Paediatrics (RCH) - Theses
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ItemInvestigating the biology of paediatric T cell acute lymphoblastic leukaemia to facilitate more effective individualised therapy( 2019)Acute lymphoblastic leukaemia (ALL) takes up the highest percentage of paediatric cancer. The treatment requires intensive chemotherapy for two to three years, as well as haematopoietic stem cell transplantation for poor-prognosis cases. Compared to B cell lineage ALL (B-ALL), T cell lineage ALL (T-ALL) has a lower 5-year event free rate, higher rate of relapse, and a worse outcome for relapsed cases. Individualised therapy, targeting at oncogenic changes in each patient, can make treatment more effective and less harmful. This requires understanding of the oncogenic biology of each individual leukaemia. We have attempted to develop a T-ALL model based on hiPSC-derived T cells, which will be in human origin, maintain normal genetic pattern, mimic in vivo T cell development, and can be massively produced for high throughput lab work. This model may make up for the shortcomings of conventional leukaemia cell lines and mouse models. This project investigates the biology of T-ALL by focusing on two novel fusion genes – TCF7-CSF1R and ETV6-CRX – identified by RNA sequencing of paediatric T-ALL patient samples. We have shown that TCF7-CSF1R is sufficient to immortalise mIL-3 dependent Ba/F3 cells. The ETV6-CRX fusion gene is anticipated to block differentiation. Establishing consistent expression of this fusion will require further optimization. The feasibility of setting up a hiPSC-derived T-ALL model was also assessed, with respect to protein expression in human T-ALL/lymphoma cell lines, hiPSC differentiation efficiency, hiPSC-derived T cell lentiviral infection rate, and cytokine withdrawal during differentiation. This project provides potential directions for improvement of methods for exogenous gene expression, such as the usage of CRISPR-Cas9 based techniques to introduce gene modifications for fusion genes such as ETV6-CRX that are difficult to express, particularly in hiPSC-derived T cells that have a low viral infection level. The T cell differentiation protocols also need to be optimised to make the T cell production easier and efficient. Detailed functional assay during T cell differentiation needs to be conducted in the future. In this thesis, Chapter 1 presents the background of this project; a literature review introducing human haematopoietic system, in vivo thymocyte development, paediatric TALL, novel oncogenic fusion-related genes, and in vitro T cell generation; the aims and hypothesis. Chapter 2 introduces the methods and materials used in this project. Chapter 3 presents the identification, cloning, and expression of novel fusion genes. Chapter 4 investigates the ability of these novel fusion genes to support cell survival and proliferation in conventional Ba/F3 cell line. Chapter 5 assesses the feasibility of setting up the hiPSC-derived T-ALL model. Chapter 6 makes a discussion on the results and concludes the whole project.
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ItemA tool for risk profiling and accurate prognostication in paediatric glioma integrating clinical features with epigenetics: it is time to move on from the binary classification( 2016)Paediatric glioma, the most common group of brain tumours in children, encompasses a wide range of entities with highly variable prognoses. Gliomas are grouped by histopathological features into high and low grade glioma but this classification does not take into account many established and emerging risk factors in this disease. Research into the molecular features of these lesions has shown that histology does not always correlate with biology and, where they differ, molecular features are usually superior at predicting outcome. Risk classifications have been developed for other paediatric malignancies which combine clinical, radiological, pathological and molecular factors to predict disease risk and prognosis. A comprehensive risk classification has not been published for paediatric glioma despite many risk factors being established in this disease. Using a clinical cohort of all paediatric glioma treated at a single institution (Royal Children’s Hospital, Melbourne, Australia) over an 18 year period a database was developed incorporating clinical, radiological, pathological and treatment factors. Where sufficient tumour tissue was available genome-wide methylation analysis was performed. The results of this were processed and evaluated by an established cluster analysis algorithm. Breaking the cohort into clinically appropriate subgroups, risk factors for disease progression and death were determined and prognosis estimated for distinct groups. A tool for robust risk profiling and prognostication was established with 5 main risk groups and 10 subgroups. Patients in the very low risk group have a predicted 100% overall survival and the majority require no treatment. In contrast there was a group of patients with 100% mortality within a short space of time where attempts at curative therapy are futile and may be deleterious to quality of life. The intermediate risk group contained a number of entities with a prognosis genuinely falling between that of low grade glioma and that of high grade glioma. Histopathological diagnosis retained prognostic importance for some, but not all, groups and methylation analysis was shown to have a significant role to play, particularly in high grade lesions or where diagnosis was unclear. This is likely to become part of routine care in the future and its place is strongly supported by the analysis presented here. Risk profiling is possible in paediatric glioma with far more accuracy than the current binary classification provides. A combination of established clinical factors and emerging molecular features provides an accurate and nuanced assessment of risk and prognosis.