Pathology - Theses
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ItemThe genomic landscape of phaeochromocytoma( 2015)Phaeochromocytomas (PCC) and paragangliomas (PGL) (collectively PPGL) are rare neural crest-derived tumours originating from adrenal chromaffin cells or extra-adrenal sympathetic and parasympathetic tissues. More than a third of PPGL cases are associated with heritable syndromes involving 18 or more known genes. These genes have been broadly partitioned into two groups based on pseudo-hypoxic and receptor tyrosine kinase (RTK) signalling pathways. Many of these genes can also become somatically mutated, although up to one third of sporadic cases have no known genetic driver. Furthermore, little is known of the genes that co-operate with known driver genes to initiate and drive tumourigenesis. To explore the genomic landscape of PPGL, exome sequencing, high-density SNP-array analysis, and RNA sequencing was applied to 36 PCCs and four PGL tumours. All tumours displayed a low mutation frequency in combination with frequent large segmental copy-number alterations and aneuploidy, with evidence for chromothripsis seen in a single case. Thirty-one of forty (77.5%) cases could be explained by germline or somatic mutations or structural alterations affecting known PPGL genes. Deleterious somatic mutations were also identified in known tumour-suppressor genes associated with genome maintenance and epigenetic modulation (e.g. TP53, STAG2, KMT2D). A multitude of other genes were also found mutated that are likely important for normal neuroendocrine cell function (e.g. ASCL1, NCAM1, GOLGA1). In addition, the existing paradigm for gene-expression subtyping of PPGL was further refined by applying consensus clustering to a compendium of previously published microarray data, enabling the identification of six robust gene-expression subtypes and subsequent cross-platform classification of RNA-seq data. The majority of cases in the cohort with no identifiable driver mutation were classified into a gene-expression subtype bearing similarity to MAX mutant PPGL, suggesting there are yet unknown PPGL cancer genes that can phenocopy MAX mutations. The cross-platform classification model was then further refined to develop a 46-gene Nanostring-based diagnostic tool capable of classifying PPGL tumours into gene-expression subtypes. The strong genotype-to-subtype relationship in PPGL makes subtyping a powerful tool that can be used clinically to guide and interpret genetic testing, determine surveillance programs and aid in better elucidation of PPGL biology. In applying the diagnostic assay to a test set of 38 cases, correct classification into one of the six subtypes was achieved for 34 (90%) samples based on the known genotype to gene-expression subtype association. The observation that at least one of the six subtypes is likely defined by the presence of non-neoplastic cells led to further refinement into five, four, and three-class architectures, further improving classification accuracy. Increasingly tumour heterogeneity is being recognised as one of the most significant challenges facing modern oncology. Genomically diverse tumour regions create additional complexity in predicting treatment response and metastatic potential through biopsy. Multi-region sampling of multiple synchronous primaries from patients with a predisposing germline mutation was used to explore tumour evolution and heterogeneity in PPGL and concomitant medullary thyroid carcinoma. Evolutionary reconstruction of a single primary PPGL demonstrated periods of both branched and linear evolution resulting in a high degree of intratumoural heterogeneity. Comparison of multiple synchronous primaries provided strong evidence of convergent evolution through recurrent chromosomal aberrations, indicating these may be obligate events in tumourigenesis, and as such, may indicate potential novel therapeutic targets.
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ItemInvestigating the role of hypoxia in tumour progression in breast cancer( 2015)Metastasis is a major cause of morbidity and mortality in breast cancer patients. The molecular processes and mediators that underpin this process have yet to be completely delineated. Hypoxia, the state of reduced oxygen conditions, occurs frequently in solid tumours and is a factor of poor prognosis for patient outcome. The upregulation of HIF-1α, the main mediator of the hypoxic response pathway, has been implicated in several different facets of tumour progression, including tumour growth, angiogenesis, therapy resistance and metastasis. Hypoxia has been shown to induce Epithelial-to-Mesenchymal Transition (EMT), a highly conserved developmental program that facilitates tumour cell dissemination. It is thought that EMT is co-opted by epithelial tumour cells in order to acquire a degree of plasticity, allowing them to undergo a number of genetic, biochemical and morphological changes to adopt a mesenchymal phenotype. This results in the loss of polarity, and the gain of migratory and invasive capabilities. EMT is regulated by a core cassette of transcription factors, including Snail, Slug, Twist, Zeb1 and Zeb2. Zeb1 is the most proximal transcription factor, however, how hypoxia modulates Zeb1 expression is not known. This study demonstrates that Siah, a family of E3 ubiquitin ligases and a master regulator of HIF-1α protein expression, binds to and targets Zeb1 for proteasomal degradation. Loss of Siah2 is sufficient to cause spontaneous EMT in tumour cells derived from the PyMT murine model of breast cancer. On the other hand, EMT induction led to the decrease in Siah protein expression. This work is the first to describe a post-translational mechanism of regulation of Zeb1 and further defines the relationship between hypoxia and EMT. There are, in fact, two forms of hypoxia in a growing tumour, chronic hypoxia and intermittent hypoxia. Chronic hypoxia describes the long-term limitations on oxygen diffusion caused by abnormal tumour vasculature. While intermittent hypoxia refers to the fluctuations of oxygen tension in a tumour, caused by the aberrant and temporary closing and reopening of tumour-supplying blood vessels. The consequences of these two different types of hypoxia in breast cancer have not yet been well characterised. Using the orthotopic, syngeneic PyMT murine model of breast cancer, it was found that intermittent hypoxia-treated cells gave rise to a greater number of larger lung metastases in vivo. This was facilitated by an enhanced ability for anchorage-independent growth, increased clonogenicity, the induction of a pro-tumourigenic gene expression and secretory profile, and the increase in tumour-initiating capacity through the gain of cancer stem cell properties. RNA sequencing of hypoxia-treated cells found distinct gene expression patterns between treatment groups. While, pathway analysis revealed a marked enrichment of immune-related pathways and a downregulation of DNA replication and cell cycle pathways, by both chronic and intermittent hypoxia. Interestingly, chronic hypoxia also upregulated extracellular matrix degradation pathways, in spite of the lack of an overt EMT in cells. These results unveil novel mechanisms and pathways involved in hypoxia-mediated metastasis while highlighting the extensive effects of hypoxia signalling in cancer. Taken together, this work demonstrates the complexity of hypoxia signalling in tumour progression. Not only does it endow tumour cells with an aggressive, tumour-initiating phenotype, but it also contributes to the priming of the tumour microenvironment to be pro-inflammatory and immunosuppressive and ultimately, tumour-promoting.
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ItemFunctional relationship between tetraspanin CD151 and TSSC6 with ADP purinergic receptor, P2Y12 in platelets regulates thrombus formation and stability( 2015)Background: Platelets play a critical role in maintaining normal haemostasis and thrombosis. However, they also participate actively in fatal pathological conditions. Tetraspanins are present in many cell types and are involved in a number of cellular processes. Of these, CD151 and tumor-suppressing subchromosomal transferable fragment cDNA 6 (TSSC6) are cell surface proteins that are structurally linked to this group. A range of cells and tissue types has been demonstrated to express CD151, such as vascular, immune and haematopoietic compartments, including platelets while TSSC6 expression is restricted to haematopoietic lineage. The adenosine diphosphate (ADP) purinergic receptor P2Y12 is mainly expressed by platelets and is known to be one of the most important targets for several different clinically approved oral and intravenous antithrombotic agents. All three receptors have been shown to influence the maintenance of the sustained activation of the integrin αIIbβ3 and the regulation of growing thrombi. Aim: Preliminary observations have shown that CD151 and TSSC6 act in conjunction with binding partners, including P2Y12 receptor in platelets. Therefore, the aim of this study was to investigate if close physical proximity of CD151 and TSSC6 with P2Y12 receptor translates into a functional relationship to regulate thrombus growth and stability. Method and Results: In this study, a mouse model deficient in both receptors was achieved by genetically knocking out the CD151 and TSSC6 receptors and functionally blocking P2Y12 receptor. The latter was achieved using the clinically approved treatment Clopidogrel, which specifically blocks P2Y12 receptor through frequent oral doses. Different in vitro, ex vivo, and in vivo approaches were used to examine these relationships. The data demonstrate that CD151 and TSSC6 form functional relationships with the purinergic ADP receptor, P2Y12 but not P2Y1 receptor in the regulation of ‘outside-in’ integrin αIIbβ3–mediated platelet aggregation, clot retraction and cytoskeletal reorganisation. In addition, to examine whether treating CD151 or TSSC6 (knockout) KO mice with Clopidogrel would affect the interaction between platelets and type I collagen, the interaction was tested using an in vitro arterial physiological flow condition with a wall shear rate of 1800 seconds-1. Using this model, it was observed that platelet-collagen interactions were significantly reduced further compared to CD151 or TSSC6 KO mice or wild-type (WT) mice treated with Clopidogrel. These results confirmed the functional relationship between CD151 and TSSC6 with P2Y12 receptor. This was additionally supported by in vivo experiments using a ferric chloride (FeCl3) vascular injury model of mesenteric arterioles showing that CD151 or TSSC6 KO mice treated with Clopidogrel exhibited smaller thrombi that were very unstable with a tendency to embolise in comparison to CD151 or TSSC6 KO mice treated with sham control or WT mice treated with Clopidogrel. Conclusion: This study demonstrates a functional association between the tetraspanin superfamily members, CD151 and TSSC6 with the ADP purinergic receptor P2Y12 in platelets. In addition, it shows that P2Y12 receptor blockade in CD151 or TSSC6 KO mice leads to a further ‘outside-in’ signalling defect of integrin αIIbβ3–mediated events compared to mice with CD151 or TSSC6 deficiency or P2Y12 receptor blockade alone. Further studies will examine the interaction of P2Y12 receptor with other tetraspanin superfamily members and whether similar functional relationships exist.
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ItemThe role of infection in the aetiology of prostate cancer( 2015)An infectious aetiology for prostate cancer has been conjectured for decades but the evidence gained from questionnaire-based and sero-epidemiological studies is weak and inconsistent and a causal association with any infectious agent remains to be established. The work described in this thesis questions whether the inconsistency in evidence could be related to tumour heterogeneity (high-grade or low-grade) or the nature of infections. It was decided to focus on high-grade disease, as this the most aggressive form, and on evidence of persistent infection, as transient infection was considered unlikely to play a causal role. Quantitative molecular methods were used first to seek evidence of single-organism infection comparing high-grade with lower-grade tumours. The potential of targeted 16S rRNA gene sequencing and total RNA sequencing was then evaluated regarding its utility to characterise microbial communities within high-grade tumours. Archival tissue blocks were retrieved for 52 high-grade and 76 low-grade prostate cancers. DNA samples were extracted and screened by RealTime-PCR using validated and standardised assays for the following candidate organisms C. trachomatis, U. urealyticum, U. parvum, M. genitalium, BKV, HSV-1 and 2, P. acnes types IA, IB and II, T. vaginalis and XMRV DNA. Samples were screened for 16 HPV genotypes by PCR and flow cytometry. Prevalence of M. genitalium, U. urealyticum, and HSV was low and did not differ by tumour grade. The prevalence of P. acnes type IA, IB, II was higher than for other agents, however no evidence of an association was detected. Neither BKV, XMRV, T. vaginalis, U. parvum, C. trachomatis nor HPV DNA were detected. Given the sensitivity and specificity of the methods used for the candidate organisms, the absence or low levels of detectable microbial DNA indicate a low probability that candidates contributed to cancer risk. A massively parallel sequencing (MPS) approach was used to characterise any resident microbial communities and the relative abundance of bacterial constituents within tissue. DNA and RNA were extracted from 20 snap-frozen tissue samples from high-grade prostate tumours (10 high-grade prostate cancer cores and matched unaffected prostate tissue). Prior to sequencing, broad-range PCR was applied to DNA across three hypervariable regions (V2-V4) of the 16S rRNA gene to enrich for bacterial species. As 16rRNA gene sequencing is only able to detect bacterial/archael microorganisms, MPS was also applied to cDNA from total RNA that was extracted from the same tissue samples to detect other microorganisms (viral, bacterial and protozoal origin) that may be associated with prostate cancer. Sample cDNA was sequenced and the data were queried for 16S rRNA sequences and the presence of expressed viral genes. Partial 16S rRNA sequencing identified Enterobacteriaceae species common to all samples and P. acnes in 95%. Total RNA sequencing detected endogenous retroviruses that provided proof of concept. As this part of the study was exploratory, associations between the organisms identified and prostate cancer risk could not be ascertained. Further studies, specifically designed to detect associations between the disease phenotype and aetiological agents, are required.
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ItemInvestigating breast cancer metastasis to brain in pre-clinical mouse models of metastasis( 2015)An increasing number of advanced breast cancer patients develop overt brain metastases. This is partly due to recent advances in therapies for visceral metastases that extend life of patients but remain largely ineffective against late stage brain metastases. Among the subtypes of breast cancer, triple negative breast cancer (TNBC) is particularly aggressive and has a strong propensity to metastasise to the brain. Although TNBC are initially sensitive to chemotherapy (Keam et al., 2007), response to treatment is usually limited by the development of resistance. Moreover, current endocrine or human epidermal growth factor receptor 2 (HER2)-targeting therapies are not effective against TNBC due to the absence of estrogen receptor (ER), progesterone receptor (PR) or HER2 receptor in this tumour subtype. Unfortunately, the limited availability of mouse models that closely recapitulate the entire metastatic process from the mammary gland to the brain remains a major barrier to identifying relevant prognostic/therapeutic target genes or testing novel therapies against TNBC brain metastases under clinically relevant settings. Thus, the first aim of this project was to develop a clinically relevant and robust mouse model of breast cancer brain metastasis (4T1Br4) that gives rise to a high incidence of spontaneous brain metastases in syngeneic/immune competent animals. Phenotypic, functional and transcriptomic characterisation showed that the 4T1Br4 model is phenotypically, functionally and genetically relevant to human brain-metastatic TNBC. In particular, we found that 4T1Br4 cells are highly migratory, are more adhesive to brain-derived endothelial cells and have increased ability to transmigrate endothelial cells and invade in response to brain-derived factors compared to the parental 4T1 cells from which they are derived. We identified a cell adhesion molecule, limitrin, whose high expression is associated with the increased brain metastatic abilities of 4T1Br4 tumours. Prognostic analysis of limitrin using BreastMark analysis tool revealed that limitrin is associated with metastasis and poor clinical outcome in basal-like but not other subtypes of breast cancer. In addition, we found that limitrin promotes trans-endothelial migration in vitro, a function likely to be critical for the crossing of tumour cells through the blood-brain barrier (BBB). Lastly, we tested the efficacy of novel histone deacetylase inhibitors (HDACi) namely SB939 and 1179.4b, against mouse (4T1Br4) and human (MDA-MB-231Br) brain metastatic breast cancer cell lines. SB939 and 1179.4b potently inhibited proliferation and survival of both cell lines in vitro and inhibited 4T1Br4 tumour growth and spontaneous metastasis to bone and brain in vivo. Moreover, we observed that both HDACi have radiosensitising properties against both cell lines in vitro. In summary, we developed a clinically relevant mouse model of spontaneous TNBC brain metastasis amenable to identifying therapeutic/prognostic target genes and evaluating novel therapies against this incurable disease.
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ItemMolecular profiling of ovarian cancer to guide targeted treatment( 2015)Ovarian cancer is a complex disease composed of multiple distinct molecular and clinical subtypes. The survival rate for ovarian cancer has remained largely unchanged over the past three decades, despite the rapid advancement of the knowledge of the molecular and genetic mechanisms underlying most of the subtypes of ovarian cancer. There is, therefore, an urgent need to rapidly translate this knowledge into improved clinical outcomes for patients with ovarian cancer. There have been significant clinical responses of certain types of cancer to targeted therapies that are designed to inhibit specific molecular defects that some tumours appear to be dependent upon. To assist in allocating patients with ovarian cancer to targeted therapies, two customised assays for mutation and copy number alteration detection were developed for molecular profiling. A panel of 29 genes, which are commonly mutated in ovarian cancer, and are potentially therapeutically targeted, was selected to be screened using an amplicon-based assay, designed for next generation sequencing. Seventy six ovarian cancer cases with matched formalin-fixed paraffin- embedded tumour tissue, snap-frozen tumour tissue and blood samples were used for the assay validation and estimation of the diagnostic yield. A panel of 11 commonly copy number altered genes in ovarian cancer was also selected for screening with a herein developed method for multiplex low-level copy number detection. Furthermore, a thorough assessment and optimisation of the available and developed analysis methods was performed to ensure accurate analysis and reporting of mutations and copy number alterations. Thirty five patients with advanced ovarian cancer were tested using the developed assays as part of the ALLOCATE study, with genetic changes detected in 90.9%, demonstrating a high diagnostic yield. Molecular profiling of these cases was not only useful in identification of possible targeted treatment strategies with the aim of improving clinical outcomes, but also assisted in determining the correct diagnosis. Moreover, a novel algorithm was proposed for the prediction of individual tumour response to PARP inhibitors, a promising targeted treatment in high-grade serous ovarian cancer.
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ItemIdentification of CREB as a transcriptional regulator of glioblastoma biology( 2015)Glioblastoma (GBM) is both the most common and malignant type of brain tumour with a median survival of 7.4 months from the date of diagnosis; even following current treatment regimens of resection, radiation and chemotherapy, highlighting the need for a greater understanding of the molecular mechanisms driving tumour biology. The Cyclic-AMP Response Element Binding (CREB) protein is a kinase-inducible transcription factor which sits at a hub of pro-oncogenic signalling pathways. CREB is a critical regulator of normal brain development where it regulates proliferation, differentiation and survival. The phosphorylated form of CREB (pCREB) is overexpressed in several types of cancer where it regulates various aspects of tumour biology but little is known about the role of CREB in GBM. As such, this thesis hypothesises that pCREB is an oncogene in GBM where it is activated by multiple cancer signalling pathways and is essential for the maintenance of tumour biology. To investigate the pathways responsible for CREB activation in GBM and the potential of pCREB to serve as a prognostic biomarker, a combination of immunohistochemistry (IHC) and bioinformatics analysis were undertaken. pCREB was co-expressed in cells along with either pMAPK or pAKT in GBM specimens, implicating the PI3K/AKT and MEK/MAPK pathways in differentially activating CREB. Signalling through a MAPK-CREB axis was associated with cell proliferation whilst an AKT-CREB signalling axis was implicated in invasion. pCREB was not prognostic for survival in GBM however was associated with poor prognosis when analysed in the context of MEK/MAPK signalling. Investigation of the role of CREB in vitro confirmed the role of CREB in regulating proliferation. Knockdown of CREB resulted in slower cell cycle progression and this occurred through the transcriptional regulation of the cell cycle factor Cyclin D1. In addition, CREB was selectively regulated the activity of the PI3K/AKT pathway via a feedback mechanism which in turn regulated the expression of cell cycle factors Cyclin B1 and PCNA. In contrast to the PI3K/AKT and MEK/MAPK pathways, the cAMP/PKA pathway was not associated with CREB activation in GBM. Exogenous activation of the cAMP/PKA pathway resulted in BIM dependant apoptosis in selective GBM cell lines. CREB played a pro-survival role in this context where it inhibited BIM expression and apoptosis. To investigate the link between the PI3K/AKT pathway and CREB, hyperactivation of the PI3K/AKT pathway was induced in neural stem cells via an activating mutation of PIK3CA and loss of PTEN, leading to glioma initiation in vivo. Cells with these mutations expressed high levels of pAKT as well as CREB target genes Cyclin D1 and Cyclin B1 independently of exogenous growth factors. pCREB was expressed throughout PIK3CA/PTEN tumours and knockout of CREB in PIK3CA/PTEN tumours resulted in prolonged survival, demonstrating the key role of CREB in PI3K/AKT mediated tumour biology. Collectively this study establishes CREB as a pro-oncogenic factor in GBM where it plays a pivotal role in tumour growth and survival. These results form the basis for future research into the role of CREB in GBM and highlight the potential use of CREB as a target for future therapies.
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ItemThe mechanism of action of CuII(atsm) for the treatment of amyotrophic lateral sclerosis( 2015)Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterised by the progressive loss of motor neurons in the spinal cord, motor cortex and brain stem leading to complete paralysis and death, usually within 2-3 years of diagnosis. There is currently no cure for ALS and the only approved therapeutic is riluzole. However, its clinical efficacy is marginal with an average extension in survival of 3 months. A subset of ALS cases (~10%) can be attributed to genetically inherited mutations in a number of different genes (familial ALS). Mutations in the gene for Cu,Zn superoxide dismutase (SOD1) – an antioxidant enzyme – were the first to be identified. These mutations lead to a toxic gain of function but the exact nature of this toxicity remains largely unknown. There is evidence to suggest that mutations may cause incorrect metallation of SOD1 leading to aberrant catalytic chemistry and misfolding. Over-expression of the mutant forms of the human protein in mice gives rise to a phenotype that recapitulates many of the symptoms of the human condition including progressive paralysis and premature death. The PET imaging agent, diacetyl-bis(4-methylthiosemicarbazonato)Cu(II) [CuII(atsm)] has been shown to have therapeutic potential in one of these models – SOD1G93A mice. In addition, CuII(atsm) has also been shown to be protective in multiple models of Parkinson's disease. The purpose of this thesis was to further characterise the therapeutic potential of CuII(atsm) in a second model of ALS and to determine if its therapeutic mechanism involves modulation of Cu bioavailability in disease affected tissue. CuII(atsm) was shown to have similar therapeutic potential in the SOD1G37R model as in the SOD1G93A model. Survival extension and improvement in locomotor symptoms were dependent on the dose administered with the highest dose administered proving to be the most effective. No apparent therapeutic ceiling was reached. CuII(atsm) was also co-administered with riluzole with no apparent additive or detrimental effects. When administered alone, riluzole was not as effective at attenuating symptoms and survival as CuII(atsm). Additionally, CuII(atsm) was therapeutic even when given post-onset of a locomotor deficit. Even though severity of disease symptoms in these mice is dependent on mutant SOD1 expression levels, treatment with CuII(atsm) was shown to paradoxically increase the concentration of mutant SOD1 in the spinal cord of these mice. This was due to an increase in fully metallated holo SOD1 – the stable, non-toxic form of the enzyme. The holo SOD1 pool was increased by incorporation of Cu from CuII(atsm) into the Cu-deficient, Zn-containing SOD1 pool. Several other proteins also incorporated Cu from CuII(atsm) however, not all detectable cuproproteins were targets of CuII(atsm)-mediated Cu delivery. Preliminary results suggest that the cuproprotein targets of CuII(atsm) are involved in oxidative stress, metal homeostasis and Cu delivery to SOD1, potentially inhibiting the toxic action of metal-deficient SOD1 on mitochondria. The clinical and pathological similarities between familial and sporadic ALS suggest that similar pathological processes occur in both forms of the disease. There is evidence to suggest that SOD1 can cause disease in the absence of mutations and there is ample evidence implicating mitochondrial dysfunction in sporadic ALS as well as familial ALS. CuII(atsm) is therefore a promising therapeutic for the treatment of ALS and the results presented and mechanism proposed in this thesis position CuII(atsm) as an excellent candidate for translation into human clinical trials.
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ItemUnderstanding melanoma progression and therapy response using in vivo modelling( 2015)A fundamental limitation to improving cancer outcomes is the lack of models that faithfully recapitulate the biological and molecular characteristics of human disease. In melanoma, with the emergence of immune based therapies and targeted therapies against defined oncogenic mechanisms, the development of clinically relevant models of melanoma progression and therapy response/resistance is a higher priority than ever. This thesis examines the key features of a particular type of cancer modelling called patient-derived xenografting (PDX), in which patient cancer cells (in this case melanoma cells) are injected into immunocompromised mice in order to form tumours whose biological and molecular features can be subsequently studied. Although PDX assays have some limitations, they are being increasingly used to study human cancer biology and to test the efficacy of new therapies. For example, PDX melanomas have been used to test whether hierarchical relationships exist amongst phenotypically distinct cells in patient melanomas, with surprisingly contrasting results obtained by different groups that used different PDX assays. An important implication of these studies is the possibility that differences in PDX assay methodology might result in different conclusions about underlying disease mechanisms. Chapters 3 and 4 will address this possibility by examining the effects of various modifications to PDX melanoma assays on the ability to reveal tumourigenic potential in melanoma cells, and to test theoretical models of disease progression. These studies and the work of others highlight a broader issue regarding cancer modelling and the degree to which individual approaches can be used to infer features of actual human disease, including disease-related outcomes of patients such as metastasis, survival and response/resistance to therapy. Although PDX melanoma assays may assist in the prediction of patient prognosis and therapy responses (Quintana, Piskounova et al 2012), extended testing of these possibilities is lacking. Chapter 5 will thus describe a series of direct comparisons of disease-related features of matched melanomas growing in patients and in PDX assays.
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ItemInvestigating stemness and plasticity models as sources of intra-tumoural heterogeneity in Glioblastoma multiforme( 2015-09-09)Glioblastoma mutiforme (GBM) is a heterogeneous tumour of the brain with a poor prognosis. Genome-wide profiling has revealed four molecular subtypes, yet there is no significant difference in long-term survival between subtypes. Recurrence and resistance to GBM therapy is believed to be due to an underlying Glioma Stem Cell (GSC) subpopulation. To identify gene expression differences with the ability to predict patient survival, the cancer stem cell subpopulation of Patient Derived Glioma Cells (PDGCs) was isolated based on the expression of the putative stem cell marker CD133. RNA-seq libraries were prepared from six different PDGCs with the identification of 37 differentially expressed genes. Downstream characterisation of the cellular phenotypes exhibited by CD133+ and CD133- cells indicated that CD133 does not enrich for stem and malignant phenotypes. Genes coexpressed with GSCs markers were used to build a gene signature that classified patients based on a CD133 coexpression module signature (CD133-M) or a CD44 coexpression module signature (CD44-M) subtype. CD133-M tumours were enriched for the Proneural GBM subtype and correspondingly CD44-M tumours were enriched for the Mesenchymal subtype. Gene set enrichment identified proliferative pathways as activated in CD133-M and invasion/ migration pathways as enriched in CD44-M. CD133-M patients benefitted most from radiotherapy while CD44-M classified patients responded equally well to temozolomide or radiotherapy. In different PDGCs there was a culture specific equilibrium of distinct PN and MES subpopulations, potentially due to the genetic background of the original patient. The distribution of Proneural and Mesenchymal cells in the same tumour was measured in GBM sections using the expression of Olig2 and CD44 proteins as markers. Heterogeneous expression of these markers in tumours was observed, consistent with the heterogeneity observed in cell cultures. The influence of oxygen tension and chemoradiotherapy on the intra-tumoural equilibrium of PN and MES cells in PDGCs was investigated, with hypoxia inducing a MES to PN shift and chemoradiotherapy inducing a PN to MES shift respectively. The results of this study favour a cellular plasticity model over a hierarchical cancer stem cell model and is in agreement with accumulating evidence that CD133 and CD44 expression are markers of PN and MES molecular subtypes respectively. Both PN and MES subtypes coexist in the same tumours while rare cells that transiently express both CD44 and CD133 (having PN and MES properties) may be cancer stem cells. The results of this thesis suggest the tumour specific proportion of these states is determined by a combination of genetic and environmental factors. Surveillance and modulation of intra-tumoural heterogeneity could be of benefit in the clinical management of GBM.