Molecular characterizations of glioblastoma,targeted therapy,and clinical results to date

During the last decade, extensive multiplatform genome‐wide analysis has yielded a wealth of knowledge regarding the genetic and molecular makeup of glioblastoma multiforme (GBM). These profiling studies support the emerging view that GBM comprises a group of highly heterogeneous tumor types, each with its own distinct molecular and genetic signatures. This heterogeneity complicates the process of defining reliable intertumor/intratumor biological states, which will ultimately be needed for classifying tumors and for designing effective customized therapies that target resultant disease pathways. The increased understanding of the molecular pathogenesis of GBM has brought the hope and expectation that such knowledge will lead to better and more rational therapies directed toward specific molecular targets. To date, however, these expectations have largely been unrealized. This review discusses some of the principal genetic and epigenetic aberrations found in GBM that appear promising for targeted therapies now and in the near future, and it offers suggestions for future directions concerning the rather disappointing results of clinical trials to date. Cancer 2015;121:502–516. © 2014 American Cancer Society.     

Global DNA methylation reflects spatial heterogeneity and molecular evolution of lung adenocarcinomas

Lung adenocarcinoma (ADC) is the most prevalent subtype of lung cancer and characterized by considerable morphological and mutational heterogeneity. However, little is known about the epigenomic intratumor variability between spatially separated histological growth patterns of ADC. In order to reconstruct the clonal evolution of histomorphological patterns, we performed global DNA methylation profiling of 27 primary tumor regions, seven matched normal tissues and six lymph node metastases from seven ADC cases. Additionally, we investigated the methylation data from 369 samples of the TCGA ADC cohort. All regions showed varying degrees of methylation changes between segments of different, but also of the same growth patterns. Similarly, copy number variations were seen between spatially distinct segments of each patient. Hierarchical clustering of promoter methylation revealed extensive heterogeneity within and between the cases. Intratumor DNA methylation heterogeneity demonstrated a branched clonal evolution of ADC regions driven by genomic instability with subclonal copy number changes. Notably, methylation profiles within tumors were not more similar to each other than to those from other individuals. In two cases, different tumor regions of the same individuals were represented in distant clusters of the TCGA cohort, illustrating the extensive epigenomic intratumor heterogeneity of ADCs. We found no evidence for the lymph node metastases to be derived from a common growth pattern. Instead, they had evolved early and separately from a particular pattern in each primary tumor. Our results suggest that extensive variation of epigenomic features contributes to the molecular and phenotypic heterogeneity of primary ADCs and lymph node metastases.     

Editor's choice: GBM's multifaceted landscape: highlighting regional and microenvironmental heterogeneity

Gliomas are a heterogeneous group of tumors that show variable proliferative potential, invasiveness, aggressiveness, histological grading, and clinical behavior. In this review, we focus on glioblastoma multiforme (GBM), a grade IV glioma, which is the most common and malignant of primary adult brain tumors. Research over the past several decades has revealed the existence of extensive cellular, molecular, genetic, epigenetic, and metabolic heterogeneity among tumors of the same grade and even within individual tumors. Evaluation of different tumor types has shown that tumors with advanced grade and clinical aggressiveness also display enhanced molecular, cellular, and microenvironmental heterogeneity. From a therapeutic standpoint, this heterogeneity is a major clinical hurdle for devising effective therapeutic strategies for patients and challenges personalized medicine.In this review, we will highlight key aspects of GBM heterogeneity, directing special attention to regional heterogeneity, hypoxia, genomic heterogeneity, tumor-specific metabolic reprogramming, neovascularization or angiogenesis, and stromal immune cells. We will further discuss the clinical implications of GBM heterogeneity in the context of therapy.     

Increased expression of EphA2 correlates with adverse outcome in primary and recurrent glioblastoma multiforme patients

Glioblastoma multiforme (GBM) is the most aggressive form of brain tumor characterized by excessive angiogenesis. The dismal prognosis of patients with GBM warrants the development of new targeting therapies based on novel molecular markers. The EphA2 receptor tyrosine kinase plays a pivotal role in tumor angiogenesis and an increased expression in glioma patients has recently been reported. In this study, we investigated the expression of EphA2 in human normal brain, primary and recurrent GBM and correlated it with clinical pathological parameters and patient's outcome. In addition, intratumor microvascular density was quantified by immunostaining for the endothelial cell marker, von Willebrand factor. A different intensity of the membranous and cytoplastic expression of EphA2 was observed in the 40 primary and recurrent samples of GBM analyzed but not in the normal brain. A high level expression of EphA2 was demonstrated in 24 (60%) of the primary and recurrent GBM analyzed. The increased expression of the EphA2 protein was significantly associated with the adverse outcome of GBM patients (p<0.01 for overall survival). The data presented in this study define the expression pattern of EphA2 in both primary and recurrent glioblastoma and suggest an important role of EphA2 in the pathogenesis of GBM. The EphA2 may be used as a surrogate marker to screen patients for tyrosine kinase inhibitor therapy.     

Gene expression profiling of gliomas: merging genomic and histopathological classification for personalised therapy

The development of DNA microarray technologies over the past decade has revolutionised translational cancer research. These technologies were originally hailed as more objective, comprehensive replacements for traditional histopathological cancer classification systems, based on microscopic morphology. Although DNA microarray-based gene expression profiling (GEP) remains unlikely in the near term to completely replace morphological classification of primary brain tumours, specifically the diffuse gliomas, GEP has confirmed that significant molecular heterogeneity exists within the various morphologically defined gliomas, particularly glioblastoma (GBM). Herein, we provide a 10-year progress report on human glioma GEP, with focus on development of clinical diagnostic tests to identify molecular subtypes, uniquely responsive to adjuvant therapies. Such progress may lead to a more precise classification system that accurately reflects the cellular, genetic, and molecular basis of gliomagenesis, a prerequisite for identifying subsets uniquely responsive to specific adjuvant therapies, and ultimately in achieving individualised clinical care of glioma patients.     

Combined HSV-TK/IL-2 gene therapy in patients with recurrent glioblastoma multiforme: biological and clinical results

Following our pilot clinical study of combined IL-2/HSV-TK gene therapy for recurrent glioblastoma multiforme (GBM), we extended the protocol to a larger population of patients and evaluated safety, feasibility, and biological activity of treatment. A total of 12 patients received intratumor injection of retroviral vector-producing cells (RVPCs), followed by intravenous ganciclovir (GCV). Treatment was well tolerated with only minor adverse events. Transduction of tumor cells was demonstrated in tumor biopsies. A marked and persistent increase of intratumor and plasma Th1 cytokine levels was demonstrated after RVPC injection. At magnetic resonance imaging evaluation, two patients had a partial response (including a patient showing disappearance of a distant noninjected tumor mass), four had a minor response, four had stable disease, and two had progressive disease. The 6- and 12-month progression-free survival rates were 47 and 14%, respectively. The 6- and 12-month overall survival rates were 58 and 25%, respectively. In conclusion, the results of our clinical protocol of gene therapy for recurrent GBM, based on combined delivery of a suicide and a cytokine gene, demonstrate that intratumor injection of RVPCs was safe, provided effective transduction of the therapeutic genes to target tumor cells, and activated a systemic cytokine cascade, with tumor responses in 50% of cases.     

Proscillaridin A is cytotoxic for glioblastoma cell lines and controls tumor xenograft growth in vivo

Glioblastoma is the most frequent primary brain tumor in adults. Because of molecular and cellular heterogeneity, high proliferation rate and significant invasive ability, prognosis of patients is poor. Recent therapeutic advances increased median overall survival but tumor recurrence remains inevitable. In this context, we used a high throughput screening approach to bring out novel compounds with anti-proliferative and anti-migratory properties for glioblastoma treatment. Screening of the Prestwick chemical library® of 1120 molecules identified proscillaridin A, a cardiac glycoside inhibitor of the Na⁺/K⁺ ATPase pump, with most significant effects on glioblastoma cell lines. In vitro effects of proscillaridin A were evaluated on GBM6 and GBM9 stem-like cell lines and on U87-MG and U251-MG cell lines. We showed that proscillaridin A displayed cytotoxic properties, triggered cell death, induced G₂/M phase blockade in all the glioblastoma cell lines and impaired GBM stem self-renewal capacity even at low concentrations. Heterotopic and orthotopic xenotransplantations were used to confirm in vivo anticancer effects of proscillaridin A that both controls xenograft growth and improves mice survival. Altogether, results suggest that proscillaridin A is a promising candidate as cancer therapies in glioblastoma. This sustains previous reports showing that cardiac glycosides act as anticancer drugs in other cancers.     

Activated EGFR signaling increases proliferation,survival, and migration and blocks neuronal differentiation in post-natal neural stem cells

Recent evidence supports the notion that transformation of undifferentiated neural stem cell (NSC) precursors may contribute to the development of glioblastoma multiforme (GBM). The over-expression and mutation of the epidermal growth factor receptor (EGFR), along with other cellular pathway mutations, plays a significant role in GBM maintenance progression. Though EGFR signaling is important in determining neural cell fate and conferring astrocyte differentiation, there is a limited understanding of its role in NSC and tumor stem cell (TSC) biology. We hypothesized that EGFR expression and mutation in post-natal NSCs may contribute to cellular aggressiveness including enhanced cellular proliferation, survival and migration. Stable subclones of C17.2 murine NSCs were transfected to over-express either the wild-type EGFR (wtEGFR) or its most common mutated variant EGFRvIII. Activated EGFR signaling in these cells induced behaviors characteristic of GBM TSCs, including enhanced proliferation, survival and migration, even in the absence of EGF ligand. wtEGFR activation was also found to block neuronal differentiation and was associated with a dramatic increase in chemotaxis in the presence of EGF. EGFRvIII expression lead to an increase in NSC proliferation and survival, while it simultaneously blocked neuronal differentiation and promoted glial fate. Our findings suggest that activated EGFR signaling enhances the aggressiveness of NSCs. Understanding the regulatory mechanisms of NSCs may lend insight into deregulated mechanisms of GBM TSC invasion, proliferation, survival and resistance to current treatment modalities.     

Bevacizumab in high-grade gliomas: past,present, and future

The survival of patients with high-grade gliomas (anaplastic gliomas and glioblastoma) remains poor despite current treatment modalities. However, an enhanced understanding of gliomagenesis is supporting the development of targeted molecular therapies with the potential for improving clinical outcomes. Glioblastoma (GBM) is characterized by extensive microvascular proliferation and the production of large amounts of VEGF. Bevacizumab is a humanized IgG1 monoclonal antibody that selectively binds with high affinity to human VEGF and neutralizes VEGF’s biologic activity. Preclinical data indicate that angiogenesis is essential for the proliferation and survival of GBM cells. A number of studies have evaluated the outcomes of both newly diagnosed and recurrent GBM patients with bevacizumab in a prospective manner. Here, we discuss the role of bevacizumab in the treatment of anaplastic gliomas and GBM in the recurrent and upfront setting.     

Intratumoral heterogeneity: pathways to treatment resistance and relapse in human glioblastoma

Intratumoral heterogeneity (ITH) has increasingly being described for multiple cancers as the root cause of therapy resistance. Recent studies have started to explore the scope of ITH in glioblastoma (GBM), a highly aggressive and fatal form of brain tumor, to explain its inevitable therapy resistance and disease relapse. In this review, we detail the emerging data that explores the extensive genetic, cellular and functional ITH present in GBM. We discuss current experimental models of human GBM recurrence and suggest harnessing new technologies (CRISPR-Cas9 screening, CyTOF, cellular barcoding, single cell analysis) to delineate GBM ITH and identify treatment-refractory cell populations, thus opening new therapeutic windows. We will also explore why current therapeutics have failed in clinical trials and how ITH can inform us on developing empiric therapies for the treatment of recurrent GBM.     

Gene expression profiling in cervical cancer: an exploration of intratumor heterogeneity.

PURPOSE: To explore intratumor heterogeneity in gene expression profiles from patients with cervical cancer. EXPERIMENTAL DESIGN: A total of 33 biopsies were obtained from 11 patients, sampling between two and five different areas for each tumor. The extracted RNA was hybridized onto the Affymetrix U133 Plus 2.0 oligonucleotide chip. The variance of expression within a patient (W), between patients (B) and the total variance (T = W + B) were calculated for each ProbeSet, and the ratio W/T was used as a measure of intratumor heterogeneity. Gene Ontology functional analysis was done to assess the function of genes that had high W/T (top 10%) and low W/T (bottom 10%) values. RESULTS: In total, 448 ProbeSets (2.2% of the total) had W/T < 0.10, indicating low intratumor heterogeneity, and 537 ProbeSets (2.7% of the total) had W/T > 0.90, indicating high intratumor heterogeneity. In total 14,473 ProbeSets (72.4%) had higher intertumor than intratumor heterogeneity (W/T < 0.5). Genes with low intratumor heterogeneity were characterized by a statistically significant enrichment of immune-related functions (P < 0.0001). Genes with high intratumor heterogeneity were characterized by a significant tendency towards nuclear localization and nucleic acid binding (both P < 0.0001). For genes with W/T > 0.5, more than six biopsies would be required to minimize the intratumoral heterogeneity to <0.15; if W/T is 0.3 to 0.4, four biopsies are required; and for low W/T of 0.16 to 0.3, only two to three biopsies would be needed. CONCLUSION: Although the intratumor heterogeneity was low for the majority of the tested ProbeSets, for many genes, multiple biopsies are required to obtain a reliable estimate of gene expression.     

Long-term follow-up on National Cancer Institute Phase I/II study of glioblastoma multiforme treated with iododeoxyuridine and hyperfractionated irradiation.

PURPOSE: We report the results of the final phase I/II program in glioblastoma (GBM) multiforme patients using only hyperfractionated irradiation and intravenous iododeoxyuridine (IdUrd). METHODS: For a decade we investigated halogenated pyrimidine radiosensitizers in an effort to exploit the potential for differential uptake of thymidine analogs between proliferating tumor and normal brain tissues. Trials began with bromodeoxyuridine (BrdUrd) but were changed to IdUrd when the latter proved less photosensitizing. A series of dose-escalating pilot trials led to treatment at a maximum-tolerated dose (MTD) of IdUrd of 1,000 mg/m2/d for two separate 14-day courses, one during the initial radiation field and one during the cone down. The radiotherapy also evolved over time and was hyperfractionated in all cases reported. Over 5 years we accrued 45 patients into the final hyperfractionated, 1,000 mg/m2/d scheme. We report here results on only the patients with minimum follow-up of 1 year (90% had at least 2 years of follow-up) or until death. RESULTS: The results do not indicate a significant benefit for use of sensitizers, as compared with other contemporary and aggressive types of radiation treatment. The median survival has been 11 months, with a 2-year actuarial survival of 9%. As yet, there are no survivors at 3 years. Tumor biopsies at craniotomy showed relatively low sensitizer incorporation. CONCLUSION: The failure of radiosensitizers combined with radiation therapy to show major benefit may be due to patient selection but appears also to be related to the combined problems of poor drug penetration/uptake into tumor, tumor-cell heterogeneity, and a high inherent cellular radioresistance of GBM.     

Immunohistochemical analysis‐based proteomic subclassification of newly diagnosed glioblastomas

Recent gene expression and copy number profilings of glioblastoma multiforme (GBM) by The Cancer Genome Atlas (TCGA) Research Network suggest the existence of distinct subtypes of this tumor. However, these approaches might not be easily applicable in routine clinical practice. In the current study, we aimed to establish a proteomics‐based subclassification of GBM by integrating their genomic and epigenomic profiles. We subclassified 79 newly diagnosed GBM based on expression patterns determined by comprehensive immunohistochemical observation in combination with their DNA copy number and DNA methylation patterns. The clinical relevance of our classification was independently validated in TCGA datasets. Consensus clustering identified the four distinct GBM subtypes: Oligodendrocyte Precursor (OPC) type, Differentiated Oligodendrocyte (DOC) type, Astrocytic Mesenchymal (AsMes) type and Mixed type. The OPC type was characterized by highly positive scores of Olig2, PDGFRA, p16, p53 and synaptophysin. In contrast, the AsMes type was strongly associated with strong expressions of nestin, CD44 and podoplanin, with a high glial fibrillary acidic protein score. The median overall survival of OPC‐type patients was significantly longer than that of the AsMes‐type patients (19.9 vs 12.8 months). This finding was in agreement with the Oncomine analysis of TCGA datasets, which revealed that PDGFRA and Olig2 were favorable prognostic factors and podoplanin and CD44 were associated with a poor clinical outcome. This is the first study to establish a subclassification of GBM on the basis of immunohistochemical analysis. Our study will shed light on personalized therapies that might be feasible in daily neuropathological practice. (Cancer Sci, doi: 10.1111/j.1349‐7006.2012.02377.x, 2012)     

Overexpression of c-Met is Associated with Poor Prognosis in Glioblastoma Multiforme: A Systematic Review and Meta-Analyses

Objective: The aim of this study is to evaluate the association of c-Met overexpression with survival of glioblastoma multiforme (GBM) patients. Methods: A systematic review with meta-analyses was conducted on related articles from PubMed, EBSCOhost, Scopus, and Cochrane databases with last updated search on October 31, 2020. A total of 7 studies regarding c-Met overexpression and overall survival (OS) and/or progression free survival (PFS) are included in this study. Results: All studies used immunohistochemistry to examine the expression of c-Met protein. The results showed that the positive rate of c-Met overexpression was detected in approximately 33,9% - 60,5% of GBM patients. c-Met overexpression was related to worse OS (HR: 1,74; 95% CI: 1,482-2,043; Z=6,756; p<0,001) and PFS (HR: 1,66; 95% CI: 1,327-2,066; Z=4,464; p<0,001) in GBM patients. Low heterogeneity of subjects was found in both OS and PFS analyses, I2 values were 7,8% and 0,0%, respectively. Conclusion: In conclusion, c-Met overexpression is significantly related to shorter OS and PFS in GBM patients, so c-Met can be considered as a potential prognostic indicator in GBM.     

MGMT promoter methylation is predictive of response to radiotherapy and prognostic in the absence of adjuvant alkylating chemotherapy for glioblastoma

Hypermethylation of the O⁶-methylguanine-DNA-methyltransferase (MGMT) gene has been shown to be associated with improved outcome in glioblastoma (GBM) and may be a predictive marker of sensitivity to alkylating agents. However, the predictive utility of this marker has not been rigorously tested with regard to sensitivity to other therapies, namely radiation. To address this issue, we assessed MGMT methylation status in a cohort of patients with GBM who underwent radiation treatment but did not receive chemotherapy as a component of adjuvant treatment. Formalin-fixed, paraffin-embedded tumor samples from 225 patients with newly diagnosed GBM were analyzed via methylation-specific, quantitative real-time polymerase chain reaction following bisulfite treatment on isolated DNA to assess MGMT promoter methylation status. In patients who received radiotherapy alone following resection, methylation of the MGMT promoter correlated with an improved response to radiotherapy. Unmethylated tumors were twice as likely to progress during radiation treatment. The median time interval between resection and tumor progression of unmethylated tumors was also nearly half that of methylated tumors. Promoter methylation was also found to confer improved overall survival in patients who did not receive adjuvant alkylating chemotherapy. Multivariable analysis demonstrated that methylation status was independent of age, Karnofsky performance score, and extent of resection as a predictor of time to progression and overall survival. Our data suggest that MGMT promoter methylation appears to be a predictive biomarker of radiation response. Since this biomarker has also been shown to predict response to alkylating agents, perhaps MGMT promoter methylation represents a general, favorable prognostic factor in GBM.     

Bevacizumab-based therapy in relapsed glioblastoma: rationale and clinical experience to date

Relapsed glioblastoma (GBM) has an extremely poor prognosis and remains an invariably fatal disease, with a median overall survival of 6–7 months. Despite numerous clinical trials over the past 20–30 years, treatment options for relapsed GBM remain limited. In recent years, significant research efforts have focused on the use of antiangiogenic therapies for the treatment of GBM. Bevacizumab is a humanized monoclonal antibody that specifically inhibits the proangiogenic VEGF, with well-established clinical efficacy in a number of solid malignancies, which is now under investigation for the treatment of GBM. In this review, we discuss the available data regarding bevacizumab-based therapy in relapsed GBM, highlighting its potential and ongoing challenges in this difficult-to-treat disease.     

胶质瘤干细胞放射抗拒和放射增敏蛋白质组学研究进展

经过包括放疗在内的多学科治疗,多形性胶质母细胞瘤中位生存期始终停留在1年左右。胶质瘤干细胞基因组和蛋白质组的异质性是影响预后的根本原因,依据蛋白质组学制定针对关键放射抗拒蛋白的增敏研究有望改善多形性胶质母细胞瘤预后。本文通过Pubmed等数据库查阅了近十年来的相关文献,系统地讨论了各种常用蛋白质定量技术、用于数据处理的工具及其在胶质瘤干细胞放射抗拒和放射增敏中的进展。     

RNA interference targeting survivin exerts antitumoral effects in vitro and in established glioma xenografts in vivo

Malignant glioma represents the most common primary adult brain tumor in Western industrialized countries. Despite aggressive treatment modalities, the median survival duration for patients with glioblastoma multiforme (GBM), the highest grade malignant glioma, has not improved significantly over past decades. One promising approach to deal with GBM is the inactivation of proteins essential for survival or progression of glioma cells by means of RNA interference (RNAi) techniques. A likely candidate for an RNAi therapy of gliomas is the inhibitor of apoptosis protein survivin. Survivin is involved in 2 main cellular processes-cell division and inhibition of apoptosis. We show here that stable RNAi of survivin induced polyploidy, apoptosis, and impaired proliferation of human U343-MG, U373-MG, H4, and U87-MG cells and of primary glioblastoma cells. Proteome profiler arrays using U373-MG cells identified a novel set of differentially expressed genes upon RNAi-mediated survivin knockdown. In particular, the death receptor TRAIL R2/DR5 was strongly upregulated in survivin-depleted glioma cells, inducing an enhanced cytotoxic response of allogeneic human NK cells. Moreover, an experimental in vivo therapy using polyethylenimine (PEI)/siRNA complexes for survivin knockdown efficiently blocked tumor growth of established subcutaneous U373-MG tumors and enhanced survival of NMRI(nu/nu) mice orthopically transplanted with U87-MG cells. We conclude that survivin is functionally relevant in gliomas and that PEI-mediated exogenous delivery of siRNA targeting survivin is a promising strategy for glioblastoma therapy.     

Inhibition of Necl-5 (CD155/PVR) reduces glioblastoma dispersal and decreases MMP-2 expression and activity

Patients afflicted with glioblastoma (GBM) have poor survival due to dispersive invasion throughout the brain. Necl-5, a cell surface receptor for vitronectin, is expressed in GBM but not normal brain. In several GBM cell lines Necl-5 promotes migration and invasion but the mechanism is poorly understood. In this study, we show that knockdown of Necl-5 by RNAi results in markedly decreased invasion of A172 GBM cells in a 3-dimensional matrix. There is a concomitant decrease in the expression and activity of matrix metalloproteinase-2 (MMP-2), a known factor in GBM invasion and disease severity. Knockdown of Necl-5 diminishes basal activation of Akt, an established mediator of MMP-2 expression in gliomas. Knockdown of Necl-5 also limits the maximal Akt activation in response to vitronectin, which requires the activity of Integrin-linked kinase (ILK). During migration, Necl-5, Akt and ILK co-localize at focal contacts at the leading edge of the plasma membrane, suggesting that these molecules may act to integrate Akt signaling at the leading edge to induce MMP-2 expression. By virtue of its restricted expression in GBM and its role in invasion, Necl-5 may be an attractive target for limiting MMP-2 production in glioblastoma, and therefore limiting dispersal.     

胶质瘤干细胞放射抗拒和放射增敏蛋白质组学研究进展

经过包括放疗在内的多学科治疗,多形性胶质母细胞瘤中位生存期始终停留在1年左右。胶质瘤干细胞基因组和蛋白质组的异质性是影响预后的根本原因,依据蛋白质组学制定针对关键放射抗拒蛋白的增敏研究有望改善多形性胶质母细胞瘤预后。本文通过Pubmed等数据库查阅了近十年来的相关文献,系统地讨论了各种常用蛋白质定量技术、用于数据处理的工具及其在胶质瘤干细胞放射抗拒和放射增敏中的进展。