Centre for Cancer Research - Theses
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ItemInvestigation of the tumour microenvironment of prostate cancer( 2018)One in seven men in Australia is at risk of developing prostate cancer before the age of 75. This disease is a leading cause of male death worldwide, with a mortality rate of 62 men per 100,000. Treatments for prostate cancer exist, including surgery, radiotherapy and androgen deprivation therapy. However, the results achieved by the combination of these therapies can lead to variable outcomes, mainly due to the genetic heterogeneity of tumour cells and/or emergence of resistance. In contrast to the cancer cells, the non-cancerous portion of the tumour microenvironment, such as immune cells, fibroblasts and endothelial cells, is a genetically stable target that has a key role in cancer development. Improving our knowledge of the genetic and molecular interactions existing between cancer cells and other non-cancerous cell populations, both in primary or metastatic prostate cancer will provide new key insights in the biology of the disease and give new treatment opportunity.
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ItemImmune markers to predict response to neoadjuvant chemoradiotherapy and identifying methods to incorporate immunotherapy in locally advanced rectal cancer( 2018)The standard of care for locally advanced rectal cancer (T3-4 +/- N+) is neoadjuvant chemoradiotherapy (CRT) followed by total mesorectal excision (TME). However, this has been challenged recently with increasing interest and trials assessing the efficacy and safety of avoiding TME. This concept is known as the “watch and wait” strategy, if patients were deemed to have clinical complete response (cCR). The current limitation is the risk of local tumour regrowth rate between 20-30%, as cCR is not equivalent to pathological complete response (pCR). Therefore, this thesis describes the development of a novel immune cytotoxic assay for measuring patient-matched cytotoxic T cell-mediated killing of rectal cancer organoids. Subsequently a proof of principle prospective observational study was conducted, showing in those patients with pCR, their cytotoxic T cell-mediated killing were highest when compared to non-pCR. Furthermore, this was a stark difference without overlapping of 95% confidence interval when compared to the partial and non-responding T cell-mediated killing of rectal cancer organoids. At the other end of the spectrum, those that had failed to achieve any response to neoadjuvant therapy will not have any other therapeutic option left to increase their tumour response rate. Promising emerging therapies employing immunotherapy by check-point inhibition and/or targeted-vaccine are now highly relevant, especially inhighly immunogenic colorectal cancer such as microsatellite instability high subset due to high somatic mutation. A similar tumour microenvironment has been documented after induction radiotherapy, with success of check-point inhibition shown only in mouse models. Using the immune cytotoxic assay, this thesis demonstrates the increased in patient-matched T cell-mediated killing of rectal cancer organoids in the presence of check-point inhibition. This opens another avenue to explore the utility of immunotherapy using a T cell-organoid model, with the potential for investigating and identifying novel markers to immune resistance.
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ItemInterplay between MDM4 and P53 in breast cancer( 2018)Breast cancer (BC) is one of the most prevalent cancer types and second leading cause of cancer death among Australian women. BC is a genetically heterogeneous disease, divided into sub-types that differ in their aggressiveness, metastatic potential and response to treatment. Notably, relapsing triple negative BC (TNBC) have limited therapeutic options and a poor survival. A key driver of metastatic TNBC is the mutated form of p53. P53 is a major tumour suppressor, and plays a pivotal role in the cellular response to stress conditions. The function of the key tumour suppressor protein p53 is compromised frequently in cancers, either by direct mutation or by its deregulation. The latter is achieved largely by two key negative regulators: MDM2, its E3 ligase, and MDM4 a potent inhibitor of p53’s transcriptional activities. This thesis describes the interplay between p53, both wild-type (wt-p53) and mutant forms, and its regulator MDM4 in multiple subtypes of BC. In chapter 3 of this thesis, we identified MDM4 as a potent negative regulator of wt-p53 in BC, and its depletion impedes the growth of BC cells, both in vitro and in vivo experimental models. MDM4 knockdown (KD) reduces tumour progression in vitro and in vivo by activating the cell cycle inhibitor p21 in a p53-dependent manner. Our screen of BC samples in a tissue microarray and cell lines revealed high levels of MDM4 expression not only in wt-p53 expressing BC, but also in basal-like and luminal BCs that express mutant p53 (mt-p53). This prompted us to test the contribution of MDM4 to the oncogenesis of BC that expresses mt-p53, which is described in chapter 4. Surprisingly, we have demonstrated that KD of MDM4 in a range of BC cell lines expressing mt-p53 across multiple subtypes impedes their growth in culture. The therapeutic potential of this approach was further validated using XI-011, a small molecule inhibitor of MDM4 in vitro in TNBC cell lines. In a search for the underlying mechanism of growth inhibition in response to MDM4 depletion, we have established the involvement of p27 protein, a key cell cycle inhibitor. The inverse correlation between the protein expression of p27 and MDM4 was found in our basal-like BC tissue microarray supporting the relationship between p27 and MDM4 in these patients. Mutations in p53 not only result in loss of wt-p53’s tumour suppressive functions, but importantly certain hot spot mutations also acquire Gain Of Functions (GOFs), which drive oncogenesis, and in particular metastatic disease. These render mt-p53 expressing cells addicted to the expression and function of mt-p53. This opens an exciting therapeutic opportunity to target mt-p53 in these cancers. In chapter 5 we tested the effect of the first in class wt-p53 reactivating drug, APR-246, which is currently in multiple clinical trials. We rationalized that combining APR-246 treatment with MDM4 targeting would potentiate this treatment by protecting the ‘reactivated wt p53’ to suppress tumour growth. A combinatorial therapeutic strategy using APR-246 to reactivate mt-p53 and XI-011, which targets MDM4, was very effective in the inhibition of TNBC growth in vitro. We also demonstrated the potency of XI-011 and APR-246 combination to inhibit the growth of cell line xenografts and patient-derived xenografts in vivo. Overall, this thesis demonstrates an essential role for MDM4 in the establishment of oncogenesis in BC cells expressing wt-p53 and mt-p53 and defines MDM4 as an attractive therapeutic target in both single and combinatorial therapy to treat mt-p53 expressing BC. The efficacy of MDM4 targeting in mt-p53 cancers challenges current dogma and opens the window for a novel therapeutic approach in these difficult to treat BCs.
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ItemNovel strategies to enhance the therapeutic efficacy of chimeric antigen receptor T cell therapy against solid cancerAdoptive cell therapy using chimeric antigen receptor (CAR) T cells has shown great success in haematological malignancies, leading to recent approval of two CD19-specific CAR T cell products by the Food and Drug Administration (FDA) for the treatment of B cell Acute Lymphoblastic Leukaemia (ALL) and non-Hodgkin lymphoma. However, despite this high success in CD19+ malignancies, clinical trials for solid cancers have yielded only limited efficacy to date. This discrepancy is thought to be due to, in part, the immunosuppressive tumour microenvironment and antigen heterogeneity in solid tumours. Therefore strategies to overcome these challenges are necessary for CAR T cells to be effective in solid malignancies. In this thesis, we have investigated two approaches aimed at augmenting CAR T cell therapy in a solid cancer setting. The first approach involved the activation of a costimulatory pathway known as CD137 (4-1BB). Targeting this pathway using an agonist α-4- 1BB antibody has been reported to induce robust co-stimulatory signals for diversifying and enhancing T cell responses, which led us to hypothesise that combining CAR T cells and α-4- 1BB antibody would result in improved therapeutic responses. Herein, we utilised a human- Her2 self-antigen mouse model to assess the therapeutic efficacy of Her2-specific CAR T cells against Her2-expressing solid tumours. We report that the use of an α-4-1BB antibody significantly increased CAR T cell anti-tumour responses against two established Her2+ solid tumours. Our analysis revealed an increase in expression of IFNγ and the proliferation marker Ki67 in intratumoural CAR T cells following treatment with α-4-1BB antibody. Strikingly, we also demonstrated that treatment with an α-4-1BB antibody greatly decreased host immunosuppressive cells at the tumour site, including regulatory T cells and myeloidderived suppressor cells, which correlated with an increased therapeutic response. Interestingly, further investigation in immunocompromised RAG-/- mice showed reduced synergistic effects following the combination therapy, suggesting a contribution of endogenous T cells in mediating these effects. Furthermore, we observed an increased frequency of dendritic cells (DCs) and the endogenous T cells exhibiting an effector CD44hi CD62Llow phenotype in tumour-draining lymph nodes following α-4-1BB antibody stimulation, further supporting the potential involvement of the endogenous immune cells. Taken together, these data indicate an important contribution of the endogenous immune system in enhancing overall CAR T cell efficacy, and thus formed the basis for our second approach that aimed to improve CAR T cell therapy by harnessing the endogenous DC population. Herein, we genetically modified Her2-specific CAR T cells to constitutively secrete a cytokine Flt3L, that is known to be critical for the differentiation of an important subset of DCs, the cross-presenting Batf3-dependent XCR1+ IRF8+ CD103+ DCs. We hypothesised that inducing the differentiation of these CD103+ DCs would increase the overall therapeutic efficacy of CAR T cells. We successfully generated Flt3L-secreting CAR T cells, and demonstrated that CAR T cell-derived Flt3L was able to induce expansion of CD103+ DCs and enhance their T cell stimulatory capacity. Adoptive transfer of Flt3L-secreting CAR T cells led to a significant enhancement of tumour growth inhibition, and subsequently enhanced mouse survival compared to the control CAR T cells. Similarly, using a TCR-based adoptive cellular therapy approach, namely the transfer of OT-I T cells against an OVAexpressing tumour, led to superior anti-tumour responses, particularly with the addition of poly I:C adjuvant that resulted in the most significant suppression of tumour growth. In summary, results presented herein demonstrate the promising potential of combining CAR T cells with immunomodulatory agents for enhancing the efficacy of CAR T cells against solid tumours. Our results indicate that in addition to directly augmenting CAR T cell activity, immune-modulating agents are able to enhance the therapeutic efficacy of CAR T cells by modulating the endogenous immune response. Our studies have established a strong rationale for combining CAR T cells and immunomodulatory agents in the treatment of solid cancer, and therefore warrant clinical investigation of these combination therapies.
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ItemTumour immune microenvironment, genomewide copy number aberrations and their interactions in malignant pleural mesothelioma( 2017)Malignant pleural mesothelioma (MPM) is a rare but deadly malignancy. Despite predictions, worldwide incidences have not significantly diminished and available therapeutic modalities have failed to improve outcomes. There are some indications that immune based therapies and targeted therapies may hold promise but suitable phenotypes have been hard to identify. This thesis describes investigations we conducted into the tumour immune microenvironment, the copy number aberrations (CNAs) and the interaction between the two in MPM. The findings presented in this thesis elucidate the complexity of the tumour immune microenvironment in MPM. We establish MPM to be immunogenic but also demonstrate that the immune characteristics vary amongst tumours mostly with tumour histological subtype. We reveal sarcomatoid MPMs to be associated with high lymphocytic infiltration but also greater expression of immune checkpoint receptors and their ligands. In addition, we also exhibit the prognostic implications associated with some of these markers. The genome wide copy number analyses described in this thesis disclose that although copy number aberrations are the commoner of genomic alterations in MPM, their extent and pattern vary amongst tumours. We describe their clinical and pathological associations. We confirm most previously described prominent CNAs but more importantly uncover new ones which may be important in MPM prognostication and therapeutics. We find that greater degree of genomic instability confers a worse prognosis in MPM independent of histology but neither degree of genomic instability nor any specific CNA correlates with the immunological milieu. Our findings contribute towards the understanding and development of potential prognostic and predictive markers for immunotherapy in MPM. Further, they also reveal potential targets for development of targeted therapies in this disease where none was thought feasible.
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ItemReducing outcome disparities for rural Victorians with colorectal cancer: understanding pathways to treatment and informing policy.( 2017)In Victoria, rural patients with colorectal cancer have poorer outcomes than urban counterparts. To date, research and policy initiatives have focused on addressing potential variation in cancer treatment. However, pre-treatment delays may also be important. Policy change is required to reduce disparities for rural patients with cancer. Determining potential models of rural cancer care and understanding the policymaking process could enhance the success of future policies to address geographic cancer disparities. The overarching aim of the research program was to develop evidence to inform potential health system policies to reduce outcome disparities for colorectal cancer patients in rural Victoria. There were three research objectives: 1. Compare rural and urban patient pathways and experiences to diagnosis and treatment for those with colorectal and breast cancer in Victoria; 2. Identify potential models of rural cancer care that could inform future policies; and 3. Understand how large-scale health service policies in cancer care develop and are implemented. The research used a multi-phase, mixed methods design in five studies. The first phase examined rural and urban patient pathways to colorectal or breast cancer diagnosis and treatment in two studies. Breast cancer patients were included as a comparison group since outcomes are equivalent for women across Victoria. In the first study, interviews with 43 patients showed that rural and urban patients with either cancer experienced relatively similar pathways, though some had difficulty accessing GPs and longer time to specialist referral. Qualitative results informed hypotheses tested in a quantitative study of time intervals to treatment. Survey data from 922 patients with colorectal (n=433) and breast cancer (n=489), 621 GPs and 370 specialists were supplemented with cancer registry data. In quantile regression analyses, the time from first symptom or screening test to treatment was significantly longer for rural than urban patients with colorectal cancer, but not breast cancer. This was likely driven by longer diagnostic intervals for rural patients with colorectal cancer. In the second phase, patient interview data were re-analysed to explore experiences of choice of cancer treatment provider. Although most patients had limited involvement in choosing a provider, decision-making considerations were more complex for rural than urban patients. Studies in the final phase were a scoping review of rural cancer models of care and an interview study with 13 local and international key informants regarding the policymaking process. Across 47 reviewed articles, telehealth models were most common. Navigator and alternative provider models were identified for the pre-treatment period, but very few studies measured time to cancer care, and none assessed clinical cancer outcomes. Developing and implementing cancer policies at national or state-level was found to involve specific change mechanisms, such as stakeholder collaboration and evidence-use, influenced by the physical, political and temporal context. Based on these results, it is recommended that policies to address rural–urban inequities in colorectal cancer patient outcomes in Victoria focus on the diagnostic interval. Initiatives such as GP endoscopy, waiting time reporting or diagnostic centres should be investigated, acknowledging the potential impact of context in policy-change. The current research provides a baseline to assess the impact of future policies, and a starting-point for further research to understand policy development and implementation in cancer care.