Gelareh Zadeh

Loss of p53 cooperates with K‐ras activation to induce glioma formation in a region‐independent manner

Gliomas are recognized as a heterogeneous group of neoplasms differing in their location and morphological features. These differences, between and within varying grades of gliomas, have not been explained solely on the grounds of an oncogenic stimulus. Interactions with the tumor microenvironment as well as inherent characteristics of the cell of origin are likely a source of this heterogeneity. There is an ongoing debate over the cell of origin of gliomas, where some suggest a progenitor, while others argue for a stem cell origin. Thus, it is presumed that neurogenic regions of the brain such as the subventricular zone (SVZ) containing large numbers of neural stem and progenitor populations are more susceptible to transformation. Our studies demonstrate that K‐rasG12D cooperates with the loss of p53 to induce gliomas from both the SVZ and cortical region, suggesting that cells in the SVZ are not uniquely gliomagenic. Using combinations of doxycycline‐inducible K‐rasG12D and p53 loss, we show that tumors induced by the cooperative actions of these genes remain dependent on active K‐ras expression, as deinduction of K‐rasG12D leads to complete tumor regression despite absence of p53. These results suggest that the interplay between specific combinations of genetic alterations and susceptible cell types, rather than the site of origin, are important determinates of gliomagenesis. Additionally, this model supports the view that, although several genetic events may be necessary to confer traits associated with oncogenic transformation, inactivation of a single oncogenic partner can undermine tumor maintenance, leading to regression and disease remission. GLIA 2013;61:1862–1872

Contrasting in vivo and in vitro fates of glioblastoma cell subpopulations with amplified EGFR.

Despite the high incidence of EGFR amplification in patient glioblastoma multiforme (GBM) tissues, only a single GBM cell line, of the many described in the literature, is known to contain and maintain amplified EGFR. Because EGFR mutations in GBM manifest primarily, if not exclusively, in amplified form, it follows that the availability of cell lines with mutation of endogenous EGFR would also be in short supply. In fact, there are no GBM cell lines harboring the common EGFR mutants described in patient GBMs. These observations suggest that in vivo environments select for EGFR amplification, whereas in vitro environments, specifically cell cultures, select against this gene alteration. To contrast directly the fates of EGFR amplification in vivo and in vitro, as well as to examine potential relationships between EGFR amplification and mutation, we have established and maintained GBM explants as xenografts by serial passaging in nude mice. Analysis of EGFR copy number and EGFR mutation status in 11 patient tumors and their corresponding xenografts, as well as the monitoring of EGFR copy number during the establishment of a GBM cell line from a xenograft with amplified EGFR, indicated that selection for EGFR amplification is an in vivo phenomenon. Furthermore, our data indicated that EGFR mutation occurs only in tumors with EGFR amplification and showed that the selection of amplified mutant EGFR over amplified wild‐type EGFR as a xenograft occurred rapidly and completely during tumor propagation.

Glioblastoma: pathology, molecular mechanisms and markers

Recent advances in genomic technology have led to a better understanding of key molecular alterations that underlie glioblastoma (GBM). The current WHO-based classification of GBM is mainly based on histologic features of the tumor, which frequently do not reflect the molecular differences that describe the diversity in the biology of these lesions. The current WHO definition of GBM relies on the presence of high-grade astrocytic neoplasm with the presence of either microvascular proliferation and/or tumor necrosis. High-throughput analyses have identified molecular subtypes and have led to progress in more accurate classification of GBM. These findings, in turn, would result in development of more effective patient stratification, targeted therapeutics, and prediction of patient outcome. While consensus has not been reached on the precise nature and means to sub-classify GBM, it is clear that IDH-mutant GBMs are clearly distinct from GBMs without IDH1/2 mutation with respect to molecular and clinical features, including prognosis. In addition, recent findings in pediatric GBMs regarding mutations in the histone H3F3A gene suggest that these tumors may represent a 3rd major category of GBM, separate from adult primary (IDH1/2 wt), and secondary (IDH1/2 mut) GBMs. In this review, we describe major clinically relevant genetic and epigenetic abnormalities in GBM—such as mutations in IDH1/2, EGFR, PDGFRA, and NF1 genes—altered methylation of MGMT gene promoter, and mutations in hTERT promoter. These markers may be incorporated into a more refined classification system and applied in more accurate clinical decision-making process. In addition, we focus on current understanding of the biologic heterogeneity and classification of GBM and highlight some of the molecular signatures and alterations that characterize GBMs as histologically defined. We raise the question whether IDH-wild type high grade astrocytomas without microvascular proliferation or necrosis might best be classified as GBM, even if they lack the histologic hallmarks as required in the current WHO classification. Alternatively, an astrocytic tumor that fits the current histologic definition of GBM, but which shows an IDH mutation may in fact be better classified as a distinct entity, given that IDH-mutant GBM are quite distinct from a biological and clinical perspective.

Methylation of the TERT promoter and risk stratification of childhood brain tumours: an integrative genomic and molecular study

BACKGROUND Identification of robust biomarkers of malignancy and methods to establish disease progression is a major goal in paediatric neuro-oncology. We investigated whether methylation of the TERT promoter can be a biomarker for malignancy and patient outcome in paediatric brain tumours. METHODS For the discovery cohort, we used samples obtained from patients with paediatric brain tumours and individuals with normal brain tissues stored at the German Cancer Research Center (Heidelberg, Germany). We used methylation arrays for genome-wide assessment of DNA. For the validation cohort, we used samples obtained from several tissues for which full clinical and follow-up data were available from two hospitals in Toronto (ON, Canada). We did methylation analysis using quantitative Sequenom and pyrosequencing of an identified region of the TERT promoter. We assessed TERT expression by real-time PCR. To establish whether the biomarker could be used to assess and predict progression, we analysed methylation in paired samples of tumours that transformed from low to high grade and from localised to metastatic, and in choroid plexus tumours of different grades. Finally, we investigated overall survival in patients with posterior fossa ependymomas in which the identified region was hypermethylated or not. All individuals responsible for assays were masked to the outcome of the patients. FINDINGS Analysis of 280 samples in the discovery cohort identified one CpG site (cg11625005) in which 78 (99%) of 79 samples from normal brain tissues and low-grade tumours were not hypermethylated, but 145 (72%) of 201 samples from malignant tumours were hypermethylated (>15% methylated; p<0.0001). Analysis of 68 samples in the validation cohort identified a subset of five CpG sites (henceforth, upstream of the transcription start site [UTSS]) that was hypermethylated in all malignant paediatric brain tumours that expressed TERT but not in normal tissues that did not express TERT (p<0.0001). UTSS had a positive predictive value of 1.00 (95% CI 0.95-1.00) and a negative predictive value of 0.95 (0.87-0.99). In two paired samples of paediatric gliomas, UTSS methylation increased during transformation from low to high grade; it also increased in two paired samples that progressed from localised to metastatic disease. Two of eight atypical papillomas that had high UTSS methylation progressed to carcinomas, while the other six assessed did not progress or require additional treatment. 5-year overall survival was 51% (95% CI 31-71) for 25 patients with hypermethylated UTSS posterior fossa ependymomas and 95% (86-100) for 20 with non-hypermethylated tumours (p=0.0008). 5-year progression-free survival was 86% (68-100) for the 25 patients with non-hypermethylated UTSS tumours and 30% (10-50) for those with hypermethylated tumours (p=0.0008). INTERPRETATION Hypermethylation of the UTSS region in the TERT promoter is associated with TERT expression in cancers. In paediatric brain tumours, UTSS hypermethylation is associated with tumour progression and poor prognosis. This region is easy to amplify, and the assay to establish hypermethylation can be done on most tissues in most clinical laboratories. Therefore the UTSS region is a potentially accessible biomarker for various cancers. FUNDING The Canadian Institute of Health Research and the Terry Fox Foundation.

Radiosurgery for basal ganglia, internal capsule, and thalamus arteriovenous malformation: clinical outcome.

OBJECTIVE:Radiosurgery is accepted as the first option for treating deep arteriovenous malformations (AVMs), although the clinical outcome in this subgroup of brain AVMs is not well studied. The objective of this study is to review our experience with radiosurgical treatment for these AVMs. METHODS:Between October 1989 and December 2000, 45 patients with deep AVMs (including basal ganglia, internal capsule, and thalamus) underwent stereotactic radiosurgery. Three patients were lost to follow-up and therefore were excluded from this study. Patient characteristics and outcomes were collected and analyzed. The obliteration prediction index and the radiosurgery-based AVM score were calculated and tested. RESULTS:Forty-two patients were followed up for a median of 39 months (range, 25–90 mo; mean, 45.8 mo). The median maximum AVM diameter during the radiosurgery was 1.8 cm (range, 0.9–4.0 cm; mean, 2.07 cm), and the median AVM volume was 2.8 cm3 (range, 0.2–18.3 cm3; mean, 4.74 cm3). The mean marginal dose was 16.2 Gy (median, 15 Gy), and the median maximum dose was 22.4 Gy (range, 16.6–30 Gy). The AVM cure rate after the first radiosurgical treatment, using angiography- and magnetic resonance imaging-confirmed obliteration, was 61.9%. The predicted obliteration using the obliteration prediction index was 60%. Eight patients developed radiation-induced complications (19%). The deficit was transient in three patients (7.1%) and permanent in five patients (11.9%). The risk of postradiosurgical hemorrhage in this cohort was 9.5% for the first year, 4.7% for the second year, and 0% thereafter. Excellent outcome (obliteration plus no new deficit) was achieved in 70% of the patients in the group with radiosurgery-based AVM score less than 1.5 compared with 40.9% in the group with radiosurgery-based AVM score greater than 1.5% (P = 0.059). CONCLUSION:Radiosurgery for deep AVMs has a satisfactory obliteration rate and acceptable morbidity, considering the risk of hemorrhage without treatment and the risk of morbidity associated with other treatment modalities.

Glioblastoma, a Brief Review of History, Molecular Genetics, Animal Models and Novel Therapeutic Strategies

Glioblastoma (GBM) is the most common and lethal primary brain tumor. Over the past few years tremendous genomic and proteomic characterization along with robust animal models of GBM have provided invaluable data that show that “GBM”, although histologically indistinguishable from one another, are comprised of molecularly heterogenous diseases. In addition, robust pre-clinical models and a better understanding of the core pathways disrupted in GBM are providing a renewed optimism for novel strategies targeting these devastating tumors. Here, we summarize a brief history of the disease, our current molecular knowledge, lessons from animal models and emerging concepts of angiogenesis, invasion, and metabolism in GBM that may lend themselves to therapeutic targeting.

ANGIOGENESIS IN NERVOUS SYSTEM DISORDERS

ANGIOGENESIS IS A crucial requirement for embryonal development and new vessel formation during adult life. Various disease processes such as cancer, ischemia, vascular malformations, and inflammatory processes also depend on pathological angiogenesis. A better understanding of the complex and coordinated interactions among various angiogenic pathways involved in pathological angiogenesis is necessary to improve our therapeutic approaches to the various disease processes observed in the central nervous system. This review summarizes the current understanding of the role of principal angiogenic factors relevant to neurosurgical abnormalities.

Targeting the Tie2/Tek receptor in astrocytomas.

Tie2 is an endothelial cell-specific receptor tyrosine kinase, whose activation is positively and negatively modulated by angiopoietin-1 and angiopoietin-2, respectively. Angiopoietin-mediated modulation of Tie2 activation contributes to normal vessel development and stability, however, its role in tumor angiogenesis is not well known. We investigated the role of Tie2 activation in malignant astrocytomas, a common and highly vascularized primary human brain tumor. We found that Tie2 expression and activation increases with increasing malignancy grade of astrocytomas. Inhibition of Tie2, using a kinase-deficient Tie2 construct, decreases growth of malignant human astrocytoma subcutaneous and intracranial xenografts. Tie2 inactivation disrupted the tumor vascularity, with a decrease in microvascular density, increased presence of abnormally dilated vessels, and loss of interaction between endothelial cells and surrounding smooth muscle cells, all collectively resulting in increased tumor cell apoptosis. Overall, these findings strongly suggest that Tie2 activation contributes significantly to astrocytoma tumor angiogenesis and growth. We postulate that targeting Tie2 activation, either independently or in conjunction with other anti-angiogenic therapies, such as against vascular endothelial growth factor, is of potential clinical interest.

Study of the biodistribution of fluorescein in glioma-infiltrated mouse brain and histopathological correlation of intraoperative findings in high-grade gliomas resected under fluorescein fluorescence guidance

OBJECT Intravenous fluorescein sodium has been used during resection of high-grade gliomas to help the surgeon visualize tumor margins. Several studies have reported improved rates of gross-total resection (GTR) using high doses of fluorescein sodium under white light. The recent introduction of a fluorescein-specific camera that allows for high-quality intraoperative imaging and use of very low dose fluorescein has drawn new attention to this fluorophore. However, the ability of fluorescein to specifically stain glioma cells is not yet well understood. METHODS The authors designed an in vitro model to assess fluorescein uptake in normal human astrocytes and U251 malignant glioma cells. An in vivo experiment was also subsequently designed to study fluorescein uptake by intracranial U87 malignant glioma xenografts in male nonobese diabetic/severe combined immunodeficient mice. A genetically induced mouse glioma model was used to adjust for the possible confounding effect of an inflammatory response in the xenograft model. To assess the intraoperative application of this technology, the authors prospectively enrolled 12 patients who underwent fluorescein-guided resection of their high-grade gliomas using low-dose intravenous fluorescein and a microscope-integrated fluorescence module. Intraoperative fluorescent and nonfluorescent specimens at the tumor margins were randomly analyzed for histopathological correlation. RESULTS The in vitro and in vivo models suggest that fluorescein demarcation of glioma-invaded brain is the result of distribution of fluorescein into the extracellular space, most likely as a result of an abnormal blood-brain barrier. Glioblastoma tumor cell-specific uptake of fluorescein was not observed, and tumor cells appeared to mostly exclude fluorescein. For the 12 patients who underwent resection of their high-grade gliomas, the histopathological analysis of the resected specimens at the tumor margin confirmed the intraoperative fluorescent findings. Fluorescein fluorescence was highly specific (up to 90.9%) while its sensitivity was 82.2%. False negatives occurred due to lack of fluorescence in areas of diffuse, low-density cellular infiltration. Margins of contrast enhancement based on intraoperative MRI-guided StealthStation neuronavigation correlated well with fluorescent tumor margins. GTR of the contrast-enhancing area as guided by the fluorescent signal was achieved in 100% of cases based on postoperative MRI. CONCLUSIONS Fluorescein sodium does not appear to selectively accumulate in astrocytoma cells but in extracellular tumor cell-rich locations, suggesting that fluorescein is a marker for areas of compromised blood-brain barrier within high-grade astrocytoma. Fluorescein fluorescence appears to correlate intraoperatively with the areas of MR enhancement, thus representing a practical tool to help the surgeon achieve GTR of the enhancing tumor regions.

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.