Pathology - Theses
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ItemActivated platelets and antibody opsonization as tumor markers: novel avenues for cancer diagnosis, targeted therapy and monitoring therapeutic outcomes( 2018)With over 100 types of cancer known, the study presented in this thesis introduces novel means of targeting a wide range of cancers, rather than a specific cancer antigen. Here, unique cancer targets, such as components of the tumor microenvironment and monoclonal antibody opsonization are investigated as new approaches for cancer diagnosis and targeted therapy. The first part of the study investigates the possibility of targeting activated platelets in the tumor microenvironment as a novel cancer diagnostic target. I showed the feasibility of using a single-chain antibody, which targets the activated form of GPIIb/IIIa, the most abundant platelet-specific receptor on the platelet surface as a possible tool for cancer diagnosis using PET/CT, fluorescence imaging and ultrasound. The second part of the investigation aims to further expand the utility of the single-chain antibody as an antibody-drug conjugate for cancer therapy. Using a mouse metastasis model of triple negative breast cancer, I showed that the activated platelet targeting single-chain antibody, conjugated to Auristatin E, a clinically available chemotherapy agent, was successful in reducing tumor growth and preventing metastasis development. The final part of the thesis describes the development of an FcγRIIIa receptor dimer, which has been engineered to selectively bind avidly to multimeric Fc complexes. This binding mimics the engagement of FcγRIIIa on effector cells, such as on NK cells, with antibody-coated cells that leads to antibody-mediated target killing. Here, using a mouse xenograft model of B cell lymphoma treated with Rituximab and triple negative breast cancer adenocarcinoma, treated with an EGFR receptor antibody, I show that the FcγRIIIa receptor dimer, labeled with a near-infrared contrast agent, could be used to specifically image antibody opsonization of tumor cells in vivo.
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ItemDissecting the role of laminin and Stat3 in colorectal cancer( 2018)Colorectal cancer (CRC) represents the 3rd most common causes of cancer-related death in the world, with increasing number of cases diagnosed every year in Australia. CRC is known to be associated with chronic inflammation, which induces responses in the tumour microenvironment (TME) including remodelling of the extracellular matrix (ECM) and aberrant activation of the key signalling effector Stat3. ECM remodelling is a crucial cellular event during tumour invasion and an essential step towards metastatic progression and/or tumour recurrence metastatic sites. Laminins are among the key ECM proteins that have been shown to regulate tumour invasion, and this study examined whether this could involve a role for laminins in the regulation of cancer stem cells (CSCs). CSCs represent a rare tumour cell subpopulation with enhanced self-renewal properties and a demonstrated role in tumour initiation and progression and therapy resistance. Here, we characterized the role of laminin-521 (LN-521), one of the laminin isoforms with a known function in maintaining normal stem cells, in the regulation of colorectal CSCs in vitro. Our results show that LN-521 promotes CSC self-renewal and invasion abilities and activates downstream signalling including Stat3. We identify the integrin isoforms mediating this effect, and provide results to suggest that LN-521 may contribute to poor survival of CRC patients, supporting its potential use of LN-521 as a prognostic marker for CRC. The impact of aberrant activation of Stat3 on primary tumour initiation and growth is well-described, but the consequences of it on the behaviour of metastatic CRC remain poorly understood. We therefore investigated the functions of Stat3 in metastatic tumours and their TME. This was performed by silencing Stat3 within tumour cells and/or within mice recipients in an orthotopic mouse allograft model of liver metastasis, the most prevalent type of CRC metastasis in patients. Our data indicate that Stat3 knockdown correlates with prolonged survival, reduced metastatic size, and decreased expression of genes implicated in tumour progression, which supports Stat3 being a potential therapeutic target for treating metastatic CRC.
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ItemThe role of next generation sequencing in the management of haematological malignancies( 2018)Next generation sequencing comprises a rapidly-evolving cohort of technologies which enable detection of genetic variants present in DNA or RNA. In the context of haematological malignancies, such variants may have diagnostic, prognostic or therapeutic relevance. The scope of diagnostic-grade genetic testing is increasing as the underlying molecular landscape of haematological malignancies is increasingly well-characterised and becomes further embedded in clinical management. Multiple conventional methods for the detection of somatic single-gene variants are already embedded in standard-of-care. The technical performance of next generation sequencing when compared to these existing methods is of interest, since this determines in which instances it can be used to supplement or replace the status quo. This work focuses on acute myeloid leukaemia and systemic mastocytosis, since detecting variants in these malignancies presents several technical challenges. Acute myeloid leukaemia demands detection of a broad spectrum of molecular lesions, whereas systemic mastocytosis requires high sensitivity testing. Chapter one reviews molecular testing for haematological malignancies, including aspects of next generation-based testing relevant to diagnostics. Molecular markers currently employed in the diagnosis of acute myeloid leukaemia and systemic mastocytosis are discussed. The second chapter details methods used in the generation of data for chapters three to five. In the third chapter the selection and use of a targeted sequencing panel is discussed, testing performance is compared to conventional molecular methods using data generated from 30 clinical samples. Limitations of panel-based testing are discussed, including sensitivity, detection of specific variant types, and relevant bioinformatic and analytical issues. In chapters four and five, proof of principle is demonstrated for two novel next generation sequencing library generation methods, addressing the issues of structural variant detection ‘HEPTAD’ and high sensitivity single nucleotide variant detection ‘LNA PCRbrary’, respectively. Theory, optimisation and testing results for a combination of 49 clinical samples and cell lines using HEPTAD are discussed. The technique is applied to detect chromosomal translocations involving RUNX1, BCR, ABL, RARA, MLL/KMT2A, a chromosomal inversion involving CFBF, and KMT2A partial tandem duplications. The method detects structural variants in diagnostic samples with 100% sensitivity but requires further optimisation for minimal residual disease testing. Successful prospective application in a clinical scenario where orthogonal FISH testing was performed is detailed. LNA PCRbrary’s application to the detection of KIT D816V single nucleotide variants in peripheral blood is discussed. Suboptimal results when testing RNA derived from clinical samples are detailed, but promising results when testing cell line DNA, and significant potential for further optimisation and testing. In conclusion this work shows that NGS does have a role in the management of haematological malignancies, but technical challenges remain, including high sensitivity detection of single nucleotide variants, minimal residual disease monitoring of structural variants and gene targets which are difficult to amplify. Some of these issues are in part addressed in this work, but careful validation of testing sensitivity and specificity is required when introducing such NGS assays into clinical diagnostics.
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ItemInvestigation of clustered hypermutation in cutaneous melanoma( 2017)Cutaneous melanoma is a malignant neoplasm that arises from melanocytes in the skin, and is a significant cause of morbidity and mortality, especially in Australia. Next generation sequencing studies of cutaneous melanoma have confirmed the role of UV mutagenesis in its aetiology and demonstrated the high somatic mutation burden in this disease. To date, the majority of genomic analyses of cutaneous melanoma have limited their evaluation of somatic mutations to protein coding regions in order to detect cancer driver mutations. The recent discovery of driver mutations within the TERT promoter however has shown that biologically significant somatic events occur within non-coding regions of the melanoma genome. This thesis set out to further explore the presence, timing and biology of promoter-based somatic mutations in cutaneous melanoma. In this thesis, a systematic analysis of mutations in cutaneous melanoma demonstrated enrichment of somatic mutations within active promoters. Furthermore, somatic mutations were located within sequence motifs matching binding sites for ETS and Sp1 transcription factors. The number of promoter mutations within a tumour correlated with clinicopathological markers of chronic UV exposure and overall somatic mutation load. Strikingly, the same promoter mutations were detected in other cutaneous malignancies. In order to evaluate the potential of promoter mutations as biomarkers, a highly sensitive assay was developed to detect TERT promoter mutations, the most prevalent and biologically plausible promoter mutation. Using this assay on a series of benign melanocytic naevi identified TERT promoter mutations in 2/17 samples, providing evidence that these driver mutations occur in benign neoplasms with implications for the interpretation of TERT mutations in borderline clinical samples. Microdissection of melanomas with coexistent radial and vertical growth phases demonstrated that a majority of promoter mutations are early events in melanomagenesis. TERT promoter mutations are shown to sometimes occur late in melanoma development and show coexistent subclones. In addition, somatic promoter mutations were detected in non-neoplastic skin adjacent to melanoma. Finally, a somatic mutation load assay was designed and evaluated on clinical melanoma samples. An analysis of publically available melanoma exome data demonstrated a subgroup of highly mutated BRAF/NRAS wildtype cutaneous melanomas. The assay was tested on clinical samples and was shown to be incapable of accurate determination of mutation load in melanoma due to technical limitations. In conclusion, the results of this thesis demonstrate a novel somatic mutational process at work in cutaneous melanoma represented by hypermutation within specific promoters, and evaluates these, along with the previously described TERT promoter mutation, as potential biomarkers. In so doing, these findings contribute to understanding of the biology, aetiology and role of somatic promoter mutations, critical knowledge for their potential future use in clinical assays.
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ItemInvestigating pathogenic mechanisms associated with prion protein and α-synuclein misfolding( 2017)Neuronal loss and the aggregation of misfolded prion protein (PrPSc) and α-synuclein (αsyn) in the central nervous system are hallmarks of prion and synucleinopathy disorders (such as Parkinson’s disease, multiple system atrophy, dementia with Lewy body), respectively. PrPSc is unusual because it is the major component of prions; ‘proteinacious infectious particles’ that are the causative agent in prion disease. Many features of prion disease have been directly attributed to PrPSc including its ability to propagate, causing disease in susceptible animals and be transmissible. Another feature of PrPSc is strain variability; where the conformation of the misfolded species ascribes the clinical profile of disease that develops. Neurotoxicity is also intimately associated with the generation of PrPSc, however, the precise mechanisms underlying its development in prion disease are not resolved. Mounting evidence suggests that αsyn shares similar pathogenic mechanisms to PrP in terms of its ability to misfold, propagate and cause disease in susceptible animals. Like PrP, the mechanisms associated with the toxicity of misfolded αsyn are not well defined. This thesis studied the pathogenic mechanisms of PrP and αsyn misfolding with particular interest in how these proteins cause neurotoxicity through the development of in vitro and ex vivo systems that model pathogenic aspects of their respective disorders. A potential neurotoxic mechanism of misfolded αsyn was found using the protein misfolding cyclic amplification (PMCA) assay to produce a heterogeneous population of misfolded species and their pathogenicity examined in cultured neuronal cells. Misfolded αsyn was found to bind to the lipid cardiolipin that is virtually exclusively expressed in mitochondria. Extensive analysis revealed misfolded αsyn causes hyperactive respiration in the mitochondria of live cells without causing any functional deficit. This robust data gives strong support for the mitochondrion as a target for misfolded αsyn and reveals a potential up-stream pathogenic pathway of neuronal cell loss in Parkinson’s disease. Next, a model of prion disease was established using organotypic brain slice cultures. Upon infection with a mouse-adapted human prion strain, brain slices were shown to propagate protease resistant PrP and develop neurotoxicity. The relevance of protein misfolding in these systems was assessed using the small molecule Anle138b; which has been previously reported to alter misfolding of PrP and αsyn, and be efficacious to prion infected animals and animal models of PD. This molecule was found to modulate the misfolding of both proteins, which in the case of PrP, led to a rescue of prion-induced neuronal loss in organotypic brain slice cultures. Collectively this work robustly shows misfolded αsyn and PrP can cause substantial alterations to neurons they are exposed to. Findings from this work contribute to our general understanding on these proteins in disease and highlight both similarities and differences in their misfolding and pathogenicity.
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ItemDefining the function of Amyloid Precursor Protein dimerisation in neuritogenesis( 2017)Amyloid Precursor Protein (APP) is known to be primarily involved with Alzheimer’s disease; however APP is also involved with neurogenesis, synaptic plasticity and neuroprotection. Many factors can bind to APP to affect its function and processing. APP can also bind to other membrane bound APP, known as APP dimerisation. We hypothesise that dimerisation of APP can affect the biological actions of APP. This study aims to determine the effect APP dimerisation has on neurite outgrowth and elucidate its mechanism of action. We found that APP dimerisation can reduce neurite outgrowth by modulating extracellular and intracellular signals regulating neurite outgrowth. We determined the effect APP dimerisation has on APP neurite outgrowth by using APP dimerisation mutants, G33I and L17C (these cause decreased and increased APP dimerisation, respectively), transfected into SH-SY5Y cells. To determine if APP dimerisation utilises extracellular signalling to modulate neurite outgrowth, condition media treatment of APP dimerisation mutants was used to determine if a secreted factor was responsible for modulating neurite outgrowth. Intracellular mechanisms including APP localisation, RhoA activity and miRNA expression were investigated. The localisation of APP can affect its function therefore; immunofluorescent imaging was used to determine its localisation. RhoA GTPase is known to negatively regulate neurite outgrowth, therefore RhoA activity was determined using an ELISA based assay that was specific for activated RhoA. The Ion Torrent Next Generation Sequencing system was also used to determine the differential expression of miRNA in the APP dimerisation mutants and these were confirmed by qRT-PCR. APP-L17C mutants inhibit neurite outgrowth by inhibiting the secretion of a neurite outgrowth promoting factor as APPwt condition media treatment rescues neurite outgrowth. APP dimerisation also caused perinuclear APP aggregates which colocalised within the endoplasmic reticulum. RhoA activity is increased in the APP-L17C mutant, and treatment with condition media decreases RhoA activity which correlated with neurite outgrowth. APP dimerisation also affects several miRNA expressions. The miR-125a, miR-135b, miR-34a species, which have a known role to play in the regulation of neuritogenesis, were down regulated and transduction of an miR-34a restored neurite outgrowth back to wild type levels. In conclusion, APP dimerisation reduces neurite outgrowth and can mediate its effects by inhibiting a secreted factor, modulating APP localisation, increasing RhoA activity and modulating the expression of certain miRNAs involved in neurite outgrowth.
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ItemNetwork approaches to understanding biomarker biology( 2017)Common non-communicable diseases such as cardiovascular diseases, chronic respiratory diseases, and diabetes are the leading causes of premature mortality and ill-health worldwide. These are complex diseases that take multiple decades to manifest with myriad genetic, lifestyle, and environmental risk factors. The last decade has seen rapid technological advancements and falling costs to high-throughput omic profiling platforms enabling large-scale studies into the molecular differences between healthy individuals and those that will later develop disease. Epidemiological studies of omic data in population cohorts have identified many new biomarkers of future disease risk and mortality. The end-goal of biomarker research is not only to identify individuals at increased risk of disease, but also to find ways to intervene to reduce that risk. Identification and characterisation of the biological processes these biomarkers participate in or reflect is a fundamental step in the process of clinical translation. This thesis explores the use of network-based approaches for identification and characterisation of aberrant biological processes associated with biomarkers using population cohort multi-omic data. A scalable and efficient method for robust statistical assessment of network module preservation and reproducibility is developed: NetRep. Multi-omic data from three large population biobanks are analysed to identify and characterise biological processes associated with elevated GlycA levels; a heterogeneous NMR signal that has been of recent interest as a biomarker for long-term risk of cardiovascular disease, type II diabetes, and premature mortality. I found that elevated GlycA levels corresponded with the presence of sub-clinical inflammation and increased coordinated expression of a reproducible gene coexpression network module indicative of neutrophil activity. Accordingly, analysis of a population cohort with linked electronic health records showed that elevated GlycA levels had long-term consequences for increased risk of severe infections up to 14-years in the future. To fine-map the GlycA biomarker, I developed accurate imputation models for predicting concentrations of three of the five glycoproteins contributing to the GlycA signal from population-based serum NMR data: alpha-1-acid glycoprotein (AGP), alpha-1 antitrypsin (A1AT), and haptoglobin (HP). Imputation of these three glycoproteins in two large population cohorts with linked electronic health records revealed elevated A1AT had the most severe long-term ramifications for future disease and mortality risk over an 8-year follow-up period. In total, this thesis shows the utility of leveraging population-based omic data for elucidating biomarker biology and provides a useful framework to guide future studies of new and established biomarkers for future disease risk.
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ItemApplication of bayesian networks to a longitudinal asthma study( 2016)Asthma is a highly prevalent and often serious condition causing significant illness and sometimes death. It typically consumes between 1-3% of the medical budget in most countries and imposes a disease burden on society comparable to schizophrenia or cirrhosis of the liver. Its causes are as yet unknown but a significant number of risk factors, covering such diverse factors as viral infections during infancy, blood antibody titres, mode of birth and number of siblings have been identified. In recent years there has been increasing recognition of the role played by the microbiome in human health, with a growing understanding that our relationship with the microbes that colonise the different parts of the human body is symbiotic. Disruptions in the microbiome have been implicated in diseases such as obesity, autism and auto-immune diseases, as well as asthma. At the same time there has been an increasing awareness in asthma research that its multi-faceted and multi-factorial nature requires more sophistication than statistical association and regression. In this spirit we employ Bayesian networks, whose properties render them suitable for representing time-direct or even causal relationships, to gain insight into the nature of asthma. We begin with an example of the simplest Bayesian networks, a linear classifier, with which we predict outcomes in the fifth year-of-life according to the statistical distribution of variables from the first two years-of-life. (The qualification linear refers to the neglect of correlation and interaction among the predictive variables.)While classifiers have long been used for prognosis and diagnosis, we use them to identify useful asthma subtypes, called endotypes. Different endotypes often require different treatments and management programs, and driven by different biological factors. These different factors provide different predictors, and a predictor which separates one endotype from the healthy may not do so for a different endotype. We use this to mathematically construct an indicator of when a given predictor is exclusively predictive of a given endotype. Our so-called “exclusivity index” is quantitatively precise, unlike a significance threshold. The Cohort Asthma Study, whose longitudinal data we analyse, includes the relative abundances of genera present in the nasopharyngeal microbiome. In an apparent diversion, we use qq-plots to indicate relationships between the infant microbiome and fifth-year wheeze- and atopy- status. Interestingly, the relative abundance of Streptococcus under certain circumstances was found to be highly predictive of one of the endotypes we identified in the preceding chapter. Finally, we address the problem of mapping out the complicated interactions among multiple variables. Our model is an adaption of a package originally designed for inferring gene-interaction networks, called ARTIVA. This was a non-trivial matter requiring us to augment the discrete data values in order to make them compatible with the underlying mathematics of ARTIVA’s algorithm. With questions from the asthma literature and the posterior probabilities output by ARTIVA, we were guided to networks of the interactions between atopy, wheeze and infection, and could see the difference in the development of immunity-related variables between those who went on to exhibit wheeze in the fifth year-of-life and those who did not. Our model yielded networks indicating that sensitivity to viral infection is an effect and not a cause of atop and wheeze.
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ItemNorepinephrine transporter gene silencing by MeCP2 in postural tachycardia syndrome( 2016)The Postural Tachycardia Syndrome (POTS) is characterized by the clinical symptoms of orthostatic intolerance, light-headedness, tachycardia and syncope or near syncope with assumption of upright posture. While the aetiology remains largely unknown, faulty neuronal reuptake of the sympathetic nervous system signaling neurotransmitter has been implicated. The action of norepinephrine (NE) is terminated, in part, by its uptake into presynaptic noradrenergic neurons by the plasma-membrane NE transporter (NET) which is encoded by the SLC6A2 gene. The effectiveness of NE reuptake relies on the capacity of NET to tightly recapture NE released by sympathetic nerves, this being approximately 90% for the heart. Since inter-individual variation in NET function is likely to be common and, in light of the clinical importance of the transporter’s central role in NE regulation, rather than querying genetic variation, we investigated gene-environment interactions to determine which biological processes may be prime targets for SLC6A2 regulation. Experimental evidence from our group has previously shown chromatin-modifying events associated with SLC6A2 repression may augment epigenetic regulation in POTS. SLC6A2 repression is associated with substitution of active histone modifications with repressive modifications and binding of Methyl CpG binding protein-2 (MeCP2) co-repressor complex. MeCP2 is commonly assumed to function mainly as a silencing factor at methylated DNA sequences. Interestingly, DNA methylation is unremarkable between the case and control cohort. In context to these findings and given that non-coding RNAs (ncRNAs) interactions can induce structural changes to chromatin to regulate transcription, we hypothesized that MeCP2 binding is dependent on its interaction with ncRNAs. The purpose of this study was to investigate SLC6A2 silencing with the specific aim to understand epigenomic regulation as a mean to reactivate expression. We developed a novel RICh-seq (RNA of Isolated Chromatin combined with sequencing) method to identify SLC6A2 promoter bound RNAs. Analysis of RICh-seq data identified let-7i associated with SLC6A2 promoter with elevated expression in POTS patients. We show that let-7i miRNA interacts with MeCP2 co-repressor to silence SLC6A2 activity in POTS subjects. Furthermore, we demonstrate that in POTS subject’s histone modifying drugs such as histone deacetylase inhibitors and inhibitor for EZH2 (Enhancer of zeste homologue 2) restores epigenetic modifications associated with SLC6A2 gene expression. Our results represent the first pre-clinical description for the gain of NET function in POTS and a previously unknown target of pharmacological therapy.
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ItemCuproenzyme dysfunction in the pathogenesis of amyotrophic lateral sclerosis and multiple sclerosis( 2016)Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterised by the selective death of motor neurons within the spinal cord and brain. Although the aetiology of the disease is not well understood, inherited genetic mutations account for a small proportion of cases, with Cu,Zn-superoxide dismutase (SOD1) mutations being the most extensively studied. Effective treatment options for ALS do not exist, however, pre-clinical outcomes indicate that therapeutically modulating copper bioavailability in the central nervous system (CNS) may be a feasible treatment strategy for ALS. Therefore, the initial objective of this study was to investigate the significance of copper dyshomeostasis in the progression of a mutant SOD1 mouse model of ALS. We hypothesised that age-related changes to cuproenzymes progress with disease symptoms in SOD1G37R mice compared to age-matched non-transgenic littermates and mice overexpressing wild-type human SOD1. To test this hypothesis, locomotor performance was assessed to track disease progression, then CNS and peripheral tissues were collected at distinct stages of disease for biochemical analyses. Data presented in Chapter 3 provide evidence for copper malfunction in the CNS of ALS mice and indicate that copper malfunction is an early feature of the disease which worsens as symptoms progress. Specifically, a disconnect exists between the abundance and copper-dependent activity of cuproenzymes SOD1 and ceruloplasmin. Next, the therapeutic significance of these changes to SOD1 and ceruloplasmin were assessed. In Chapter 4, data show that overexpressing CTR1 or treating ALS model mice with the copper compound CuII(atsm) extends survival and improves copper bioavailability to SOD1 and ceruloplasmin in the CNS. To ascertain the relevance of outcomes in a broader disease context, we next assessed human cases of sporadic ALS. Data presented in Chapter 5 show that SOD1 and ceruloplasmin dysfunction detected in mice is also evident in sporadic ALS. Significantly, changes to ceruloplasmin are associated with changes to iron homeostasis, where diminished copper- dependent ceruloplasmin activity may contribute to iron overload in the ALS-affected motor cortex and decreased transferrin bound iron in the cerebrospinal fluid. As such, we propose that changes to copper-dependent ceruloplasmin activity in ALS may be the mechanistic basis for two ALS biomarkers and represent the first biochemical evidence for the feasibility of treating ALS, including sporadic ALS, by therapeutically improving copper bioavailability to CNS cuproenzymes. Multiple sclerosis (MS) is a disease characterised by CNS demyelination, with evidence suggesting a link between demyelination and limited copper bioavailability. This is supported by data presented in Chapter 7 from both ALS model mice and MS-affected CNS tissue. We also show that changes to copper, SOD1, ceruloplasmin and myelin-associated proteins are common to ALS and MS, and that modulating copper bioavailability may provide a therapeutic intervention. Overall, data presented in this thesis indicate that: copper malfunction is a feature of ALS and MS; copper malfunction evident in sporadic cases of ALS are recapitulated in mutant SOD1 mouse models of familial ALS; and perturbations to the copper-dependent ceruloplasmin activity may be important to iron accumulation in the ALS-affected motor cortex. The therapeutic implications of these observations are discussed.