Clinical Pathology - Theses
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ItemOncogenes in KRAS Wild Type Pancreatic Cancer( 2019)Pancreatic ductal adenocarcinoma (PDAC) is an invasive cancer, ranked the fourth most prevalent cause of cancer related death. Somatic genetic alterations are primary drivers of PDAC. 93% of patients have activating mutations in the master oncogene, KRAS. Several studies have investigated the mutational landscapes of pancreatic cancer. However, comprehensive studies of KRAS wild type pancreatic tumours are limited. Hence the process of initiation and progression of this cancer remains to be discovered and may be associated with genes that have not been identified. The current project aims to identify oncogenes in KRAS wild type Pancreatic cancer. It also aims to identify whether these oncogenes are from the MAPK pathway or independent of it. The genomic and transcriptomic landscapes of KRAS wild type PDAC were verified. In the absence of KRAS mutation, alternative oncogenes were found. In the genomic data analysis, two cohorts were analysed including 70 samples in the KRAS wild type cohort and 571 in the KRAS mutant cohort. In the absence of KRAS mutation, tumours were found to be rare as were (i) Oncogenic BRAF in-frame deletions, hotspot alterations and oncogenic fusions (known and novel) (frequency of 17%), oncogenic GNAS hotspot mutation (frequency of 12%) and somatic alterations in RET (7% frequency). Recurrent copy number gains (CNV >4) were observed in MYC (23% frequency), CDK6 (16% frequency), AKT2 (16% frequency), KDM6A (14% frequency), EGFR (12% frequency), RICTOR (12% frequency), MET (11% frequency), FGFR1(10% frequency), FGF3 (9% frequency) and FGF4 (9% frequency) in the KRAS wild type cohort. Other low frequency fusion events in the MAPK pathway include: RET-CCDC6; ROS1-SLC4A4; BRAF-SND1; BRAF-SDK1; TRIM24-BRAF; STK4-SLC13A3; ARHGAP24-MAPk10; BRAF-BRAF; STMN1-CDK5RAP3, and; SLC4A4-RASGRF1. Two independent differential expression analyses were performed on the RNA-seq of KRAS wild type versus KRAS mutant Pancreatic Adenocarcinomas, generated by the Australian and Canadian ICGC- pancreatic cancer Consortium consisting of RNA-seq from 88 and 224 bulk tumour samples respectively. Pathway analysis showed that the Calcium signalling pathway was over-expressed in both the Australian and Canadian wild type.This up-regulation of the Calcium signalling pathway in the whole cohort of KRAS wild type is consistent with GNAS mutation in the genomic analysis of the KRAS wild type cohort. Additionally, the MAPK signalling pathway shows no difference throughout the whole cohort of KRAS wild type. Together, these findings at the genomic level reveal that the MAPK signalling pathway is the dominant pathway in the KRAS wild type cohort. The results of comparing RNA expression in two groups of KRAS wild type and KRAS mutant analysis suggest that one oncogene has been substituted for another oncogene in the MAPK pathway, creating an interruption in the MAPK pathway. The lack of differences between KRAS mutant and KRAS wildtype in the MAPK pathway could suggest that the MAPK pathway is up-regulated in both sets, resulting in a lack of difference in the expression.
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ItemThe genetics of gene expression: from simulations to the early-life origins of immune diseases( 2019)Human complex traits and diseases are often highly polygenic. Genome-wide association studies (GWAS) have been successful in identifying the underlying genetic components. However, challenges still remain and one of them is the biological interpretation of these findings. Genetic variants that are associated with diseases or traits are enriched in regulatory regions of the genome, suggesting that they may have a role in the regulation of intermediate molecular phenotypes, such as mRNA gene expression. Studies investigating the genetic architecture of gene expression variation, or expression quantitative trait loci (eQTLs), have aided the interpretation of GWAS findings by providing potential mechanisms through which the genetic variants contribute to higher-order phenotypes. In addition, eQTLs identified in disease-relevant tissues, or those that are specific to certain cell types or conditions are more informative in disease pathogenesis. This thesis first explored eQTL study design and analysis choices using extensive, empirically driven simulations with varying sample sizes, true effect sizes, and allele frequencies of true eQTLs. False discovery rate (FDR) control applied to the entire collection of tests had inflated FDR of genes with eQTLs (eGenes) in most scenarios; in contrast, hierarchical correction procedures had well-calibrated FDR. Significant eQTLs with low allele frequencies identified using small sample sizes were enriched for false positives. Overestimation of eQTL effect sizes was common in scenarios with low statistical power, and a bootstrap method (BootstrapQTL) which can lead to more accurate effect size estimation was developed. Based on the insights of the eQTL simulation study, optimal strategies were selected for the following eQTL analysis in two types of neonatal immune cells (monocytes and T cells) under resting and stimulated conditions. A great proportion of cis-eQTLs were specific to a certain cell type or condition, and the majority of them were observed only upon stimulation. Response eQTLs (reQTLs), with effects on gene expression modified by immune responses, were identified for 31% of the eGenes in monocytes and 52% of the eGenes in T cells. Trans-eQTL effects that were mediated through expression of cis-eGenes were observed. Lastly, integrative analyses were performed, using the early-life eQTLs, as well as GWAS variants associated with immune-related diseases obtained from external large cohorts. Significant overlaps between neonatal eQTLs and postnatal disease-associated variants were observed. Some cell type- or condition-specific cis-eQTLs colocalised with disease associations, suggesting that the potential risk genes involved in disease pathogenesis are linked to the stimulation of certain immune cells. Causal effects of genes were evaluated using Mendelian randomisation, and changes in expression levels (e.g. BTN3A2) were identified to have causal associations with multiple immune-related diseases. Taken together, it demonstrates that the early-life genetic variants and gene expression might contribute to later disease development. In conclusion, this thesis provides a strong evidence base for eQTL study design and guidance for analysis strategies in future studies. The characterisation of genetic regulation of neonatal immune responses and the interaction between regulatory variants and stimulatory conditions is a useful resource, and generates insights on the early-life origins of immune-related diseases that develop later in life.
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ItemViral and host inflammation and host immunity in the pathogenesis and therapy of tumours caused by Kaposi Sarcoma herpesvirus( 2018)Kaposi sarcoma associated herpesvirus (KSHV), also known as human herpesvirus-8, is the aetiological agent of three tumours: Kaposi sarcoma, a form of multicentric Castleman disease (KSHV-MCD), and primary effusion lymphoma (PEL). These tumours are predominantly seen in people with acquired immunodeficiencies, including HIV. The burden of KSHV-associated tumours is particularly significant in resource-limited settings including Africa. Current therapies are limited by an incomplete understanding of disease pathogenesis, in particular the role of inflammation and impaired immunity in tumorigenesis. This work examines the inflammatory pathogenesis and associated clinical characteristics of KSHV-MCD, showing for the first time that virally encoded and human cytokines, viral and human IL-6, cooperate in disease activity and have an independent effect on clinical features. It then demonstrates the role of functional imaging in patients with KSHV-MCD, showing a distinctive pattern of 18FDG-PET changes associated with disease activity and severity, and further illuminating pathogenesis by demonstrating that the underlying abnormalities are systemic. These findings are used as the basis for a prospective exploration of a novel syndrome of KSHV-associated inflammation distinct from KSHV-MCD, the KSHV-inflammatory cytokine syndrome, defining for the first time its clinical, imaging, virological and immunological parameters in relation to KSHV-MCD and HIV. Finally, integrating this understanding of the pathogenic role of immunity and inflammation, a prospective interventional evaluation of an oral immune modulator, pomalidomide, as therapy of symptomatic KS is explored. This shows that this agent is well tolerated, with an overall response rate of 73% (95% CI 50-89%), and correlative studies explore its impact on underlying immune dysfunction. Taken together, these results have a significant impact on our understanding of KSHV-associated tumours. They have implications for rational therapy development, including approaches applicable in resource-limited setting and therapies tailored to the inflammatory manifestations and immune predispositions that have emerged as characteristic of these tumours.
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ItemCharacterising the role of selective Set7 inhibitors in transcriptional regulation and cytokine production in human microvascular endothelial cells( 2019)Set7 is a lysine methyltransferase which catalyses the transfer of a monomethyl group to lysine 4 of histone H3 (H3K4me1). Set7 is also associated with regulation of different non-histone proteins including nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB). Recent reports have implicated Set7 in the activation of pro- inflammatory genes in human vascular endothelial cells in response to hyperglycemia. In addition, several studies have demonstrated that Set7 is required for NF-κB dependent tumor necrosis factor alpha (TNF-α) induced inflammation. Vascular inflammation can drive endothelial dysfunction, and is involved in the development of cardiovascular disease (CVD). Manipulation of Set7 methyltransferase activity by genetic deletion or pharmacological inhibition has been reported to regulate cytokine signalling in human microvascular endothelial cells (HMEC-1). Selective Set7 inhibitors have recently been developed, but their impact on vascular inflammation remains unknown. In this project, HMEC-1 cells were used to assess the Set7 inhibitors, PFI-2 and cyproheptadine hydrochloride mediated regulation under basal conditions and during TNF-α induced inflammation. Using RNA sequencing and gene set enrichment analysis (GSEA), Set7 inhibitors upregulated the expression of genes involved in the control of cholesterol Sterol Regulatory Element-Binding Proteins (SREBP), cholesterol and fatty acyl biosynthesis. On the other hand, pharmacological inhibition of Set7 by PFI-2 and cyproheptadine hydrochloride reduced the expression of genes implicated in cell cycle and smooth muscle cell contraction. Given its contribution to pro-inflammatory gene activation, we hypothesized that targeting Set7 activity may reduce the burden of vascular inflammation. Transcriptome profiling by RNA sequencing and GSEA studies showed PFI-2 and cyproheptadine hydrochloride can attenuate TNF-α induced gene expression. Set7 inhibitors suppressed the expression of genes implicated in cytokine and interferon signalling and NF-κB activation. Having shown that pharmacological inhibition of Set7 activity attenuates TNF-α driven gene expression, we proposed that PFI-2 and cyproheptadine hydrochloride could suppress TNF-α induced cytokine release. To test this hypothesis, inflammatory insult was induced and the impact of Set7 inhibitors was assessed by cytokine array. We detected robust attenuation in the secretion of various pro-inflammatory cytokines including IL-1b, IL-6, TNF-α and IFN- ɣ. Moreover, pharmacological Set7 inhibitors attenuated TNF-α stimulated production of key chemokines such as IL-8, MCP-1 and RANTES. PFI-2 and cyproheptadine hydrochloride also decreased release of growth factors and cytokines associated with allergic inflammation. We show, for the first time, Set7 inhibition attenuates induced cytokine expression and secretion. This work will lead to new therapeutic opportunities to address vascular inflammation.