Surgery (RMH) - Theses
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ItemCirculating miRNAs as a novel biomarkers and intercellular regulators in glioma( 2018)Glioma is the most common intracranial malignant cancer despite the rarity. Glioblastoma (GBM, grade IV) has very dismal outcomes where fewer than 20% of patients survive beyond 5 years. Low Grade Glioma (LGG, grade II), on the other hand, frequently occurs in a younger population with a variable outcome that depends on their individual genetic alterations. A tissue-based biopsy requires an invasive operation for every glioma patient and it is the gold standard diagnosis. In monitoring the patients for recurrence, MRI has the evitable limitation that it could not differentiate pseudo-progression after irradiation treatment for glioma patients. Therefore, a non-invasive biomarker, in addition to tissue biopsy and MRI, is urgently needed to provide more precise cancer care and management with glioma patients. In fact, cancer cells including glioma cells, release microRNA (miRNA) into the microenvironment and peripheral circulation, which allows physicians and scientists the ability to detect the disease status by a blood test. In addition, microRNA (miRNA) displays accuracy in diagnosing and monitoring other cancer patients by analysis of miRNA abundance. To investigate the diagnostic capacity of circulating miRNA in glioma, a meta-analysis of previous relevant studies was performed. Circulating miRNA provided an over 90% accuracy of diagnosing glioma patients from healthy controls. A bioinformatic analysis revealed seven IDH1 mutation-associated miRNAs and these miRNA signatures were down-regulated in IDH1 mutant glioblastoma due to the hypermethylation in their promoter areas. Our proof-of-concept cohort recruited 91 glioma patients and 17 healthy controls and new circulating miRNAs were identified with remarkable diagnostic capacity. However, no circulating miRNA showed an association with IDH1 mutation in our cohort. As most circulating miRNAs were packaged and delivered by extracellular vesicles (EVs), serial cell experiments were conducted to examine the function of these miRNA-enriched EVs. Glioma Stem Cells-derived EVs significantly promoted glioma cell proliferation, cell migration and radiation resistance. This effect was largely caused by the degradation of PTEN and activation of AKT in the recipient cells through miRNAs. Collectively, these works established circulating miRNA as a clinical biomarker where it provides a complementary tool for glioma management. These results also formed the basis of future research into the role of EVs in glioma and highlighted the potential therapeutic target of EVs to glioma.
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ItemThe role of quiescence in treatment resistance and malignancy in glioblastoma multiforme( 2018)Glioblastoma multiforme (GBM) represents the most malignant incarnation of glial tumours – a World Health Organisation (WHO) grade IV brain malignancy. GBM is the most common primary brain tumour in adults, accounting for 78% of all malignant central nervous system (CNS) tumours, and affecting 2-3 people per 100,000 in Europe and North America, with an average survival of only 14.6 months. Despite continued research and incremental advances in imaging, surgery, and chemoradiotherapy, patient survival has stagnated in the past decade, with several promising lines of investigation failing to fully deliver on their anticipated translational outcomes. Recent advances in genetic sequencing and computational biology have allowed the simultaneous comparison of large numbers of patient cancer cell genomes and identified several GBM subtypes. It is hoped that such stratification will one day allow clinicians to tailor treatments specific to each GBM subtype as has already happened in cancers like medulloblastoma. However, despite best efforts, GBM remains highly aggressive, infiltrative, and treatment-resistant, rendering it incurable by current treatment modalities. Invasion of tumour cells into normal brain prohibits a surgical cure, while a high cancer stem cell (CSC) component resists treatment with radiation and temozolomide (TMZ) – both of which are more effective against rapidly dividing cells – and relapse remains the rule. Molecular mechanisms underlie GBM’s treatment resistance, and elucidating the key drivers that garner inherent resistance to the quiescent, stem-like fraction of cells that lead to treatment failure therefore presents as an exciting area of research that may uncover new potential drug targets that improve patient outcomes. This study has shown that the proliferation rate of GBM cells is spectral, approximating a positively skewed normal distribution, with highly proliferative cells at one end and quiescent cells at the other. The quiescent cell fraction was subsequently shown to be inherently more resistant to chemoradiotherapy than the proliferative fraction. The quiescent fraction also displayed increased size, complexity, rates of migration and invasion, secretion of extracellular matrix-degrading enzymes, and invadopodia activity than their proliferative counterparts. Similarly, quiescent cells proliferated slower as intracranial tumours but displayed significantly greater invasive properties than a subset of proliferative cells grown in vivo. mRNA expression analysis revealed the genetic signature that underpins the disparity in proliferation rate between quiescent and proliferative cells, and the putative genes that are responsible for the malignant properties identified in both populations. This body of work has uncovered the inherently dichotomous treatment response of quiescent and rapidly dividing GBM cells, as well as the difference in their abilities to migrate and invade. This study has also shed light on the fundamental molecular mechanisms that are at the root of treatment resistance and malignancy in this disease. It is hoped that this expression signature will help to inform future studies and treatments that target these differences and make GBM less of a death sentence and more of manageable, chronic disease.
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ItemBiomarkers of tumour vascularity in high grade glioma( 2016)Australia has one of the highest cancer incidences in the world, and while brain cancer is a relatively rare diagnosis, its impact on the Australian healthcare system is significant. Despite being responsible for only 1.4% of all cancer diagnoses, brain cancer has an overall impact of disease as measured by DALYs similar to much more prevalent malignancies such as melanoma and leukaemia. A young median age of diagnosis and poor 5-year survival both contribute to this. No other cancer group is responsible for a greater premature loss of life, with an average of 12 years lost per person. High grade gliomas, particularly glioblastoma multiforme, are by far the most common brain cancers, and remains incurable and highly malignant. Glioblastoma multiforme is one of the most vascular cancers found in humans, and the degree of this vascularity (as seen histologically) is known to correlate with brain tumour prognosis. More recently there has been renewed interest in tumour vascularity, with the emergence of the anti-angiogenic therapy as the newest modality of cancer treatment. Some of these agents are currently under trial in brain cancer patients in Australia. Despite its prognostic value and its potential utility perhaps as a surrogate endpoint in anti-angiogenic therapy, histopathological assessment of tumour vascularity has never been routinely used in clinical practice. This is due to its invasive nature of procurement, as well as documented concerns regarding sampling errors and inter-observer variability. These concerns would be potentially negated by the identification of appropriate biomarker of tumour vascularity. This thesis investigated candidate biomarkers of tumour vascularity in an effort to identify clinically useful tools to help in the management of high grade glioma patients. Biomarkers from each marker category were represented – molecular (serum vascular endothelial growth factor), cellular (circulating endothelial cells and their progenitors), and functional (perfusion magnetic resonance imaging).
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ItemGlioma stem cells: novel signalling pathways and potential therapeutic advances( 2013)High grade glioma continues to portend a dismal prognosis for patients, despite ongoing, albeit incremental, advances in surgery, irradiation and chemotherapy. This has necessitated an overhaul in the understanding of disease pathogenesis, and the search for a superior model for screening of small molecule therapeutic agents, especially in light of the poor translational outcomes generated from work on traditional, serum-fed, glioma cell lines. The cancer stem cell hypothesis has revolutionised neuro-oncological research, and the discovery of the glioma stem cell (and in particular, its characteristic in vitro gliomasphere-producing qualities) has led to new avenues of thinking, in terms of glioma proliferation, invasiveness and mechanisms of chemo- and radioresistance. Coupled with this has been further characterisation of the genetic constitution of high grade gliomas, which show Pi3k-related aberrations in up to 90% of such lesions, related mainly to EGFR amplification, EGFRvIII expression and PTEN mutation. This study has led to the production of sixteen gliomasphere-producing (or glioma stem cell) clones, from the collection of sixty-six individual specimens, which included lower grade lesions. It was confirmed that this set of stem cell lines exhibited all of the trademark characteristics of a tumour stem cell, characterised by a potential for self-renewal, pluripotency, proliferation, and tumour formation upon xenograft transplantation. Western blot analysis confirmed that thirteen of the sixteen reproducible stem cell clones demonstrated hyperphosphorylated Akt. This led to an investigation of suitable Pi3k inhibitors via a series of in vitro lactate dehydrogenase cytotoxicity assays, which showed that the N10-substituted phenoxazine, Akt inhibitor Akt X, was the only agent which demonstrated effective in vitro cytotoxic potential against glioma stem cells. Interestingly, Akt X was shown to be more efficacious against those glioma stem cell clones which failed to express PTEN protein on Western blot analysis, and this result was found to be statistically significant. Further in vitro LDH cytotoxicity assays revealed glioma stem cells were largely resistant to conventional irradiation and temozolomide, but this was reversed by Akt X, confirming this agent’s potential as a successful stem cell-specific agent in the clinical management of high grade glioma patients. Extensive exome sequencing analysis confirmed the presence of a number of genetic variants in the glioma stem cell clones, whose putative effect and expression concerns signalling pathways not commonly reported in conjunction with gliomagenesis in the past, including the WNT/-catenin and SWH pathways. Western blot analysis and immunohistochemistry demonstrated that the glioma stem cell clones overexpress YAP, a protein whose activation is normally suppressed via the intact SWH pathway, identifying it as a prospective, glioma stem cell-specific oncoprotein, and target for future development in pharmacotherapeutics.
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ItemRadiological and molecular factors associated with seizures in patients with supratentorial gliomas( 2013)Tumour associated epilepsy (TAE) is a common and disabling symptom experienced by patients with supratentorial gliomas. The pathogenesis of TAE is likely to involve a complex interplay between macroscopic anatomical factors, molecular factors and individual patient factors. The overarching hypothesis of this thesis is that patients with TAE have tumours with different radiological, molecular and genetic features compared to those without TAE. Furthermore, identification of these features may allow the identification of patients who are at increased risk of the development of TAE, and provide tailored, individualized treatment of these patients. An improved understanding of the genetic and molecular features associated with TAE may also inform the development of novel therapeutic strategies for these patients. There is an increasing body of evidence implicating glutamate, the most abundant neurotransmitter in the mammalian central nervous system, in the pathogenesis of TAE. Magnetic resonance spectroscopy (MRS) provides a non-invasive method to quantify brain metabolites in-vivo, but has not yet been validated for glutamate. In this thesis we firstly demonstrate that MRS quantification of glutamate has a modest, but statistically significant, correlation with concentrations of glutamate measured from tumour biopsy specimens. We then demonstrate that these MRS measurements of glutamate are useful in predicting not only glioma grade, but also the incidence of TAE. We also demonstrate that patients with low-grade gliomas and TAE are more likely to have larger tumours than patients without TAE. The opposite is found in patients with high-grade gliomas and TAE. There is also evidence that common pathological processes, including glutamate excitotoxicity, may be involved in TAE and neurodegenerative conditions such as Alzheimer’s disease, the sequelae of traumatic head injury and idiopathic epilepsy. A number of molecular factors associated with neurodegeneration and excitotoxicity are therefore investigated for their association with TAE. Phosphorylation of tau was found to be lower in patients with TAE compared to those without TAE. Over-expression of glycogen synthase kinase (GSK3β) was found to correlate with TAE, as was loss of glutamic acid decarboxylase 67 (GAD67), potentially reflecting a selective loss of inhibitory interneurons. Finally, the expression of the IDH1-R132H mutation, the most common mutation in low-grade gliomas, is shown to correlate with TAE. In conclusion, TAE results from a complex interaction of patient, environmental and tumour factors, including glutamate excitotoxicity, selective loss of inhibitory interneurons and IDH1-R132H expression. Not only do the results presented in this thesis suggest that TAE differs from other epileptic syndromes, but also that the mechanisms may differ between low and high-grade gliomas. Collaboration between neurosurgeons, neurologists, radiologists, pathologists and basic scientists will be essential for further investigation of this debilitating disease.
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ItemNeurobiological factors predisposing to tumour associated seizures( 2010)Tumour associated seizures (TAS) are a common, disabling co-morbidity of gliomas. The pathogenesis of TAS and its associated implications for survival remain poorly understood. Elevations in glutamate levels, due to alterations in expression of membrane transporters, have been reported in gliomas in vitro. As glutamate is highly epileptogenic and cytotoxic we examined whether elevations of glutamate levels and alterations in transporters were specifically associated with TAS and survival. Our comprehensive study of two large cohorts of patients (290 in total) is the first human evidence showing associations between preoperative seizure risk and clinicopathological and molecular factors in patients with glioma. We found the clinicopathological factors that were independent predictors for preoperative TAS to be earlier age at diagnosis, temporal lobe location of tumour and tumour types of anaplastic astrocytoma or those with oligodendroglial components. A strong predictor for postoperative TAS was preoperative TAS adding credence to the philosophy “seizures beget seizures”. We found of the molecular factors, raised glutamate levels in tumour and peritumoural tissue as well as altered expression of astrocytic glutamate transporters EAAT2 and xCT all to be significantly associated with preoperative TAS. We also found neuronal HCN channelopathies, particularly downregulation of peritumoural HCN2 expression, to be associated with seizures due to glioma. This is further support for the concept of peritumoural tissue holding the key to TAS pathogenesis and we suggest it should be a subject for further investigation. These findings strongly support the “glutamate hypothesis” which proposes that glioma, due to dysfunction of the glial glutamate transporters among other factors, produces abnormally high levels of glutamate which then leads to excitotoxic changes in surrounding neuronal networks that ultimately manifest as seizures. Looking for further insights into the prognosis of glioma, we have also assessed the effects of preoperative seizures and raised glutamate levels on survival. This has suggested a positive influence. Postoperative seizures on the other hand, were associated with worse survival. Through a pilot study, we also explored the feasibility of using MRS for glutamate as a non invasive clinical diagnostic and monitoring tool in the management of TAS. We did not find any statistical significance of spectroscopically derived glutamate levels with TAS but the study did highlight some improvements in design required for a larger validation study in the future. This is still a promising prospect and could lead the way in the non invasive workup of people with TAS. The importance of the study of epilepsy and tumours hinges on the therapeutic and diagnostic contributions that are possible. Understanding the underlying mechanism of TAS, particularly the significance of the peritumoural tissue in this process, could revolutionize current drug treatments as well as redefine surgical objectives for the patient with TAS.