclusions: Combining the radiological features and IDH1 status of a tumor allows more accurate prediction of survival outcomes in glioblastoma patients. The complementary roles of genetic changes and radiological features of tumors should be considered in future studies., (© The Author(s) 2015. 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.)

Notch signaling can promote tumorigenesis in the nervous system and plays important roles in stem-like cancer cells. However, little is known about how Notch inhibition might alter tumor metabolism, particularly in lesions arising in the brain. The gamma-secretase inhibitor MRK003 was used to treat glioblastoma neurospheres, and they were subdivided into sensitive and insensitive groups in terms of canonical Notch target response. Global metabolomes were then examined using proton magnetic resonance spectroscopy, and changes in intracellular concentration of various metabolites identified which correlate with Notch inhibition. Reductions in glutamate were verified by oxidation-based colorimetric assays. Interestingly, the alkylating chemotherapeutic agent temozolomide, the mTOR-inhibitor MLN0128, and the WNT inhibitor LGK974 did not reduce glutamate levels, suggesting that changes to this metabolite might reflect specific downstream effects of Notch blockade in gliomas rather than general sequelae of tumor growth inhibition. Global and targeted expression analyses revealed that multiple genes important in glutamate homeostasis, including glutaminase, are dysregulated after Notch inhibition. Treatment with an allosteric inhibitor of glutaminase, compound 968, could slow glioblastoma growth, and Notch inhibition may act at least in part by regulating glutaminase and glutamate., (© 2015 UICC.)

Introduction: The expression of vascular endothelial growth factor (VEGF) is a common genetic alteration in malignant gliomas and contributes to the angiogenesis of tumors. This study aimed to investigate the anatomical specificity of VEGF expression levels in glioblastomas using voxel-based neuroimaging analysis., Methods: Clinical information, MR scans, and immunohistochemistry stains of 209 patients with glioblastomas were reviewed. All tumor lesions were segmented manually and subsequently registered to standard brain space. Voxel-based regression analysis was performed to correlate the brain regions of tumor involvement with the level of VEGF expression. Brain regions identified as significantly associated with high or low VEGF expression were preserved following permutation correction., Results: High VEGF expression was detected in 123 (58.9 %) of the 209 patients. Voxel-based statistical analysis demonstrated that high VEGF expression was more likely in tumors located in the left frontal lobe and the right caudate and low VEGF expression was more likely in tumors that occurred in the posterior region of the right lateral ventricle., Conclusion: Voxel-based neuroimaging analysis revealed the anatomic specificity of VEGF expression in glioblastoma, which may further our understanding of genetic heterogeneity during tumor origination. This finding provides primary theoretical support for potential future application of customized antiangiogenic therapy.

Glioblastoma multiforme (GBM) is a highly aggressive brain tumor, with dismal survival outcomes. Recently, cancer stem cells (CSCs) have been demonstrated to play a role in therapeutic resistance and are considered to be the most likely cause of cancer relapse. The identification of CSCs is an important step toward finding new and effective ways to treat GBM. Tenascin-C (TNC) protein has been identified as a potential marker for CSCs in gliomas based on previous work. Here, we have investigated the expression of TNC in tissue microarrays including 17 GBMs, 18 WHO grade III astrocytomas, 15 WHO grade II astrocytomas, 4 WHO grade I astrocytomas, and 7 normal brain tissue samples by immunohistochemical staining. TNC expression was found to be highly associated with the grade of astrocytoma. It has a high expression level in most of the grade III astrocytomas and GBMs analyzed and a very low expression in most grade II astrocytomas, whereas it is undetectable in grade I astrocytomas and normal brain tissues. Double-immunofluorescence staining for TNC and CD133 in GBM tissues revealed that there was a high overlap between theses two positive populations. The results were further confirmed by flow cytometry analysis of TNC and CD133 in GBM-derived stem-like neurospheres in vitro. A limiting dilution assay demonstrated that the sphere formation ability of CD133(+)/TNC(+) and CD133(-)/TNC(+) cell populations is much higher than that of the CD133(+)/TNC(-) and CD133(-)/TNC(-) populations. These results suggest that TNC is not only a potential prognostic marker for GBM but also a potential marker for glioma CSCs, where the TNC(+) population is identified as a CSC population overlapping with part of the CD133(-) cell population.

During normal development, heterogeneous expression of Notch ligands can result in pathway suppression in the signal-sending cell, a process known as lateral inhibition. It is unclear if an analogous phenomenon occurs in malignant cells. We observed significant induction of Notch ligands in glioblastoma neurospheres and pancreatic carcinoma cells cultured in low oxygen, suggesting that this phenomenon could occur around hypoxic regions. To model lateral inhibition in these tumors, the ligand Jagged1 was overexpressed in glioblastoma and pancreatic carcinoma cells, resulting in overall induction of pathway targets. However, when ligand high and ligand low cells from a single line were co-cultured and then separated, we noted suppression of Notch pathway targets in the former and induction in the latter, suggesting that neoplastic lateral inhibition can occur. We also found that repression of Notch pathway targets in signal-sending cells may occur through the activity of a Notch ligand intracellular domain, which translocates into the nucleus. Understanding how this neoplastic lateral inhibition process functions in cancer cells may be important in targeting ligand driven Notch signaling in solid tumors.

Introduction: Phosphatase and tensin homolog (PTEN) loss and epidermal growth factor receptor (EGFR) amplification are common genetic alterations in malignant gliomas. This study aimed to investigate the anatomical relationship of tumor-related PTEN and EGFR expression in 140 patients with histologically confirmed de novo glioblastoma., Methods: Preoperative magnetic resonance images were retrospectively analyzed. The lesions of each patient were segmented manually and registered to a standard brain space. Overlaying of the lesions was performed, and specific brain regions associated with PTEN loss and EGFR amplification were identified by voxel-based lesion-symptom mapping analyses., Results: A cluster located in the right frontal lobe was found to be associated with high occurrence of PTEN loss, whereas a cluster in the right parietal lobe was demonstrated to be specifically associated with high occurrence of EGFR amplification. An overlap of the two clusters was observed at the posterior portion of the right parietal lobe., Conclusions: Based on voxel-based imaging analyses, our results suggest that genetic changes during the tumorigenic process may have anatomical specificity. We hope that this identified correlation between these biomarkers and the anatomical distribution of glioblastomas will help enhance our understanding of the molecular mechanisms underlying glioblastoma development and progression.

Accumulating evidence has suggested that tumor location is linked to the genetic profile of gliomas. Therefore, the aim of this study was to investigate the anatomical characteristics of p53-mutated glioblastomas. We also sought to provide new insight into the possible niche locations of cells of glioblastoma origin. In order to accomplish this, preoperative magnetic resonance images from 163 patients with primary glioblastomas were retrospectively analyzed. All tumors were manually marked and registered to the standard space. Using the voxel-based lesion-symptom mapping approach, voxels exhibiting the strongest correlations with p53 mutants were identified. T-values of voxels that were greater than 95% of the permutations were factored into the results of the mapping analysis. Distinct anatomical characteristics between p53-mutated and wild-type glioblastomas were demonstrated using this approach. More specifically, we found that p53-mutated glioblastomas were preferentially located in the frontal lobe in the area surrounding the rostral extension of the lateral ventricles. The distinct anatomical characteristics of p53-mutated and wild-type glioblastomas provide further evidence that these gliomas arise from distinct niche locations., (Copyright © 2014 Elsevier B.V. All rights reserved.)

O6-methylguanine DNA methyltransferase (MGMT) is one of the important tumor-related biomarkers and is considered to be a prognostic factor for glioblastoma. This study aimed to investigate the anatomical location of tumor-related MGMT protein expression in histologically confirmed de novo glioblastoma multiforme (GBM). Preoperative magnetic resonance images were retrospectively examined from GBM patients. Tumor tissues were manually segmented based on the structural image of each patient and subsequently registered to a standard brain atlas. Superimposition of the tumor tissues was carried out in patients with and without epigenetic changes. We used voxel-based lesion symptom mapping (VLSM) to identify the specific brain regions that were associated with level of MGMT protein expression. The tumors with low expression of MGMT protein and those with high expression of MGMT protein showed not significant differences in tumor size on T2 imaging. The VLSM analysis demonstrated that tumors with low expression of MGMT protein were more likely to occur in the right temporal-parietal lobe, while tumors with high expression of MGMT protein occurred more often in the left frontal lobe. Based upon VLSM data, our results suggest that the epigenetic changes, which occur during tumorigenesis, may have anatomical specificity. The identified correlation between molecular biomarkers and anatomical distribution underscores the current understanding of the biological characteristics of glioblastoma.

An important problem involves isolating subpopulations of cells defined by protein markers in clinical tissue samples for proteomic studies. We describe a method termed Immunohistochemical staining, laser capture microdissection (LCM) and filter-aided sample preparation (FASP)-Assisted Proteomic analysis of Target cell populations within tissue samples (ILFAPT). The principle of ILFAPT is that a target cell population expressing a protein of interest can be lit up by immunohistochemical staining and isolated from tissue sections using LCM for FASP and proteomic analysis. Using this method, we isolated a small population of CD90(+) stem-like cells from glioblastoma multiforme tissue sections and identified 674 high-confidence (false discovery rate < 0.01) proteins from 32 nL of CD90(+) cells by LC-MS/MS using an Orbitrap Elite mass spectrometer. We further quantified the relative abundance of proteins identified from equal volumes of LCM-captured CD90(+) and CD90(-) cells, where 109 differentially expressed proteins were identified. The major group of these differentially expressed proteins was relevant to cell adhesion and cellular movement. This ILFAPT method has demonstrated the ability to provide in-depth proteome analysis of a very small specific cell population within tissues. It can be broadly applied to the study of target cell populations within clinical specimens., (© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Notch signaling has been demonstrated to have a central role in glioblastoma (GBM) cancer stem cells (CSCs) and we have demonstrated recently that Notch pathway blockade by γ-secretase inhibitor (GSI) depletes GBM CSCs and prevents tumor propagation both in vitro and in vivo. In order to understand the proteome alterations involved in this transformation, a dose-dependent quantitative mass spectrometry (MS)-based proteomic study has been performed based on the global proteome profiling and a target verification phase where both Immunoassay and a multiple reaction monitoring (MRM) assay are employed. The selection of putative protein candidates for confirmation poses a challenge due to the large number of identifications from the discovery phase. A multilevel filtering strategy together with literature mining is adopted to transmit the most confident candidates along the pipeline. Our results indicate that treating GBM CSCs with GSI induces a phenotype transformation towards non-tumorigenic cells with decreased proliferation and increased differentiation, as well as elevated apoptosis. Suppressed glucose metabolism and attenuated NFR2-mediated oxidative stress response are also suggested from our data, possibly due to their crosstalk with Notch Signaling. Overall, this quantitative proteomic-based dose-dependent work complements our current understanding of the altered signaling events occurring upon the treatment of GSI in GBM CSCs., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

We have recently demonstrated that Notch pathway blockade by γ-secretase inhibitor (GSI) depletes cancer stem cells (CSCs) in Glioblastoma Multiforme (GBM) through reduced proliferation and induced apoptosis. However, the detailed mechanism by which the manipulation of Notch signal induces alterations on post-translational modifications such as glycosylation has not been investigated. Herein, we present a differential profiling work to detect the change of glycosylation pattern upon drug treatment in GBM CSCs. Rapid screening of differential cell surface glycan structures has been performed by lectin microarray on live cells followed by the detection of N-linked glycoproteins from cell lysates using multi-lectin chromatography and label-free quantitative mass spectrometry analysis. A total of 51 and 52 glycoproteins were identified in the CSC- and GSI-treated groups, respectively, filtered by a combination of decoy database searching and Trans-Proteomic Pipeline (TPP) processing. Although no significant changes were detected from the lectin microarray experiment, 7 differentially expressed glycoproteins with high confidence were captured after the multi-lectin column including key enzymes involved in glycan processing. Functional annotations of the altered glycoproteins suggest a phenotype transformation of CSCs toward a less tumorigenic form upon GSI treatment., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

One important function of endothelial cells in glioblastoma multiforme (GBM) is to create a niche that helps promote self-renewal of cancer stem-like cells (CSLC). However, the underlying molecular mechanism for this endothelial function is not known. Since activation of NOTCH signaling has been found to be required for propagation of GBM CSLCs, we hypothesized that the GBM endothelium may provide the source of NOTCH ligands. Here, we report a corroboration of this concept with a demonstration that NOTCH ligands are expressed in endothelial cells adjacent to NESTIN and NOTCH receptor-positive cancer cells in primary GBMs. Coculturing human brain microvascular endothelial cells (hBMEC) or NOTCH ligand with GBM neurospheres promoted GBM cell growth and increased CSLC self-renewal. Notably, RNAi-mediated knockdown of NOTCH ligands in hBMECs abrogated their ability to induce CSLC self-renewal and GBM tumor growth, both in vitro and in vivo. Thus, our findings establish that NOTCH activation in GBM CSLCs is driven by juxtacrine signaling between tumor cells and their surrounding endothelial cells in the tumor microenvironment, suggesting that targeting both CSLCs and their niche may provide a novel strategy to deplete CSLCs and improve GBM treatment.

Cancer stem cells are responsible for tumor formation through self-renewal and differentiation into multiple cell types and thus represent a new therapeutic target for tumors. Glycoproteins play a critical role in determining the fates of stem cells such as self-renewal, proliferation, and differentiation. Here we applied a multilectin affinity chromatography and quantitative glycoproteomics approach to analyze alterations of glycoproteins relevant to the differentiation of a glioblastoma-derived stem cell line HSR-GBM1. Three lectins including concanavalin A (Con A), wheat germ agglutinin (WGA), and peanut agglutinin (PNA) were used to capture glycoproteins, followed by LC-MS/MS analysis. A total of 73 and 79 high-confidence (FDR < 0.01) glycoproteins were identified from the undifferentiated and differentiated cells, respectively. Label-free quantitation resulted in the discovery of 18 differentially expressed glycoproteins, wherein 9 proteins are localized in the lysosome. All of these lysosomal glycoproteins were up-regulated after differentiation, where their principal function was hydrolysis of glycosyl residues. Protein-protein interaction and functional analyses revealed the active involvement of lysosomes during the process of glioblastoma stem cell differentiation. This work provides glycoprotein markers to characterize differentiation status of glioblastoma stem cells that may be useful in stem-cell therapy of glioblastoma.

Despite progress in the treatment of glioblastoma, more than 95% of patients suffering from this disease still die within 2 years. Recent findings support the belief that cancer stem-like cells are responsible for tumor formation and ongoing growth. Here a method combining lectin microarray and LC-MS/MS was used to discover the cell surface glycoprotein markers of a glioblastoma-derived stem-like cell line. Lectin microarray analysis of cell surface glycans showed that two galactose-specific lectins Trichosanthes kirilowii agglutinin (TKA) and Peanut agglutinin (PNA) could distinguish the stem-like glioblastoma neurosphere culture from a traditional adherent glioblastoma cell line. Agarose-bound TKA and PNA were used to capture the glycoproteins from the two cell cultures, which were analyzed by LC-MS/MS. The glycoproteins were quantified by spectral counting, resulting in the identification of 12 and 11 potential glycoprotein markers from the TKA and PNA captured fractions respectively. Almost all of these proteins were membrane proteins. Differential expression was verified by Western blotting analysis of 6 interesting proteins, including the up-regulated Receptor-type tyrosine-protein phosphatase zeta, Tenascin-C, Chondroitin sulfate proteoglycan NG2, Podocalyxin-like protein 1 and CD90, and the down-regulated CD44. An improved understanding of these proteins may be important for earlier diagnosis and better therapeutic targeting of glioblastoma.

Cancer stem cells (CSCs) are thought to be critical for the engraftment and long-term growth of many tumors, including glioblastoma (GBM). The cells are at least partially spared by traditional chemotherapies and radiation therapies, and finding new treatments that can target CSCs may be critical for improving patient survival. It has been shown that the NOTCH signaling pathway regulates normal stem cells in the brain, and that GBMs contain stem-like cells with higher NOTCH activity. We therefore used low-passage and established GBM-derived neurosphere cultures to examine the overall requirement for NOTCH activity, and also examined the effects on tumor cells expressing stem cell markers. NOTCH blockade by gamma-secretase inhibitors (GSIs) reduced neurosphere growth and clonogenicity in vitro, whereas expression of an active form of NOTCH2 increased tumor growth. The putative CSC markers CD133, NESTIN, BMI1, and OLIG2 were reduced following NOTCH blockade. When equal numbers of viable cells pretreated with either vehicle (dimethyl sulfoxide) or GSI were injected subcutaneously into nude mice, the former always formed tumors, whereas the latter did not. In vivo delivery of GSI by implantation of drug-impregnated polymer beads also effectively blocked tumor growth, and significantly prolonged survival, albeit in a relatively small cohort of animals. We found that NOTCH pathway inhibition appears to deplete stem-like cancer cells through reduced proliferation and increased apoptosis associated with decreased AKT and STAT3 phosphorylation. In summary, we demonstrate that NOTCH pathway blockade depletes stem-like cells in GBMs, suggesting that GSIs may be useful as chemotherapeutic reagents to target CSCs in malignant gliomas.

Brain tumors can arise following deregulation of signaling pathways normally activated during brain development and may derive from neural stem cells. Given the requirement for Hedgehog in non-neoplastic stem cells, we investigated whether Hedgehog blockade could target the stem-like population in glioblastoma multiforme (GBM). We found that Gli1, a key Hedgehog pathway target, was highly expressed in 5 of 19 primary GBM and in 4 of 7 GBM cell lines. Shh ligand was expressed in some primary tumors, and in GBM-derived neurospheres, suggesting a potential mechanism for pathway activation. Hedgehog pathway blockade by cyclopamine caused a 40%-60% reduction in growth of adherent glioma lines highly expressing Gli1 but not in those lacking evidence of pathway activity. When GBM-derived neurospheres were treated with cyclopamine and then dissociated and seeded in media lacking the inhibitor, no new neurospheres formed, suggesting that the clonogenic cancer stem cells had been depleted. Consistent with this hypothesis, the stem-like fraction in gliomas marked by both aldehyde dehydrogenase activity and Hoechst dye excretion (side population) was significantly reduced or eliminated by cyclopamine. In contrast, we found that radiation treatment of our GBM neurospheres increased the percentage of these stem-like cells, suggesting that this standard therapy preferentially targets better-differentiated neoplastic cells. Most importantly, viable GBM cells injected intracranially following Hedgehog blockade were no longer able to form tumors in athymic mice, indicating that a cancer stem cell population critical for ongoing growth had been removed. Disclosure of potential conflicts of interest is found at the end of this article.

Various genomic alterations have been detected in glioblastoma. Chromosome 7p, with the epidermal growth factor receptor locus, together with chromosome 10q, with the phosphatase and tensin homologue deleted in chromosome 10 and deleted in malignant brain tumors-1 loci, and chromosome 9p, with the cyclin-dependent kinase inhibitor 2A locus, are among the most frequently damaged chromosomal regions in glioblastoma. In this study, we evaluated the genetic status of 32 glioblastomas by comparative genomic hybridization; the sensitivity of comparative genomic hybridization versus differential polymerase chain reaction to detect deletions at the phosphatase and tensin homologue deleted in chromosome 10, deleted in malignant brain tumors-1, and cyclin-dependent kinase inhibitor 2A loci and amplifications at the cyclin-dependent kinase 4 locus; the frequency of genetic lesions (gain or loss) at 16 different selected loci (including oncogenes, tumor-suppressor genes, and proliferation markers) mapping on 13 different chromosomes; and the possible existence of a statistical association between any pair of molecular markers studied, to subdivide the glioblastoma entity molecularly. Comparative genomic hybridization showed that the most frequent region of gain was chromosome 7p, whereas the most frequent losses occurred on chromosomes 10q and 13q. The only statistically significant association was found for 7p gain and 10q loss., (Copyright 2002 Wiley-Liss, Inc.)

New therapeutic strategies are needed to improve survival in glioblastoma (GBM) the most malignant astrocytic tumor. We evaluated: a) the genetic status of 22 GBMs by comparative genomic hybridization (CGH); b) the specific role of mutation and/or homozygous deletion of PTEN in the genesis of GBM; and c) the possible therapeutic role of PTEN against GBM, in vitro. CGH demonstrated that the most frequent region of gain was at chromosome 7p, whereas the most frequent losses occurred at chromosomes 10q and 13q. Losses at chromosome 10 were found in 36% of patients, and PTEN was mutated in 27% of the 22 GBMs, including 4 point mutations and 2 homozygous deletions. The possible therapeutic role of PTEN in GBM was also studied in a system based on retroviral infection of the GBM cell line A172, homozygously deleted at the PTEN locus. A172 growth and proliferation rate were reduced by 50% after PTEN transduction. Moreover, we showed that inhibition of cell growth occurred through the PI3K/Akt/p27 pathway. Our findings suggest that PTEN participates in the genesis of GBM, and might be further studied as a candidate therapeutic agent in other testing systems.

Malignant gliomas are highly heterogeneous brain tumors in molecular genetic background. Despite the many recent advances in the understanding of this disease, patients with adult high-grade gliomas retain a notoriously poor prognosis. Fusions involving oncogenes have been reported in gliomas and may serve as novel therapeutic targets to date. Understanding the gene fusions and how they regulate oncogenesis and malignant progression will contribute to explore new approaches for personalized treatment. By now, studies on gene fusions in gliomas remain limited. However, some current clinical trials targeting fusion genes have presented exciting preliminary findings. The aim of this review is to summarize all the reported fusion genes in high-grade gliomas so far, discuss the characterization of some of the most popular gene fusions occurring in malignant gliomas, as well as their function in tumorigenesis, and the underlying clinical implication as therapeutic targets. [ABSTRACT FROM AUTHOR]

Introduction: The expression of vascular endothelial growth factor (VEGF) is a common genetic alteration in malignant gliomas and contributes to the angiogenesis of tumors. This study aimed to investigate the anatomical specificity of VEGF expression levels in glioblastomas using voxel-based neuroimaging analysis. Methods: Clinical information, MR scans, and immunohistochemistry stains of 209 patients with glioblastomas were reviewed. All tumor lesions were segmented manually and subsequently registered to standard brain space. Voxel-based regression analysis was performed to correlate the brain regions of tumor involvement with the level of VEGF expression. Brain regions identified as significantly associated with high or low VEGF expression were preserved following permutation correction. Results: High VEGF expression was detected in 123 (58.9 %) of the 209 patients. Voxel-based statistical analysis demonstrated that high VEGF expression was more likely in tumors located in the left frontal lobe and the right caudate and low VEGF expression was more likely in tumors that occurred in the posterior region of the right lateral ventricle. Conclusion: Voxel-based neuroimaging analysis revealed the anatomic specificity of VEGF expression in glioblastoma, which may further our understanding of genetic heterogeneity during tumor origination. This finding provides primary theoretical support for potential future application of customized antiangiogenic therapy. [ABSTRACT FROM AUTHOR]

Oncolytic virotherapy is an emerging treatment modality that uses replication-competent viruses to destroy cancer cells. M1 is a naturally occurring alphavirus (Togaviridae) which shows potent oncolytic activities against many cancers. Accumulation of unfolded proteins during virus replication leads to a transcriptional/translational response known as the unfolded protein response (UPR), which might counteract the antitumor effect of the oncolytic virus. In this report, we show that either pharmacological or biological inhibition of IRE1α or PERK, but not ATF6, substantially increases the oncolytic effects of the M1 virus. Moreover, inhibition of IRE1α blocks M1 virus-induced autophagy, which restricts the antitumor effects of the M1 virus through degradation of viral protein, in glioma cells. In addition, IRE1α suppression significantly increases the oncolytic effect of M1 virus in an orthotopic glioma model. From a molecular pathology study, we found that IRE1α is expressed at lower levels in higher-grade gliomas, suggesting greater antitumor efficacy of the oncolytic virus M1. Taken together, these findings illustrate a defensive mechanism of glioma cells against the oncolytic virus M1 and identify possible approaches to enhance the oncolytic viral protein accumulation and the subsequent lysis of tumor cells. [ABSTRACT FROM AUTHOR]