Clinical Pathology - Theses

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    Heritable DNA methylation marks associated with familial breast and prostate cancer
    Hosseinpour, Mahnaz ( 2022)
    The known genetic risk factors for breast and prostate cancer account for less than 50% and 35% of the familial risk respectively. Early candidate gene studies provided evidence that DNA methylation measured in peripheral blood can mimic the effect of cancer predisposition associated with germline pathogenic variants. Key examples included the increased risk of breast cancer associated with both BRCA1 promoter hypermethylation and intragenic DNA methylation in ATM. More recently, the association between heritable DNA methylation marks and breast and/or prostate cancer risk has been reported by genome-wide studies. However, it is not clear how most of these modifications in DNA methylation mediate phenotypes resulting in increased cancer risk. In this thesis, a recently developed analytic approach, based on complex segregation analysis and family structure, was applied to identify 1,000 heritable DNA methylation marks in 57 families with multiple cases of breast and prostate cancer. Using Cox segregation analysis, we found that 229 and 140 methylation marks were associated with breast and prostate cancer respectively and 63 were associated with increased risk of both cancer types. We developed a Clustered regularly interspaced short palindromic repeats (CRISPR)-based strategy using a dCas9-DNA methyltransferase enzyme (DNMT) and a modified sgRNA containing two PP7 hairpins fused with DNMT3A in the synergistic activation mediator (SAM) system to assess whether induction of DNA methylation at these methylation sites resulted in a recognised cancer phenotype (such as DNA damage and proliferation). We first optimised CRISPR mediated DNA methylation by testing three combinations, including fusion of dCas9-DNMT3A, dCas9-DNMT3B and dCas9-DNMT1 with our modified sgRNA and found that SAM-DNMT3A which involves fusion of dCas9-DNMT3A with our modified sgRNA induced highly robust DNA methylation even compared to the previously developed approach, SunTag system, via transfection of HEK293FT cells targeting BRCA1 promoter region. In addition, we performed lentiviral transduction of human mammary breast cell lines using SAM-DNMT3A and sgRNAs targeting BRCA1, PTEN and NF1 regions and demonstrated a significant DNA methylation induction at these regions compared to GFP- sgRNA, targeting non-human genes. We found that the SAM-DNMT3A could also induce higher gene silencing in comparison with CRISPR-inhibition (CRISPRi) tool, involving dCas9-KRAB. A lentiviral delivered sgRNA pooled library, including 10 sgRNAs for each of the 1,000 heritable DNA methylation sites, was generated to perform phenotypic screens in the human mammary epithelial cells, B80-T5 and K5+/K19+ cells using SAM-DNNT3A and CRISPRi tools.We measured the effect of DNA methylation on proliferation and the DNA damage response using a PARP inhibitor synthetic lethal screen. K5+/K19+ cells, showed very low phenotypic changes. Overall, the SAM-DNMT3A tool enabled a systemic high throughput pooled screen of heritable DNA methylation sites mediating breast cancer related phenotypes including proliferation and DNA damage. The findings of this study further characterise the non-genetic component of familial risk of breast and prostate cancer, provides new opportunities to elaborate how tumorigenesis can be affected by DNA methylation and develop epigenetic therapeutics targeting these risk factors, ultimately advancing both precision prevention and medicine.
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    Genetic and Epigenetic Risk Factors for Invasive Lobular Breast Cancer
    Suman, Medha ( 2021)
    Invasive lobular breast cancer (ILBC) is the second most common histological subtype of breast cancer and accounts for 10-15% of all cases. Loss of e-cadherin protein is a hallmark of ILBC and contributes to its characteristic discohesive morphology. In addition to distinct histological features, several subtype-specific molecular and clinical features have been described. However, ILBC remains understudied relative to other breast cancer subtypes, despite its frequency. In this era of precision medicine, there is a growing interest in further refining breast cancer tumour subtyping by identifying additional discriminating molecular features. However, there is limited data to pursue this for ILBC as it is often not well-represented in study samples. For instance, the seminal work on breast cancer classification based on gene-expression levels by Perou et al., (2000) included only two ILBC cases. It is important to identify ways to refine the subtyping of ILBC tumours so that women with ILBC can benefit from a more precise treatment plan, prognosis and targeted therapy options. The main objectives of this PhD project were: i) to examine the distinguishing methylation patterns between ILBC (n=151) and non-ILBC (n=341) tumours ii) to investigate the ILBC methylome to identify methylation signatures for prognostication (n=130) and iii) to subclassify ILBC into subgroups with increased homogeneity based on their genome-wide DNA methylation profiles (ILBC, n=151, non-ILBC=341) and to further characterise these subgroups by investigating their somatic mutational signatures (n=15). Three subgroups of ILBC were defined via unsupervised cluster analysis of genome-wide DNA methylation measured using the Infinium HumanMethylation450K assay. Of these, Subgroup 1 was identified as the most distinct ILBC subgroup, characterised by a predominant hypomethylation across 27,675 CpGs compared with Subgroup 2 and across 13,067 CpGs compared with Subgroup 3. Subgroup 1 showed more similarity to the TNBC (non-ILBC) cases compared with the other two methylation-defined subgroups in terms of their genome-wide methylation pattern. Survival analysis showed that women with ILBC tumours in Subgroup 1 had the poorest overall survival when compared with women in Subgroup 2 (hazard ratio (HR): 0.59, 95% confidence interval (CI): 0.19-1.79) and Subgroup 3 (HR: 0.16, 95% CI: 0.03-0.88), after adjusting for age and year of diagnosis. Subgroup 3 had an enrichment for women who had a first-degree relative with a history of any cancer. Both Subgroup 2 and Subgroup 3 were enriched with women who had a female relative with a history of breast cancer. This suggests that women in Subgroup 2 and Subgroup 3 may be genetically or epigenetically predisposed to developing breast cancer. The somatic genetic variant profiles of the ILBC DNA methylation-defined subgroups were further investigated by performing whole-exome sequencing (WES) on five ILBC tumours representing each of the three subgroups (n=15). The mismatch repair deficiency (MMRd) associated mutational signature SBS6 was the most frequently observed mutational signature in the ILBC tumours, detected in 12/15 (80%) cases. Microsatellite instability (MSI) was also predicted in 13/15 (87%) of the cases, including all 12 tumours with SBS6. Although distinct somatic (genetic) characteristics for tumours of individual subgroups were not observed, this research highlighted the potential role of MMRd in ILBC tumourigenesis and progression. DNA methylation of ILBC was also investigated as a possible prognostic biomarker. The analysis revealed 2,771 variably methylated regions within the ILBC tumours (n=130). A pooled survival analysis of the study set and TCGA data identified APC, TMEM101, HCG4P3 and CELF2 promoter methylation as potential prognostic biomarkers for women with ILBC. Comparing the DNA methylation profiles of ILBC (n=151) and non-ILBC (n=341) tumours, 13,763 genes and 8,456 intergenic regions showing statistically significant differences in DNA methylation (false discovery rate (fdr), P-value < 0.01) were identified. Gene set enrichment analysis revealed that the differentially methylated genes were found to be involved in biological pathways related to metabolism of RNA (R-HSA-8953854), mRNA processing (GO:0006397), RNA splicing (GO:0008380), cell cycle (R-HSA-1640170) and DNA repair (GO:0006281). This study brings together several lines of evidence to indicate that distinct molecular features of ILBC can enable further subtyping, identify important features for targeted therapies (e.g., MMRd) and provide additional information for prognostication. This research identified Subgroup 1 as an important subgroup with similarities to TNBC and more aggressive clinical behaviour. Further investigation of samples from Subgroup 1 may identify additional important targets for precision medicine.
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    Characterising tumour and immunological heterogeneity in colorecal cancer
    Shembrey, Carolyn Elizabeth ( 2021)
    Despite improvements in surgical oncology and precision medicine, 5-year survival rates for those with late-stage colorectal cancer (CRC) remain extremely poor and innovative treatment strategies are needed. Although T-cell directed immunotherapies are strikingly effective in many solid cancer types, durable responses are limited to approximately 5% of all CRC patients. Accordingly, there is an urgent need to explore alternate immunotherapeutic strategies which harness other cytotoxic cell types, most notably NK cells. However, the inter-patient variability of NK cell involvement in CRC, and particularly in colorectal liver metastases (CRLMs), is poorly characterised. Moreover, very little is known regarding the influence of tumour heterogeneity on immunotherapy response, mandating the development of novel methodologies which can dissect divergent responses to immuno-, chemo- and targeted therapies at the level of individual tumour cell subpopulations. In this thesis, the scope of tumour and immunological heterogeneity in primary CRC and CRLMs was assessed from transcriptomic, spatial and functional perspectives. A CRC-specific NK cell gene signature which infers the NK cell load of individual tumours from bulk RNAseq data was designed and validated. Differential expression analysis revealed that tumours with high evidence of this NK cell signature were characterised by the upregulation of chemotactic and cytolytic transcriptional programs. Furthermore, amongst patients with primary CRC, those with high NK scores were shown to have better survival outcomes in two independent cohorts. Focussing on metastatic disease, Cell type Identification by Estimating the Relative Subsets of RNA Transcripts (CIBERSORT) analysis revealed significant variance in terms of immune cell composition between CRLMs and adjacent normal (AdjN) liver tissue. Using a novel mIHC panel to quantify the scope of NK cell infiltration and NK cell ligand expression, it was determined that neoadjuvant chemotherapy increased NK cell penetrance of CRLM tissue, restoring NK cell load to levels comparable with the AdjN liver. Furthermore, NK cell densities were comparable in the AdjN liver and CRLM tissue of patients who partially responded to chemotherapy whereas nonresponders showed preferential accumulation in the AdjN liver. Preliminary data is also presented regarding the establishment of a NK cell versus patient-derived organoid (PDO) co-culture killing assay which can be used to assess the functional activity of NK cells against CRLMs. Lastly, a lineage tracing technique termed optical barcoding (OBC) was employed to study the relationship between intra-tumour heterogeneity and treatment response. Stably marking heterogenous populations of CRC cells with unique fluorescent signatures enabled the real-time identification and tracking of different cellular subpopulations under pharmacological selective pressures. Moreover, optimising the fluorescent protein panel allowed the maximum theoretical subpopulation resolution for the OBC technique to be achieved, greatly improving throughput as compared with previously published fluorescent barcoding techniques. Collectively, the results provided herein have deepened our understanding of tumour and immunological heterogeneity in primary CRC and CRLMs, and, by introducing several novel technologies, have laid strong foundations for future studies.
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    Oncogenes in KRAS Wild Type Pancreatic Cancer
    Ahmadloo, Somayeh ( 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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    The genetics of gene expression: from simulations to the early-life origins of immune diseases
    Huang, Qinqin ( 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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    Viral and host inflammation and host immunity in the pathogenesis and therapy of tumours caused by Kaposi Sarcoma herpesvirus
    Polizzotto, Mark ( 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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    Characterising the role of selective Set7 inhibitors in transcriptional regulation and cytokine production in human microvascular endothelial cells
    Al-Sarray, Sameer A. Jadaan ( 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.