Sir Peter MacCallum Department of Oncology - Theses
Permanent URI for this collection
Search Results
1 - 10 of 59
-
ItemThe role of USP9X in Low-Grade Serous Ovarian Cancer( 2023)Low-grade serous ovarian carcinoma (LGSOC) is a rare histotype of epithelial ovarian cancer (EOC), and accounts for approximately 3-5% of diagnosed EOC cases. LGSOC is characterised by wildtype TP53 expression, frequent aberrance in the RAS/RAF signalling pathway, and relative genomic stability which in part explains LGSOC resistance to current standard-of-care platinum-based chemotherapeutics. Current chemotherapy strategies for LGSOC have predominantly been driven by that of the far more common high-grade serous subtype. Optimal cytoreductive surgery is challenging, given that the majority of LGSOC diagnoses are at a late stage where the cancer has metastasised from the primary site. Recently, alternative therapy strategies including targeted therapy of the RAS/RAF pathway have shown efficacy against tumours, but further characterisation into potential novel drivers of this disease is required to expand the treatment repertoire for patients suffering from this disease. Previous sequencing studies elucidated USP9X as one such potential driver of LGSOC. USP9X is a deubiquitinase involved in protein turnover. The gene has been implicated as both oncogenic and tumour-suppressive depending on the cancer type being investigated. In the context of LGSOC, little is known as to the role that this gene has in disease development. This thesis evaluated 121 LGSOC cases, 71 sequenced via targeted sequencing, 49 through whole exome sequencing, and 1 by whole genome sequencing. Sequencing results identified USP9X mutations at a frequency of 14%, and as the most frequently mutated non-RAS/RAF gene in the assessed cohort. Interrogation into the allelic status of these mutations revealed more than half of the mutations were inactivating, suggesting a tumour-suppressive function; USP9X was elucidated to follow a classical two-hit tumour suppressor model. Gene knockdown and knockout experiments on LGSOC cell lines highlighted a potential perturbance to clonogenic survival, but not to migration and proliferation. Mass spectrometry analysis on USP9Xnull LGSOC cell lines identified the molecular chaperone BAG3 as a likely direct substrate of USP9X, and the deubiquitinase as a potential regulator of the mTORC signalling pathway. Assessment of the global proteomic perturbations as a result of USP9X downregulation suggested the downstream consequences of USP9X suppression are likely to be decreased cell adhesion, and potentially increased cell migration and invasion.
-
ItemReducing the morbidity and mortality of ovarian cancer through prevention( 2022)Ovarian cancer (OC) remains a major cause of morbidity and mortality in women. With the current absence of effective screening and early detection methods, prevention of OC through risk-reducing salpingo-oophorectomy remains the most effective means of reducing OC morbidity and mortality. However, a significant challenge lies in identifying the women at greatest risk of OC and enabling their access to risk reducing interventions before cancer occurs. This study aimed to explore pathways to facilitate cancer prevention in at risk individuals that are not currently available in the clinical setting. In Chapter 3 of this thesis, I describe the TRACEBACK study - a novel approach to OC prevention through finding previously unrecognised carriers of pathogenic germline variants (PVs) among women with high grade non-mucinous ovarian cancer (HGNMOC) who had missed the opportunity for genetic testing. I piloted three methods of ascertainment and evaluated sequenced DNA of tissue samples from over 1000 women with HGNMOC, including some under a waiver of consent. The findings were returned to the women (probands) if they were alive, or if deceased the results were returned to a family member. PVs were detected in 119 probands and to date, 56 results have been returned. Although logistically and ethically complex, I have demonstrated the feasibility of this approach to find previously unknown carriers of PVs. The success of TRACEBACK as a cancer prevention initiative hinges on the subsequent uptake of cascade genetic testing and use of risk reducing strategies by family members. Therefore, the acceptability and value of TRACEBACK to probands and their family members was explored in Chapter 4 of this thesis, through qualitative interviews performed as part of the TRACEBACK.Evaluation study. In this pilot study, eight interviews were performed. While participants were shocked by the TRACEBACK contact irrespective of their method of ascertainment or consent process, all were unanimously positive in their perceptions of the value of the project and were appreciative and grateful that a project of this type was being conducted. Chapter 5 of this thesis explores the utility of polygenic risk scores (PRS) in characterising OC risk and response to treatment. I demonstrated that a one unit increment in PRS increases the risk of high grade serous ovarian cancer (HGSOC) by 38% (OR 1.38, p value <0.001). This study gives further validation to the role that PRS is likely to have in OC clinical risk prediction models in the future. Interestingly, this study revealed that a high PRS may be negatively impacting OC treatment response. My research has demonstrated that identification of at-risk individuals through the TRACEBACK study and examination of PRS are likely to facilitate OC prevention, and provide important steps toward reducing the morbidity and mortality of OC. The genetic testing of samples from deceased patients provides a novel model for risk reduction in cancers where there is a substantial frequency of pathogenic germline variants, including triple negative breast cancer.
-
ItemStrategies for Preventing Opportunistic Infections in Patients with Haematological Malignancies and Haematopoietic Cell Transplantation (HCT)( 2022)Opportunistic infections are a major cause of morbidity and mortality in patients with haematological malignancies and haematopoietic cell transplantation (HCT) recipients due to impaired immune function. The risks for, and aetiologies of infections in this patient population are rapidly evolving with the development of new therapies, which produce profound and prolonged immunosuppression. Prevention of these infections is essential, however there remains a knowledge gap in the specific treatment-related infection risk factors with new and emerging therapies as well as the optimal application of current infection prevention modalities. This thesis addresses the optimisation of strategies used to prevent opportunistic infections in patients with haematological malignancies and HCT, particularly infections associated with immunosuppression in this population, including fungal and viral infections. The thesis has four primary aims: 1) to describe the rate and patterns of infection related mortality (IRM) in allogeneic HCT recipients; 2) to assess risk groups for cytomegalovirus (CMV) infection, CMV viral kinetics and CMV related mortality after allogeneic HCT; 3) to assess the viral kinetics of Epstein-Barr virus (EBV) DNAemia after allogeneic HCT to predict EBV-related post-transplant lymphoproliferative disorder (PTLD), and; 4) to examine the preventative strategies used for invasive fungal disease (IFD) in patients with haematological malignancies and HCT, including: the safety, tolerability and effectiveness of a novel antifungal prophylaxis agent, SUBA-itraconazole (S-ITZ); the usage and drug level attainment of a commonly used, existing antifungal agent, voriconazole (VCZ); and; the IFD incidence and risk factors associated with the use of a contemporary chemotherapy regimen, “CLAG-M”. To address Aim 1, a national scale, retrospective analysis of IRM after allogeneic HCT in Australia over a 6-year period was conducted (Chapter 2). The study demonstrated that IRM was the leading cause of early post-HCT mortality in adults and children. Infectious aetiologies reported included bacterial, fungal, and viral. The findings of this study illustrate the critical importance of better strategies to prevent infections and were the foundation of the thesis, which was then separated into a literature review and original research studies for each of the opportunistic infections: CMV, EBV and IFD. To address Aim 2, a retrospective single-site Australian study to determine pre-transplant predictors of CMV reactivation, clinically significant infection and disease was conducted (Chapter 4). The study also investigated post-transplant kinetics of CMV and associated survival. The study identified pre-transplant predictors for clinically significant CMV infection and CMV related mortality (D-/R+ serology and unrelated donor stem cell source with antithymocyte globulin), as well as patterns of viral load kinetics to optimise pre-emptive CMV therapy. These findings identified priority groups for allocation of new CMV prevention medications and provide evidence-based thresholds for interventions to treat CMV reactivation. To address Aim 3, a retrospective study of two major HCT institutes in Australia was conducted, evaluating the role of viral kinetics of EBV-DNAemia after allogeneic HCT to predict EBV-PTLD (Chapter 6). The study demonstrated that EBV-DNAemia >10,000 IU/mL was the strongest predictor for the development of EBV-PTLD and also described the kinetics of EBV-DNAemia to guide pre-emptive therapy with rituximab. This is the first Australian study published to provide evidence-based recommendations for EBV-PTLD prevention therapy in the course of EBV reactivation. To address Aim 4, a series of studies were conducted to investigate knowledge gaps in IFD prevention strategies in patients with haematological malignancies and allogeneic HCT. Firstly, a retrospective analysis of two Australian HCT centres using the novel antifungal, S-ITZ, for primary IFD prophylaxis after allogeneic HCT was conducted (Chapter 8). S-ITZ prophylaxis in HCT recipients without grade II-IV acute graft-versus-host-disease (aGVHD) resulted in a low incidence of breakthrough proven/probable IFD at approximately 1.0%. Additionally, S-ITZ was well tolerated and achieved therapeutic levels in most patients. Next, an analysis of an established mould-active antifungal, VCZ, was conducted at a major tertiary United States (US) transplant centre, to evaluate the utilization and therapeutic level attainment of VCZ among HCT and chimeric antigen receptor (CAR)-T cell recipients (Chapter 9). This study identified that the current usage of VCZ was common, however therapeutic drug level attainment was poor with approximately 50% patients therapeutic at first measurement. Indications for VCZ consisted mostly for the empiric or directed treatment of IFD, however for the use of VCZ for prophylaxis was also common. The final analysis was a single site retrospective study at a major tertiary cancer centre in the US, investigating the incidence and patterns of IFD with the use of a novel induction chemotherapy regimen for acute leukaemia, “CLAG-M” (Chapter 10). The study demonstrated that the rate of IFD was high with an incidence of approximately 20% overall. The only risk factor identified for reducing IFD was the use of mould active antifungal prophylaxis, which when initiated within 14 days from the start of chemotherapy resulted in an IFD incidence of approximately 7.5%. Collectively, these antifungal studies provide valuable information on refining antifungal prophylaxis strategies in previously unpublished haematological malignancy and HCT populations. In summary, this thesis has explored critical knowledge gaps in relation to the management of CMV, EBV-PTLD and IFD in hematological malignancy and HCT populations. The research has provided insights into patterns, risk factors and novel prevention strategies for these opportunistic infections, and highlighted future research needs in these fields. This information can be used to update infection prevention protocols and guidelines, as well as guide future research, to improve outcomes for this vulnerable patient population.
-
ItemCirculating tumour DNA to guide precision medicine in solid malignancies( 2022)The primary objective of precision oncology is to improve outcomes for patients with cancer by individualising diagnosis and treatment. In order to achieve this, it is essential to obtain accurate molecular profiles of individual tumours, which are currently determined from analysis of tumour tissue. Advances in next-generation sequencing (NGS) technology has allowed a more robust and accurate characterisation of the tumour genome, which has led to the identification of somatic alterations to guide the selection and individualisation of treatment. However, obtaining adequate tissue material for molecular analysis is often not feasible due to the invasive nature of tissue biopsy and the risk of complications. Furthermore, significant challenges remain due to tumour heterogeneity and capturing serial genomic information is important to understand the molecular mechanisms of response and resistance to therapy. Liquid biopsies, which includes the analyses of circulating nucleic acids present in blood, are emerging as a minimally invasive tool for molecular testing, cancer detection, and disease monitoring in solid malignancies. Circulating tumour DNA (ctDNA) detected in patient’s blood is derived from cancer cells and can mitigate the limitations of tumour biopsy. This thesis focuses on understanding the clinical utility of ctDNA analysis in patients with early-stage melanoma, advanced non-small cell lung cancer (NSCLC) and advanced colorectal cancer (CRC) undergoing therapy. Using a suite of sequencing technologies, this work has demonstrated that comprehensive genomic analysis can be applied at different time-points of a patient’s cancer management. Firstly, the presence of post-operative ctDNA was predictive of poorer outcomes in patients with stage III melanoma post curative surgery. Importantly, ctDNA was shown to be a useful tool for monitoring minimal residual disease (MRD), with the ability to detect relapse prior to radiographic assessment. In the metastatic disease setting, early ctDNA dynamics in patients with EGFR positive NSCLC and BRAF V600E mutant CRC undergoing treatment with targeted therapies served as a robust biomarker of treatment response and provided important insights into mechanisms of treatment resistance. Notably, the use of longitudinal ctDNA analysis in a case of RET rearranged NSCLC demonstrated the utility of ctDNA in monitoring clonal evolution in real-time, highlighting the potential of precision-guided patient management. Together, these data reveal important insights into the molecular mechanisms of resistance to novel combination targeted therapies and provide a comprehensive overview of the diverse clinical applications of ctDNA to guide precision oncology.
-
ItemDiagnosis and treatment of infections in patients with haematologic malignancies: examining the role of FDG-PET/CT imaging in the rational use of antimicrobials in neutropenic fever( 2022)Infections are one of the leading causes of morbidity and mortality in acute leukaemia and haematopoietic stem cell transplant (HSCT) patients. The highest risk for infection is during the neutropenic period post intensive chemotherapy for acute leukaemia and conditioning for transplantation, and these infections typically manifest as neutropenic fever (NF), often without clear localising signs or symptoms of the source. The aetiology of NF in these patients is frequently not known, leading to prolonged and costly empiric antimicrobial therapies and adverse outcomes. This thesis aims to improve the detection of causes of NF and hence guide antimicrobial rationalisation in acute leukaemia and HSCT recipients through employing the novel technique of 18F-fluorodeoxyglucose positron-emission tomography combined with low-dose CT (FDG-PET/CT). In support of this major aim, the thesis also aims to describe antimicrobial prescribing in this complex area, assess utility of FDG-PET/CT specifically in invasive fungal infection (a key differential diagnosis in high-risk NF), and assess the feasibility of employing FDG-PET/CT as a diagnostic for NF in Australian hospitals. My review of antimicrobial prescribing in NF showed that use of very broad-spectrum antibiotics and addition of gram positive agents was often inappropriate, likely reflecting clinician uncertainty about NF aetiology and concern about ongoing fever and its consequences in vulnerable patients. To explore whether improved diagnostics could aid in better targeting of antibiotics, the functional metabolic FDG-PET/CT scan was compared with the standard CT scan in persistent or recurrent NF and showed a higher rate of antimicrobial rationalisation in the FDG-PET/CT group without a signal of inferior clinical outcomes. This study suggested that FDG-PET/CT may be an important tool for aiding antimicrobial stewardship in high-risk NF. In a dedicated study, FDG-PET/CT also performed better than conventional CT in assessing the extent and response to treatment of invasive fungal disease (IFD). Finally, for assessment of the feasibility of introducing FDG-PET/CT to the diagnostic work-up of high-risk NF, the attitudes and access to FDG-PET/CT scanning were assessed among Australian haematology and infectious diseases clinicians, showing that despite lack of funding, FDG-PET/CT was often used for this indication, with fair on-site access, although timeliness of this access for an urgent indication was suboptimal. There was a clear appetite to utilise FDG-PET/CT more if dedicated funding were available. This body of work shows that antimicrobial prescribing in high-risk haematology patients can be improved using FDG-PET/CT and that clinicians recognise its value but have limited access. Future studies will examine the cost-effectiveness of FDG-PET/CT with data derived from this thesis.
-
ItemExploring the immune landscape in penile squamous cell carcinoma and establishing preclinical models to explore therapeutic options( 2022)Penile squamous cell carcinoma (SCC) is a rare disease that has demonstrated little improvement in survival over the last 6 decades. The current management of penile cancer is for surgical management of the primary penile lesion, whilst advanced penile SCC requires multimodal management, including surgery and combination systemic therapy. Given its rarity, there have been few substantial advances in our understanding of the molecular and genomic drivers of penile SCC, especially for patients with advanced or relapsed disease. Progress in identifying new treatment options for patients with penile SCC have been hindered by a lack of understanding of the underlying biology of the disease and a paucity of appropriate pre-clinical models. This thesis has focused on addressing these deficiencies in addition to assessing the patterns of care and oncological outcomes of a contemporary Australian cohort of patients with penile SCC. Due to the infrequency of penile SCC, I also assessed the quality of the information readily available to patients on the internet which has become an increasingly common source of information accessed by both patients and clinicians. Exploration of the immune landscape in penile SCC was undertaken to assess the immune infiltrate and its spatial relationship to tumour. In addition, the establishment and characterisation of a panel of human penile SCC tumoroids and cell lines across the spectrum of disease stage and HPV status created a platform on which to further explore and facilitate investigation into potential new therapeutic options in this disease.
-
ItemInvestigating the regulation of metabolism by oncogenic transcription factors in liver cancer( 2022)Liver cancer remains one of the most lethal cancers worldwide. Therefore, understanding the pathogenesis of liver cancer is imperative to develop appropriate interventions to combat the disease. Although liver cancer is a heterogeneous disease at the genetic level, one of the unifying features is the deregulation of transcription factors. These transcription factors reprogram metabolism to promote liver tumourigenesis by mechanisms that are still poorly understood. In this thesis, I examined the role that two oncogenic transcription factors play in regulating metabolism and promoting liver growth. Firstly, I investigated the molecular mechanism by which the Hippo pathway nuclear effector, Yes-associated protein (Yap) reprograms lipid metabolism to promote hyperplastic liver growth. Secondly, I studied the effects of commonly amplified oncogene Myc, factor in regulating nucleotide metabolism to promote dysplastic liver growth. Thirdly, I examined the consequence of simultaneous expression of Yap and Myc in the liver to promote tumourigenesis. The central aim of my PhD project was to determine the role that oncogenic transcription factors, namely Yap and Myc play in regulating metabolism during pre-malignant growth and tumorigenesis. Liver cancer occurs in the context of chronic liver disease, where several stages of liver disease eventually develop into liver cancer. In this thesis, I examined the pre-malignant and tumour initiation stages of liver cancer using the zebrafish as an in vivo model. I asked several fundamental questions: 1) What are the key features of the liver tissue following activation of oncogenic transcription factors? 2) What metabolic pathways are regulated by oncogenic transcription factors to promote liver growth? 3) Can these metabolic pathways be targeted to suppress oncogenic liver growth? Using the zebrafish as an in vivo model, I found that: 1) Yap transcriptionally activates serum and glucocorticoid-regulated kinase 1 (SGK1), a regulator of the phosphoinositide 3-kinase (PI3K)- mechanistic target of rapamycin target complex 1 (mTORC1) pathway to promote activation of sterol regulatory-element binding protein (SREBP) lipogenic program. Consequently, this leads to hyperplastic liver growth, which was also characterised by lipid droplet (LD) accumulation in the hepatocytes. 2) Myc transcriptionally activates inosine monophosphate dehydrogenase (IMPDH) to promote de novo guanosine triphosphate (GTP) biosynthesis. This metabolic change promoted dysplastic liver growth, characterised by nucleolus expansion and hepatocyte dedifferentiation. 3) Simultaneous activation of Yap and Myc promote rapid liver tumourigenesis. Classical Yap and Myc gene signatures are upregulated in the liver tumour, which is sensitive to the standard of care chemotherapy, sorafenib. While activation of a single oncogene was not sufficient to induce tumourigenesis, activation of Yap or Myc was able to recapitulate aspects of pre-malignant liver growth. Specifically, hepatocyte-specific Yap activation promoted a fatty liver-like phenotype (steatosis). On the other hand, hepatocyte-specific Myc activation promoted dysplasia, similar to that typically found in the dysplastic nodules of cirrhotic livers. Simultaneous Yap and Myc activation caused rapid liver tumour development that models the initiation of liver tumorigenesis. All in all, I showed that oncogenic transcription factors reprogram metabolism to promote liver growth and tumourigenesis. I revealed that de novo lipogenesis (DNL) and de novo GTP biosynthesis are metabolic vulnerabilities in Yap or Myc driven cancer, respectively.
-
ItemCharacterisation of Novel Candidates in the Regulation of Lymphangiogenesis and Lymphatic Vessel Remodelling( 2022)The lymphatic vasculature is a key component of the vascular system, as it is essential for tissue fluid homeostasis, immunity and fatty acid absorption. The malfunction of lymphatic vessels is seen in numerous disorders and diseases, including cancer, infection and lymphoedema. In cancer, the growth and remodelling of lymphatic vessels can facilitate tumour metastasis and correlates with disease progression and low survival. Insufficient lymphatic vessel development or direct damage to lymphatic vessels results in lymphedema while the correct formation of new lymphatic vessels is an important stage of wound healing. Considering the overall importance of lymphatics, understanding their key regulatory molecules and signalling pathways involved in formation and growth of lymphatics is important to develop novel therapeutic targets and approaches. Lymphatic vessels can be classified as blind-ended initial lymphatics, pre-collecting lymphatics and collecting lymphatics, and all are lined on the luminal side by a single layer of cells, known as lymphatic endothelial cells (LECs). Both the remodelling of existing lymphatic vessels and the generation of new lymphatic vessels (lymphangiogenesis) rely on the migration of LECs, however the molecular mechanisms and related signalling pathways are still not completely characterised. A previous genome-wide small interfering RNA (siRNA) migration screen of primary human dermal LECs revealed multiple candidate genes that regulated LEC migration in vitro. These candidates were then compared with LEC-specific differentially expressed genes derived from a microarray study of mouse lymph nodes undergoing lymphatic remodelling. Based on these findings, this study undertook to a shortlist and extensively characterised some of these candidate genes and uncovered their mechanisms of regulating LEC migration. To identify key genes regulating LEC migration, validated genes from the genome-wide screen were re-screened and a selection of candidates was analysed on the basis of their related pathway information and expression in different datasets using bioinformatics and data valorisation protocols. Secondly, a scratch wound migration screen of a small list of candidates was performed using siRNA, and six genes were validated due to their strong and reproducible effects on migration of LECs. Thirdly, their expression in LECs and function in migration and proliferation were investigated with small molecule inhibitors or agonists. Eventually, vitamin D receptor (VDR) was identified as playing an important role in LEC migration. VDR is the key component of the vitamin D signalling pathway and plays an important role in tissue homeostasis, while being less explored in vascular biology. This thesis undertook various in vitro and in vivo analyses to define the functional role and regulation of VDR in LEC signalling and biology. By siRNA analysis, VDR was shown to be a key regulator of LEC migration. Interestingly, human neonatal dermal LECs expressed low levels of a specific VDR isoform, VDRX1 instead of the major isoform, VDRA, which has been characterised in other VDR responsive cells. Our findings showed that the agonist for VDR did not affect migration or proliferation of LECs, which suggested that it potentially acted in a ligand-independent manner. RNA-sequencing analysis revealed more than 1000 genes and 15 signalling pathways that were significantly changed after VDR knockdown, including increased inflammatory response and decreased endothelial to mesenchymal transition. Using Vdr knockout mice, the roles of VDR in lymphatics in both normal tissue and specific pathological conditions were investigated using specific models. VDR was involved in the development of dermal lymphatic vessels in the mouse embryo and the maturation/remodelling of dermal lymphatic vessels in 5-day old postnatal mice. Notably, VDR showed dual roles in lymphangiogenesis and lymphatic remodelling through directly modulating lymphatic sprouting and indirectly through effects on inflammatory responses during wound healing in the ear. Overall, this thesis uncovered the role of VDR in LEC migration and the growth and remodelling of lymphatics. Furthermore, LECs were found to express an under-studied isoform, VDR-X1, the unique function of which remains to be determined. The deeper analysis on the downstream signalling pathways of VDR provided insight into a potential mechanism underlying its effects on LEC migration. In all, the important role of a new regulator, VDR, in growth and remodelling of lymphatics was elucidated, presenting a potential novel therapeutic target to treat diseases associated with lymphatic dysfunction.
-
ItemNo Preview AvailableDevelopment of new methods for accurate estimation of tumour heterogeneity( 2022)It is now understood that intra-tumor heterogeneity is one of the leading determinants of therapeutic resistance and treatment failure and one of the main reasons for poor overall survival in cancer patients. However, the possibility to study this phenomenon is so far underexplored as the acquisition of multi region data sets from the different tumour sites can be ethically challenging. With circulating tumour DNA (ctDNA) used as a proxy for tumour biopsies, it is possible to analyse a snapshot of the unified heterogeneity in each patient, but there is still an unmet need for new methods to optimize the analysis of these large-scale, high-dimensional data to derive new treatment targets. The contributions of this work include the development of multiple new methods, which show that the analysis of bulk sequencing from tumour tissue and ctDNA has unrealised potential for both diagnostic and research questions. This thesis presents three distinct but related projects, which explore the analysis of tumour heterogeneity at different levels and depths, focusing on method development. First, we developed a workflow to improve the detection of somatic variants present at very low allele frequencies. When multiple samples, separated in time or space, from the same patient were available, we were able to substantially improve the detection threshold of variants. These low abundance variants are invaluable in a clinical setting, where they can indicate an arising resistance mechanism or relapse of disease. With the improved sensitivity of our method, the treatment of patients can be adjusted earlier and more accurately. We then used our new analysis workflows to explore evolutionary trajectories and resistance pathways of five lung cancer patients enrolled in the CASCADE autopsy program. In addition to analysis of somatic variants, we used copy number analysis and structural variants to contrast and compare each sample within a patient to generate phylogenies to visualise the evolutionary distances and a pseudo time scale to assess the timing of mutations. Clear genomic determinants of treatment resistance were identified for three of the five cases with non-small cell lung cancer and the diversity of these genomic mechanisms profoundly highlighted the true extent of inter-patient heterogeneity. This work included the identification of a novel genomic resistance mechanism to the drug selpercatinib, a small molecule inhibitor of REK kinase. Among the remaining two patients, treatment resistance was mediated by transformation of their disease from non-small cell lung cancer to a small cell lung cancer histological phenotype. These two cases showed distinct evolutionary trajectories compared to the other non-small cell lung cancer cases, with similarity in their nuclear and mitochondrial phylogenies, but no clear genetic determinant for the small cell transformation, highlighting the additional importance of non-genomic mechanisms which can drive resistance in this disease. Finally, we developed a method, called MisMatchFinder, to monitor tumour heterogeneity and evolution over time through ctDNA. We tailored the method to be fully tumour agnostic and enabled it to be easily applied in the clinical setting by using low-coverage whole genome sequencing. The method uses highly specialised filtering steps to enrich the tumour signal and eliminate the background noise from normal cell-free DNA and sequencing errors in these data. We showed that the method could accurately detect specific cancer-related signatures at low tumour purity and tumour burden in simulated and patient data for melanoma and breast cancer. In summary, with this work we contributed multiple new methods to study, measure and understand genetic tumour heterogeneity. This understanding is crucial for the continuous optimisation of cancer management and the development of new and effective treatments for patients.
-
ItemInvestigating mechanisms of tumourigenesis and novel therapeutic strategies in oesophageal adenocarcinoma( 2022)The prognosis for patients with oesophageal adenocarcinoma (OAC) is dismal, largely due to the late stage at which most diagnoses are made and a paucity of treatment options for metastatic disease. OAC is known to develop from a metaplastic precursor lesion called Barrett’s oesophagus. A large number of genomic changes have been found to occur across the progression of Barrett’s neoplasia, where the mutational burden increases stepwise from the initial metaplasia through to dysplastic tissue and, eventually, invasive cancer. Interestingly, the mutation spectrum is broad and genomic alterations are seemingly stochastic across the stages of progression. The exceptions to this are the tumour suppressor genes, tumour protein 53 (TP53) and SMAD4, for which genomic alterations have been identified as being specific to stages of progression. Mutation of TP53 in is frequently identified in high-grade dysplastic tissue, whilst genomic loss or mutation of SMAD4 is seen only in the invasive disease stage. SMAD4 is the central mediator of the transforming growth factor beta (TGFbeta) family of signalling pathways. The presence of SMAD4 is crucial for canonical TGFbeta-mediated cell processes, such as cell cycle regulation, and its loss in OAC constitutes a particularly poor prognosis. Previous work by our group has shown that loss of SMAD4 on a background of mutant TP53 is sufficient to drive Barrett’s tumourigenesis. However, tumour formation was preceded by a notable period of latency, suggesting that further driver events were required for transformation. This thesis examines the functional impact of loss of the tumour suppressor gene, SMAD4, in oesophageal adenocarcinoma and its precursor lesion, Barrett’s oesophagus. A combined in vitro and in vivo genome-wide CRISPR-Cas9 knockout screen identify co-operative drivers of tumourigenesis and synthetic lethal interactions in SMAD4-deficient oesophageal cancer. Transcriptomic and proteomic analyses combined with in vitro studies delineate the mechanisms of these interactions. A patient-derived organoid model of Barrett’s oesophagus was also established to further investigate these findings. In sum, this thesis delivers insight into novel roles for SMAD4 in OAC and Barrett’s tumourigenesis, identifying a SMAD4-deficiency signature that challenges the paradigm for key regulators of mitosis and genomic stability, whilst providing a new model for future exploration of this phenotype.