Sir Peter MacCallum Department of Oncology - Theses

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    Development of new methods for accurate estimation of tumour heterogeneity
    Hollizeck, Sebastian ( 2022)
    It is now understood that intra-tumor heterogeneity is one of the leading determinants of therapeutic resistance and treatment failure and one of the main reasons for poor overall survival in cancer patients. However, the possibility to study this phenomenon is so far underexplored as the acquisition of multi region data sets from the different tumour sites can be ethically challenging. With circulating tumour DNA (ctDNA) used as a proxy for tumour biopsies, it is possible to analyse a snapshot of the unified heterogeneity in each patient, but there is still an unmet need for new methods to optimize the analysis of these large-scale, high-dimensional data to derive new treatment targets. The contributions of this work include the development of multiple new methods, which show that the analysis of bulk sequencing from tumour tissue and ctDNA has unrealised potential for both diagnostic and research questions. This thesis presents three distinct but related projects, which explore the analysis of tumour heterogeneity at different levels and depths, focusing on method development. First, we developed a workflow to improve the detection of somatic variants present at very low allele frequencies. When multiple samples, separated in time or space, from the same patient were available, we were able to substantially improve the detection threshold of variants. These low abundance variants are invaluable in a clinical setting, where they can indicate an arising resistance mechanism or relapse of disease. With the improved sensitivity of our method, the treatment of patients can be adjusted earlier and more accurately. We then used our new analysis workflows to explore evolutionary trajectories and resistance pathways of five lung cancer patients enrolled in the CASCADE autopsy program. In addition to analysis of somatic variants, we used copy number analysis and structural variants to contrast and compare each sample within a patient to generate phylogenies to visualise the evolutionary distances and a pseudo time scale to assess the timing of mutations. Clear genomic determinants of treatment resistance were identified for three of the five cases with non-small cell lung cancer and the diversity of these genomic mechanisms profoundly highlighted the true extent of inter-patient heterogeneity. This work included the identification of a novel genomic resistance mechanism to the drug selpercatinib, a small molecule inhibitor of REK kinase. Among the remaining two patients, treatment resistance was mediated by transformation of their disease from non-small cell lung cancer to a small cell lung cancer histological phenotype. These two cases showed distinct evolutionary trajectories compared to the other non-small cell lung cancer cases, with similarity in their nuclear and mitochondrial phylogenies, but no clear genetic determinant for the small cell transformation, highlighting the additional importance of non-genomic mechanisms which can drive resistance in this disease. Finally, we developed a method, called MisMatchFinder, to monitor tumour heterogeneity and evolution over time through ctDNA. We tailored the method to be fully tumour agnostic and enabled it to be easily applied in the clinical setting by using low-coverage whole genome sequencing. The method uses highly specialised filtering steps to enrich the tumour signal and eliminate the background noise from normal cell-free DNA and sequencing errors in these data. We showed that the method could accurately detect specific cancer-related signatures at low tumour purity and tumour burden in simulated and patient data for melanoma and breast cancer. In summary, with this work we contributed multiple new methods to study, measure and understand genetic tumour heterogeneity. This understanding is crucial for the continuous optimisation of cancer management and the development of new and effective treatments for patients.
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    Antimicrobials in hospitalised and high-risk children: understanding and improving use
    McMullan, Brendan Joseph ( 2021)
    Infection is a near-universal human experience and is responsible for substantial child mortality across the globe, despite impressive reductions in child mortality and morbidity since the twentieth century. Antibiotics and other antimicrobial drugs have transformed our ability to prevent and treat infection. In general, these drugs are so safe, effective and widely available that overuse and inappropriate use are common. This is a cause of real problems in hospitals and the community, with unintended consequences of antimicrobial use including rising antimicrobial resistance. Antimicrobial stewardship (AMS) is aimed at improving the safety and efficacy of prescribing and has received growing attention in recent years. However, evidence to support and improve AMS for Australian children in hospitals is lacking. Australian hospitals are mandated to implement AMS programs and provide access to appropriate national and/or local prescribing guidelines. However, hospitals are under no current obligation to provide appropriately targeted AMS for the children in their care. Prior to mid-2019, national antimicrobial guidelines contained little paediatric and no neonatal advice. Since 2013, the voluntary National Antimicrobial Prescribing Survey (NAPS) has provided national reports on prescribing. However, until now, paediatric-specific data have not been reported. Compared with the literature on adult AMS, research on paediatric AMS is lacking, with few high-quality studies on interventions to improve care. This situation creates challenges for child healthcare providers and paediatric AMS program leaders, and more evidence is required to prioritise and improve care. The overall aims of this thesis are to improve the understanding of current antimicrobial use and stewardship for children in Australian hospitals and determine priorities to improve antimicrobial use now and in the future. This is achieved by analysing antimicrobial prescribing epidemiology and quality using national datasets, including national point prevalence survey and cohort study data. Chapter 1 reviews antimicrobial prescribing to children in hospitals, including in Australia. Chapter 2 presents the first analysis of paediatric antimicrobial prescribing to children in hospitals throughout Australia using NAPS data. Chapter 3 turns to high-risk groups, presenting the first nationwide analysis of prescribing for neonatal sepsis and fungal infections, again using NAPS data. Chapter 4 presents an analysis of antimicrobial prescribing in a contemporary cohort of immunocompromised children with fever and neutropenia, including prescribing quality and outcomes. Chapter 5 presents an interventional study evaluating the implementation of Australian guidelines on antibiotic duration and intravenous-to-oral switch. This is an example of the evidence translation and implementation approach needed for sustainable AMS improvement. Chapter 6 concludes the thesis, discussing the implications of the research and the paediatric AMS horizon in Australia. The analyses reported here reveal unnecessary variations in care and systemic inequities, which have implications for policy and guidelines. Non-metropolitan and non-tertiary hospitals in general provide lower-quality antimicrobial prescribing to children. This is likely to reflect decreased access to high-quality AMS resources, including guidelines and personnel, suggesting the need for systemic improvements. Neonates in Australian hospitals receive highly structured care in terms of antimicrobial choice and indications, but variations in dosing are substantial and undesirable, reflecting the lack of use of national guidelines. Prescribing for febrile neutropenia is highly diverse and often includes empiric aminoglycosides, which this research reveals are associated with real harm, suggesting the need for national guidelines to optimise care. Finally, the standard management of infections in hospitals involves excessive intravenous therapy, which is associated with unnecessarily increased hospital length of stay. As demonstrated, this can be improved with a structured AMS program, which should be available wherever children are treated in hospital. The information generated by this thesis provides new evidence on current antimicrobial prescribing practice and priorities and demonstrates the importance of utilising routinely collected data for the surveillance and improvement of paediatric AMS. Since this body of research began, national guidelines and paediatric-specific resources are now being developed, establishing new benchmarks. Along with continuous surveillance, these must be implemented appropriately to improve care. The research collaborations and networks developed during the production of this thesis will be used to support future surveillance and implementation work, which is needed to address AMS priorities in Australia and support the research and development of paediatric AMS across the globe.
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    The control of lymphatic vascular remodelling in cancer by microRNAs
    Arcucci, Valeria ( 2021)
    Metastasis is the lethal aspect of cancer for most patients. Remodelling of lymphatic vessels associated with a tumour is a key initial step in metastasis because it facilitates the entry of cancer cells into the lymphatic vasculature and their spread to lymph nodes and distant organs. Although it is clear that vascular endothelial growth factors (VEGFs), such as VEGFC and VEGFD, are key drivers of lymphatic remodelling in cancer, the means by which many signalling pathways in endothelial cells are co-ordinately regulated to drive growth and remodelling of lymphatics in cancer is not understood. In this thesis, I seek to understand the broader molecular mechanisms that control cancer metastasis through the analysis of microRNAs which act to co-ordinately regulate signalling pathways involved in complex biological responses, such as lymphatic remodelling, in health and disease. Here, using high-throughput small RNA sequencing, I found that a specific microRNA, miR-132, is up-regulated in expression in lymphatic endothelial cells (LECs) in response to stimulation with VEGFC and VEGFD. Interestingly, inhibiting the effects of miR-132 in LECs in vitro blocked proliferation and tube formation of these cells induced by VEGFC and VEGFD - LEC proliferation and tube formation are key steps in lymphatic remodelling. Moreover, I demonstrated that miR-132 is expressed in the lymphatic vessels of a subset of human breast tumours which were previously found to express high levels of VEGFD. In order to dissect the complexity of molecular regulation by miR-132 in lymphatic biology, my collaborators and I identified miR-132 target mRNAs in LECs, using high-throughput sequencing after RNA-protein cross-linking and immunoprecipitation of the Argonaute protein (Argonaute HITS-CLIP), which led us to define the miR-132-mRNA interactome in LECs. We found that this microRNA in LECs is involved in the control of many different molecular pathways mainly involved in cell proliferation and regulation of the extracellular matrix and cell-cell junctions. It is logical that miR-132 regulates such pathways given they are involved in the processes of LEC proliferation and tube formation, which I showed are dependent on miR-132 in my in vitro studies. Finally, I demonstrated that inhibiting the effects of miR-132 in a mouse ear model of lymphangiogenesis, using an antagomiR inhibitor of miR-132 coupled to cholesterol, blocked the complex remodelling of lymphatic vessels stimulated by VEGFC, in vivo. It was noteworthy that all aspects of lymphatic remodelling induced by VEGFC were restricted by inhibition of miR-132, including the enlargement, branching and sprouting of lymphatic vessels. Thus the inhibitory effect of targeting this microRNA on lymphangiogenesis and lymphatic remodelling can be considered comprehensive. The research described in this thesis identified miR-132 as a critical regulator of lymphangiogenesis and lymphatic remodelling, and delineated molecular mechanisms by which this microRNA influences these important biological processes. This work also identified new molecular pathways which are involved in modifying the lymphatic vasculature in response to key lymphangiogenic growth factors. In-so-doing, these studies identified potential therapeutic targets for drugs designed to block the growth and remodelling of tumour lymphatics, and thereby restrict the metastatic spread of cancer.
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    Pan-cancer reconstruction of clonal evolution in 1,800 patients using the discrete time-branching process
    Lara-Gonzalez, Luis Eduardo ( 2020)
    Intra and inter-tumour heterogeneity poses a challenge for associating molecular and immunohistochemical markers with clinical outcomes. Sequencing technologies has enabled detailed assessment of tumour heterogeneity, facilitating the genomic characterisation of tumours. Whilst such technologies have revealed mutational landscapes and have identified key driver alterations for tumorigenesis, pan-cancer clonal evolution reconstructions are lacking. In order to bridge this gap, I used discrete-time branching models to derive biological insights into tumour progression and reconstructed the clonal evolution in 1,800 patients, successfully linking mutations with growth patterns of disease progression. I first modified a discrete time-branching process to account for individual clonal subpopulations and derived analytical solutions for expectation and variance of both clonal and tumour expansions. Additionally, I derived the expected time for any given clone to successfully expand as \hat{\tau}, and with the use of these analytical solutions, I showed the likely driver and clonal compositions of the tumours and their phylogenies. Secondly, I generated a database of results from four different versions of time-branching process models that covered multiple parameters. Here I identified how an increase in diversity arises by both increased mutation rate and reduced fitness. I further corroborated that total number of drug resistant cells is directly proportional to lineage extinction probability (\delta) and tumour size as shown in previous studies. I also showed that this effect can be extrapolated to other types of functional passenger mutations involved in cancer-specific mortality. Moreover, I showed how commonly used sequencing cut-offs limit the accurate inference of tumour’s average selective advantage and driver mutation rate. Thirdly, I identified that a minimum distance metric can provide accurate fits of simulated cancer cell fractions to real patient tumour data. This metric showed at least 80% accuracy to identify the initial parameters of s and u and at least 40% accuracy to recover the correct evolutionary trajectory. Fourthly, I applied this fitting procedure to reconstruct the evolutionary trajectories of 1,800 tumours from different cancer sequencing studies. The best fits derived suggests that the most likely parameters for the evolution of solid tumours are high driver mutation rates and weak driver effects of fitness. Fifthly, using The Cancer Genome Atlas cohort, I identified an association between predicted degree of clonality and survival, and found branched topologies are common in malignancies with adverse prognosis. In the TRACERx non-small-cell lung cancer cohort, I identified that clonal reconstructions agreed with previously reported phylogenies. Additionally, using data from the Breast International Group 1-98, I identified the role of tumour fitness in determining clinical outcome, and the evolutionary dynamics of TP53 and PIK3CA mutations conducive to distant metastasis. Finally, using data from a metastatic melanoma patient collected through the CASCADE melanoma study, I was able to propose a pattern of dissemination from the primary to metastatic sites in the liver and brain based on the phylogenies recovered from my data-fitting procedure. This study demonstrates the power of the discrete-time branching process in reconstructing tumour evolution, and its potential to uncover insights in the dynamics of tumour growth that are missed by current methods.