Pathology - Theses
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ItemThe genomic landscape of phaeochromocytoma( 2015)Phaeochromocytomas (PCC) and paragangliomas (PGL) (collectively PPGL) are rare neural crest-derived tumours originating from adrenal chromaffin cells or extra-adrenal sympathetic and parasympathetic tissues. More than a third of PPGL cases are associated with heritable syndromes involving 18 or more known genes. These genes have been broadly partitioned into two groups based on pseudo-hypoxic and receptor tyrosine kinase (RTK) signalling pathways. Many of these genes can also become somatically mutated, although up to one third of sporadic cases have no known genetic driver. Furthermore, little is known of the genes that co-operate with known driver genes to initiate and drive tumourigenesis. To explore the genomic landscape of PPGL, exome sequencing, high-density SNP-array analysis, and RNA sequencing was applied to 36 PCCs and four PGL tumours. All tumours displayed a low mutation frequency in combination with frequent large segmental copy-number alterations and aneuploidy, with evidence for chromothripsis seen in a single case. Thirty-one of forty (77.5%) cases could be explained by germline or somatic mutations or structural alterations affecting known PPGL genes. Deleterious somatic mutations were also identified in known tumour-suppressor genes associated with genome maintenance and epigenetic modulation (e.g. TP53, STAG2, KMT2D). A multitude of other genes were also found mutated that are likely important for normal neuroendocrine cell function (e.g. ASCL1, NCAM1, GOLGA1). In addition, the existing paradigm for gene-expression subtyping of PPGL was further refined by applying consensus clustering to a compendium of previously published microarray data, enabling the identification of six robust gene-expression subtypes and subsequent cross-platform classification of RNA-seq data. The majority of cases in the cohort with no identifiable driver mutation were classified into a gene-expression subtype bearing similarity to MAX mutant PPGL, suggesting there are yet unknown PPGL cancer genes that can phenocopy MAX mutations. The cross-platform classification model was then further refined to develop a 46-gene Nanostring-based diagnostic tool capable of classifying PPGL tumours into gene-expression subtypes. The strong genotype-to-subtype relationship in PPGL makes subtyping a powerful tool that can be used clinically to guide and interpret genetic testing, determine surveillance programs and aid in better elucidation of PPGL biology. In applying the diagnostic assay to a test set of 38 cases, correct classification into one of the six subtypes was achieved for 34 (90%) samples based on the known genotype to gene-expression subtype association. The observation that at least one of the six subtypes is likely defined by the presence of non-neoplastic cells led to further refinement into five, four, and three-class architectures, further improving classification accuracy. Increasingly tumour heterogeneity is being recognised as one of the most significant challenges facing modern oncology. Genomically diverse tumour regions create additional complexity in predicting treatment response and metastatic potential through biopsy. Multi-region sampling of multiple synchronous primaries from patients with a predisposing germline mutation was used to explore tumour evolution and heterogeneity in PPGL and concomitant medullary thyroid carcinoma. Evolutionary reconstruction of a single primary PPGL demonstrated periods of both branched and linear evolution resulting in a high degree of intratumoural heterogeneity. Comparison of multiple synchronous primaries provided strong evidence of convergent evolution through recurrent chromosomal aberrations, indicating these may be obligate events in tumourigenesis, and as such, may indicate potential novel therapeutic targets.
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ItemMolecular profiling of ovarian cancer to guide targeted treatment( 2015)Ovarian cancer is a complex disease composed of multiple distinct molecular and clinical subtypes. The survival rate for ovarian cancer has remained largely unchanged over the past three decades, despite the rapid advancement of the knowledge of the molecular and genetic mechanisms underlying most of the subtypes of ovarian cancer. There is, therefore, an urgent need to rapidly translate this knowledge into improved clinical outcomes for patients with ovarian cancer. There have been significant clinical responses of certain types of cancer to targeted therapies that are designed to inhibit specific molecular defects that some tumours appear to be dependent upon. To assist in allocating patients with ovarian cancer to targeted therapies, two customised assays for mutation and copy number alteration detection were developed for molecular profiling. A panel of 29 genes, which are commonly mutated in ovarian cancer, and are potentially therapeutically targeted, was selected to be screened using an amplicon-based assay, designed for next generation sequencing. Seventy six ovarian cancer cases with matched formalin-fixed paraffin- embedded tumour tissue, snap-frozen tumour tissue and blood samples were used for the assay validation and estimation of the diagnostic yield. A panel of 11 commonly copy number altered genes in ovarian cancer was also selected for screening with a herein developed method for multiplex low-level copy number detection. Furthermore, a thorough assessment and optimisation of the available and developed analysis methods was performed to ensure accurate analysis and reporting of mutations and copy number alterations. Thirty five patients with advanced ovarian cancer were tested using the developed assays as part of the ALLOCATE study, with genetic changes detected in 90.9%, demonstrating a high diagnostic yield. Molecular profiling of these cases was not only useful in identification of possible targeted treatment strategies with the aim of improving clinical outcomes, but also assisted in determining the correct diagnosis. Moreover, a novel algorithm was proposed for the prediction of individual tumour response to PARP inhibitors, a promising targeted treatment in high-grade serous ovarian cancer.
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ItemFunctional genomics analysis of interplay between cell polarity and oncogenic RAS-MAPK signalling( 2014)The development of cancer is a complex process and progression towards malignancy requires the cooperation of multiple transformation events. This often involves mutations or aberrant expression of various oncogenes and tumour suppressors. Whilst epithelial tumours are inherently heterogeneous, and each tumour type and sub-type is molecularly distinct, one of their pervasive features is disruption to normal epithelial cell polarity and tissue architecture. Indeed it has now emerged that the polarization and differentiation of epithelial cells is innately tumour suppressive, and deregulation of epithelial polarity often correlates with invasive and metastatic disease. Conversely, evidence from experimental model systems indicates that the enforced expression of core polarity proteins such as Scribble is sufficient to potently suppress cellular transformation driven by oncogenes such as RAS. Despite an indisputable link between deregulation of the polarity network and the process of oncogenic transformation, little is known about how this network functions collectively on a systems-level within human epithelial cells, nor how engagement of this network may mediate innate mechanisms of tumour suppression. Here I have undertaken a detailed systems-level analysis to identify and characterize the interface between the global epithelial polarity network and oncogenic RAS-MAPK signalling in both normal and malignant human epithelial cells. This was achieved through a genome-scale RNAi functional genomics screen carried out in human epithelial cells coexpressing both oncogenic RAS and the polarity protein Scribble. Notably, this approach identified a robust network of cell polarity, adhesion and RAS-MAPK signalling proteins that are required for suppression of oncogenic RASV12 transformation mediated by Scribble. In direct support of these findings, a parallel analysis of the transcriptome of cells expressing RASV12 and Scribble, either alone or in combination, revealed that this network of RAS suppression cell polarity proteins are targeted for transcriptional deregulation by RAS via an ERK-FRA1 dependent mechanism. Conversely, enforced Scribble expression was sufficient to restore basal expression of these polarity genes. Together these findings demonstrate for the first time bidirectional feedback loops that serve to integrate cell polarity with RAS-MAPK signalling in the context of oncogenic transformation. Additionally, a novel role was identified for a TPL2-MAPK-JNK2-JUND stress signalling pathway in iv Scribble-mediated suppression of morphological transformation driven by RAS, suggesting that Scribble controls a human cell polarity network that constitutes a novel form of balancing crosstalk between MAPK-ERK and MAPK-JNK signalling to regulate epithelial polarization and suppress the RAS oncogene. Finally, to examine whether regulation of ERK signalling was a common property of all cell polarity regulators, I designed and applied a comprehensive siRNA library corresponding to the known human cell polarity genes and assessed their role in regulation of the sustained phase of ERK signalling using a high-content bio-image analysis screen. This analysis revealed a previously unappreciated degree of complexity and specificity regarding polarized regulation of ERK signalling, and identifies the polarity network as a novel class of highly specific spatiotemporal modulators of the ERK signalling response. Through this combination of systems-level RNAi mediated approaches, I have been able to construct a detailed working map of the cross-regulation and interplay between the RAS-MAPK-ERK signalling pathway and the broad network of genes that regulate epithelial polarity. This map will now serve as a foundation for future work aimed at understanding the functional landscape of human epithelial polarity networks in normal and malignant cells. Such future studies will serve to bridge the gap in our understanding of how polarity networks and oncogenic signalling interact to promote cancer progression.
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ItemGeneration of novel chimeric antigen receptors to enhance the specificity and activity of T cells for the adoptive immunotherapy for cancer( 2014)Adoptive immunotherapy is a promising treatment for cancer, with response rates of up to 70% in metastatic melanoma. To broaden this approach, T cells have been genetically modified to express chimeric antigen receptors (CARs) to endow T cells with anti-tumour activity capable of recognising a range of different cancer types. This approach has shown encouraging results in recent clinical trials for the treatment of haematological malignancies, however it has shown only moderate activity against solid cancers. To date, only a small number of molecules involved in T cell signaling have been incorporated into CARs, resulting in their suboptimal activity. Therefore improvements in CARs are needed in order to realise the full potential of adoptively transferred T cells. We proposed that using multiple or alternate signaling domains could enhance CAR-mediated T cell function. In this thesis, we describe the use of a DNA library of signaling molecules to investigate novel combinations of signaling molecules that could mediate enhanced CAR activity in the Jurkat T cell line and primary human T cells. A novel single-chain variable receptor was discovered comprising DAP10, CD3ζ and CD27 signaling domains that was able to trigger enhanced T cell activity in vitro and in an adoptive transfer mouse model. Clinical trials utilising CAR modified T cells have in some cases resulted in resulted in severe autoimmunity due to T cell recognition of tumour-associated antigens expressed on normal tissues. It is anticipated that as the application and efficacy of adoptive immunotherapy increases, toxicity against normal tissue will become increasingly common. To address this, we proposed that a T cell will respond less against normal tissue if endowed with a tumour-associated antigen-specific activating CAR co-localised with a chimeric inhibitory receptor (CIR) that is capable of turning off the T cell following engagement of antigen on normal tissue. We generated several novel chimeric inhibitory receptors and demonstrated expression of both CAR and CIR in T cells, which were then characterised for function against tumour-associated and normal tissue antigen expressing cell lines. In conclusion, the combination of these novel chimeric receptors may lead to a more efficacious but safer therapy for cancer.
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ItemThe identification and characterisation of novel genes involved in sensitivity to the histone deacetylase inhibitor, vorinostat( 2013)Histone deacetylase inhibitors (HDACi) are a novel class of anti-cancer agents that elicit a range of anti-tumour responses including apoptosis. Vorinostat is an FDA-approved broad-spectrum HDACi, which has achieved remarkable clinical success in some patients, particularly those with Cutaneous T cell lymphoma and Peripheral T cell lymphoma, however it remains unclear why certain patients remain unresponsive. Constitutive STAT activation, overexpression of pro-survival Bcl-2 proteins and loss of HR23B have been identified as potential biomarkers of HDACi resistance, however none have improved the clinical utility of HDACi. Therefore, the study described within aimed to further elucidate vorinostat resistance mechanisms through a functional genomics screen to identify novel genes that when knocked down by RNA interference (RNAi) sensitised cells to vorinostat-induced apoptosis. A synthetic lethal functional screen using a whole-genome RNAi library was used to identify genes that when knocked down co-operated with vorinostat to induce tumour cell apoptosis in otherwise resistant cells. The primary SMARTpool siRNA screen yielded 450 gene hits, of which 106 validated in a secondary ‘deconvolution’ screen using the four individual constituent siRNAs of each SMARTpool. Tertiary screening was conducted to evaluate specificity of these genes to co-operate with vorinostat compared to conventional chemotherapeutics. Thirteen vorinostat-resistance candidate genes were identified, which sensitised specifically to vorinostat in two or more of the four cell lines tested and these were validated in multiple independent assays for apoptosis. For ten of these genes, knockdown was greater than 90% while three appeared to be off-target. Comparative gene expression analysis was undertaken upon knockdown of vorinostat-resistance candidates, however transcriptome analysis did not identify common molecular network(s) through which these genes contributed to vorinostat resistance. Detailed analyses were conducted on two vorinostat-resistance candidates, GLI1 and to a lesser degree, PSMD13. GLI1 is a known oncogene not previously known to regulate the activity of HDACi. Treatment of vorinostat-resistant cells with the GLI small molecule inhibitor, GANT61, phenocopied the effect of GLI1 knockdown. The mechanism by which GLI1 loss of function sensitised tumour cells to vorinostat-induced apoptosis was through interactions with vorinostat to alter gene expression in a manner that favoured apoptosis. Finally, a xenograft model was established to test this combination in vivo. Together, the results presented in this thesis describe the identification and characterisation of ten vorinostat-resistance genes, eight of which had not previously been described as important for HDACi response. These ten vorinostat-resistance candidates may serve as biomarkers for stratification of patients for HDACi treatment. Furthermore, the mechanism of action of one of these candidates, GLI1, was investigated and results herein provide a strong rationale for development of GLI1 inhibitors for clinical use in combination with HDACi therapy.
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ItemRegulation of E6AP and involvement of the E6AP/PML axis in human cancer( 2013)The ubiquitin-proteasome system (UPS) plays a major role in the regulation of many cellular processes. However, aberrations in the regulation of the UPS can lead to a variety of pathological conditions. A firm link has been established between UPS and the development of cancer. Velcade, a proteasome inhibitor, is in clinical use for the treatment of diseases such as multiple myeloma. This thesis is based on our recent findings of a new regulatory pathway of tumour suppression. We discovered that the HECT E3 ligase, E6-associated protein (E6AP), is a key regulator of the tumour suppressor promeylocytic protein (PML). My thesis describes 3 studies: the regulation of E6AP by c-Abl (Chapter 3); the involvement of the E6AP-PML axis in colorectal cancer (Chapter 4) and also in prostate cancer (Chapter 5). Regulation of E6AP by c-Abl: In human papillomavirus (HPV) - infected cells, p53 is degraded by the HPV-E6/E6AP complex. Our laboratory previously demonstrated that the c-Abl tyrosine kinase protects p53 from ubiquitination and degradation by the HPV-E6/E6AP complex under stress conditions. Chapter 3 explores the underlying mechanism of this protection. We demonstrated an interaction between c-Abl and E6AP in vivo and in vitro. Activation of c-Abl by DNA damage leads to phosphorylation of E6AP. Mass spectrometric analysis revealed that tyrosine (Y) 636 within the HECT catalytic domain of E6AP is phosphorylated by c-Abl. Further computational analysis of E6AP trimerisation revealed an interaction between Y636 and glutamate (E) 544 in a neighbouring E6AP molecule. Using substitution mutants we examined the effect of Y636 phosphorylation, and its interaction with E544, on the E3 ligase activity of E6AP. We demonstrated that Y636 and E544 regulate the E3 ligase activity of E6AP, in a substrate-specific manner. Our findings suggest that in response to stress, c-Abl phosphorylates E6AP on Y636, thereby reducing the interaction with E544 leading to inhibition of E6AP E3 ligase activity. This is the first description of a post-translational modification of E6AP regulating its activity. This also provides a molecular explanation for the protection of p53 in HPV-infected cells under stress. Involvement of the E6AP-PML axis in cancer: PML protein expression have been shown to be down-regulated in multiple cancer types. Since we discovered E6AP as the E3 ligase of PML, we hypothesized that E6AP may be responsible for the loss of PML expression in colon and prostate cancers. Using a panel of colorectal cancer cell lines we did not find a significant elevation of E6AP correlating with low PML, nor were we able to restore PML expression by down-regulating E6AP (Chapter 4). On the other hand, we found an inverse correlation between E6AP and PML in a panel of prostate cancer cell lines. Down-regulation of E6AP was also associated with a reduction in cell numbers and an increased sensitization of prostate cancer cells to stress-induced death. This study supports a role for the E6AP-PML axis in prostate cancer. This is consistent with our analysis of prostate cancer samples demonstrating the poorest survival for patients with high E6AP and low PML expression (Chapter 5).
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ItemAn assay to screen for mutant p53 gain of function using high content imaging( 2012)The p53 tumour suppressor gene is mutated in approximately half of all human cancers. These mutations not only inactivate the growth inhibitory functions of wild type p53 but certain mutations also confer additional oncogenic properties. These gained functions may contribute to the increased growth rate, resistance to apoptosis, reduced chemosensitivity and increased invasiveness of mutant p53 bearing tumours. For the purposes of discovering novel mediators of mutant p53 gain of function, a multi-parameter assay was developed for future use in a high throughput siRNA screen. Mutant p53 expressing cells were demonstrated to display an increased migratory phenotype in vitro compared to p53 null cells. A wound healing endpoint assay was adapted to a 96 well format using automated liquid handling and image capture. An image analysis algorithm was designed to accurately measure cell migration in a high throughput manner. Performing a low throughput pilot screen of a panel of siRNAs demonstrated that the general migration machinery was necessary for migration. However, knockdown of known components of the mutant p53 pathway failed to provide the robustness necessary for a high throughput screen. p53 protein stability is known to be regulated at multiple levels involving a complex network of feedback loops. Unlike the case with wtp53, the regulation of mutant p53 is only partially understood. A high content assay for mutant p53 stability was developed. The immunofluorescent staining protocol was adapted to a 384 well assay and an algorithm was designed to accurately measure pixel intensity in the nucleus and cytoplasm. A statistically significant and robust downregulation of mutant p53 was measured with siRNA against mutant p53. A pilot screen in endogenous mutant p53 cell lines demonstrated a sufficiently large assay window to identify siRNAs that reduce mutant p53 stability. A genome wide siRNA screen for genes that reduce mutant p53 could uncover novel therapeutic targets, which will enable the design of new molecularly targeted therapeutics. Drugs able to impede the action of mutant p53 will be relevant to a significant proportion of human cancers.
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ItemThe role of perforin in human health and disease( 2011)Cytotoxic lymphocytes (CL) are key effectors of the immune system responsible for the elimination of intracellular pathogen-infected and transformed cancerous cells. This occurs predominantly through the perforin-dependent granule exocytosis pathway, where perforin pores formed in the target cell membrane allow the delivery of pro-apoptotic serine proteases, granzymes, into the cytoplasm of the target cell and the initiation of apoptosis. However, despite a critical role of perforin in CL biology, the physiological role of perforin in human health and disease states is not fully understood. In humans, perforin deficiency is most commonly associated with an autosomal recessive disorder, Type 2 Familial Haemophagocytic Lymphohistiocytosis (FHL), the manifestations of which suggest a role for perforin in maintaining immune homeostasis. However, unlike studies in perforin deficient mice, no evidence has yet linked defective perforin cytotoxicity with cancer susceptibility in humans. Here, a putative role for perforin in the immune surveillance of human cancers was investigated. Although most patients with bi-allelic perforin mutations develop FHL in early infancy, some individuals have been reported to develop an atypical delayed late-onset of disease. Perforin function was examined in these individuals, using surrogate assays developed in rat basophilic leukaemia cells and mouse cytotoxic T lymphocytes (CTLs). Accordingly, it was found that perforin dysfunction was associated with an increased susceptibility to haematological malignancies, providing the most compelling evidence to date for a role for perforin and thus CLs in human cancer immune surveillance. To account for the delayed age of disease onset in these patients, it was postulated that partial CL cytotoxicity protected from overwhelming FHL in early childhood. Novel assays were developed in both mouse and human natural killer (NK) cells to compare the effects of early- and late-onset disease-associated perforin mutations on CL cytotoxicity. Whilst perforin alleles associated with early-onset FHL were completely detrimental to NK cell cytotoxicity, perforin alleles associated with atypical late-onset disease retained partial activity in NK cells. By contrast, almost all these mutations were completely detrimental to CTL cytotoxicity. Hence, it appears that residual NK cell cytotoxicity, rather than CTL cytotoxicity, is responsible for protecting against overwhelming FHL in early infancy, supporting a predominant role for the loss of NK cell cytotoxicity in the pathogenesis of FHL. Furthermore, these studies revealed that partial perforin deficiency is caused by protein misfolding, providing a rationale for the development of specific drug therapies aimed at stabilising perforin structure. Finally, the case studies of two patients with clinical suspicion of FHL were presented. Sequence analysis of the patients’ perforin genes was performed in order to confirm diagnosis, resulting in the identification of two novel perforin mutations. The molecular basis of perforin dysfunction was analysed in each case using the functional assays generated in previous chapters, in combination with structural information extrapolated from the recently resolved crystal structure of mouse perforin. These studies revealed valuable insight into the role of perforin and CLs in disease pathology, including the discovery of the gain of glycosylation as a pathogenic mechanism common to perforin mutations associated with early-onset disease. In summary, the body of work presented in this thesis highlights the absolute importance of functional perforin in maintaining human health. It has also widened the range of available diagnostic assays and provided possible novel approaches for managing diseases associated with perforin deficiency.