Paediatrics (RCH) - Theses

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Subject: paediatric × Subject: AML ×

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    MicroRNA expression and genome-wide epigenetic analysis of paediatric acute myeloid leukaemia
    Morenos, Leah ( 2014)
    Introduction: Paediatric Acute Myeloid Leukaemia (AML) is the third most prevalent cancer in Australian children under the age of 14. Chromosomal and genetic lesions are commonly found within specific subtypes of paediatric AML and these help to direct treatments and prognostic predictions. However, there are no common genetic lesions across subtypes of paediatric AML and subtype-associated genetic changes may fail to induce leukaemogenesis. DNA methylation is the most commonly studied molecular alteration in cancer. Strong evidence indicates that methylation variations exist within paediatric cancer known to modify gene expression. Similarly, microRNA are small non-coding RNA that also regulate gene expression and whose dysregulation within paediatric cancer is now beginning to be appreciated. Therefore altered methylation and miRNA expression may contribute to malignancy through activating oncogenes or inactivating tumour suppressor genes, similar to genetic mutations. Furthermore, both DNA methylation and microRNA expression represent viable epigenetic mechanisms for application to clinical biomarkers of disease diagnosis, prognosis and disease tracking. At the beginning of this study, limited interrogations of genome-scale DNA methylation and microRNA expression had been conducted within paediatric AML with appropriate non-leukaemic controls. Furthermore, there has been few extensive studies evaluating the utility of current epigenetic techniques on primary clinical specimens. Therefore I chose to study genome-wide DNA methylation and large-scale miRNA expression changes in paediatric AML, as well as interrogating techniques for using archived primary patient samples. Materials and Methods: Two approaches were used to interrogate large-scale DNA methylation and microRNA expression; TaqMan OpenArray Human MicroRNA Panel (Life Technologies) was used to interrogate 5 primary paediatric AML patients with matched controls; and Illumina Infinium HumanMethylation450 BeadChip array (HM450) was used to interrogate 18 primary AML alongside matched non-leukaemic samples. Validation of AML DNA methylation alterations was conducted using the SEQUENOM MassARRAY EpiTYPER with an expanded cohort of 28 patients, in conjunction with validation of microRNA expression utilising quantitative real-time PCR (qRT-PCR) and TaqMan singleplex assays on 32 primary paediatric AML, together with matched control groups. This study also developed extensive extraction techniques for the analysis of microRNA expression within a range of samples, including archived bone marrow aspirate smear slides. Results: Genome-scale DNA methylation disruptions were characterised within paediatric AML, and identified hundreds of genes associated with disease compared to matched control samples. Interrogation of a refined subset of target genes also identified gene expression alterations within these regions, which were further associated with patient disease onset and predicted outcome. Conversely, large-scale microRNA expression disruptions were characterised within paediatric AML, whereby a small number of reliable targets were identified. Such microRNA disruption was found to be associated with DNA methylation regulation on the microRNA gene, and could also be used as reliable biomarkers to predict disease onset and patient outcome in connection with identified patient cytogenetic abnormalities. Integration of data from genome-scale DNA methylation and combined gene and microRNA expression analysis, identified common epigenetic disruptions within paediatric AML affecting known tumour suppressor genes, cytoskeletal organisation, cellular proliferation and immune function. Conclusions: The findings of this study reveal that DNA methylation alterations within paediatric AML can associate with gene disruptions with the potential to initiate and perpetuate malignant phenotypes. Likewise, microRNA deregulation can establish widespread gene disruptions as a catalyst for leukaemogenesis. Interestingly we establish that combined deregulation of epigenetic mechanisms, and the occurrence of one epigenetic mechanism working to deregulate another, may be a common feature of paediatric AML. Lastly this study identifies targets and techniques to extend such studies into the clinic and provides attractive targets for therapeutic intervention.