Sir Peter MacCallum Department of Oncology - Theses
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Coming of age with Li-Fraumeni syndrome: perspectives of young people and health professionals
This thesis is situated in the discourse of risk that defines our technology-driven modern society, wherein one’s health is constructed as a personal and moral responsibility. A key contributor to the individualisation of risk in modern medicine is genomic technology. As genomics becomes progressively normalised in mainstream society, individuals of younger and younger ages are seeking to learn of their genetic risk of disease, including cancer. Young people occupy a formative and transitional life stage with complex processes of human development, making them a unique population for which genetic services are currently ill-equipped to serve appropriately. This research contributes to a new field of genetic counselling research that aims to explore and meet the distinctive developmental needs of young people with genetic disease. This thesis comprises of three inter-related studies that explore the psychosocial implications of living with a devastating, early onset inherited cancer condition, Li- Fraumeni syndrome (LFS), from the perspectives of young people and health professionals in Australasia. Informed by a pragmatic-critical realist stance, this thesis uses a mixed-method approach divided into a qualitative and quantitative phase. The qualitative phase consists of two studies, the first is a systematic review and thematic synthesis of 39 studies investigating how young people experience inherited disease with similar implications to LFS. The second and principal study of this research is an interpretive description of how young people experience LFS in their daily lives. To develop findings, I conducted interviews with 30 adolescents and young adults (aged 15- 39 years) with, or at 50% risk of, a pathogenic germline mutation in TP53 from across Australia, and used reflexive thematic analysis. The quantitative phase consists of a survey of 43 Australasian health professionals who care for young people with LFS to document their current practices and how they meet the developmental needs of this population. The first key finding is that experiences of cancer, either familial or personal, strongly influence how young people make sense and meaning of LFS, how they consider genetic testing, and their perceptions of cancer risk and mortality. The nature of LFS in terms of its high penetrance, early onset, and varied phenotype meant young people’s experiences were unique in oncology and genetic settings and require in-depth exploration during genetic counselling. The second key finding was that young people’s growing autonomy from family complicated the process of genetic testing, which was recognised by health professionals who worked to foster their autonomous decision- making. Genetic testing and whole-body cancer risk management represented instrumental actions of control for young people to mitigate their cancer risk from LFS, a perspective shared and promoted by health professionals. Intensive risk management and risk-reducing surgery, however, was emotionally and logistically burdensome for some young people, constructing the body as a material object be scrutinised in great detail by health professionals and blurring boundaries between self and body. The third key finding was that young people felt strongly about preventing the passage of their LFS- causing genetic variant to the next generation, reporting that pre - implantation genetic diagnosis was their preferred method for having a ‘healthy’ biological family. Few had reached a life stage, however, where they appreciated the ethical, financial and logistical burdens of this technology, and many deferred reproductive decision-making until they were ‘ready’. This research argues that young people with inherited disease have unique psychosocial and clinical needs that are directly tied to the formative developmental tasks of their life stage. They require specialist youth-friendly counselling that considers and appreciates their developmental needs both during genetic testing and beyond. Youth-friendly genetic counselling must therefore be longitudinal, incorporating psychosocial check-ups as a key clinical interaction. Further, youth-friendly models of care promote the distinct value of genetic counsellors in managing rare and complex inherited conditions. As the mainstreaming of genetic services begins to outsource tasks of genetic education and pre-test counselling, the genetic counselling profession must make better use of their psychosocial counselling skills to serve high-needs populations. In this context, young people stand out as a key focus. Finally, this research argues that attending genetic counselling exposes young people to a discourse of risk bounded by an ethos of responsibility. Undergoing genetic testing, subjecting their bodies to intensive surveillance and risk-reducing surgery, and having a family with reproductive technology all stand out as the ‘right thing’ to do when living with genetic risk of disease. Yet, each presented complex psychosocial implications for young people that were intrinsically linked to the broader effects of our modern risk society defined by Beck (1992) and Giddens (1991). Young people’s decision-making and preferences during this developmentally labile life stage therefore need to be interpreted in relation to normative societal pressures that dictate expected behaviour around risk.
Investigating drug response and resistance to IDH1R132H inhibition and hypomethylating agents in AML
Mutations in epigenetic regulators frequently occur in AML. Furthermore, the epigenetic landscape is typically dysregulated in AML. Targeted epigenetic therapeutics that directly inhibit mutant epigenetic oncogenic drivers, such as AG120 inhibition of IDH1R132H, have shown clinical success for the treatment of IDH1-mutant AML. Other epigenetic therapies target wildtype epigenetic regulators, such as DNMT1 inhibition by hypomethylating agents, with the aim of reprogramming the transcriptional networks driving malignant progression. Despite some patients achieving clinical remission, many AML patients do not respond to epigenetic therapies or relapse post-treatment. Furthermore, the contribution of mutant epigenetic regulators to leukaemogenesis is relatively poorly understood. A clinically and pathophysiologically relevant murine model of IDH1R132H driven AML co-expressing oncogenic DNMT3A and Nras was utilised to investigate the cellular and molecular consequences of IDH1R132H expression in leukaemia initiation and progression. Results from this thesis suggest that IDH1R132H governs distinct leukaemic properties within the different AML cell-types; IDH1R132H drove the expression of genes that underpin HSC-like self-renewal and proliferation in leukaemic progenitor-like cells, whilst IDH1R132H most profoundly impaired the differentiation capacity of leukaemic immature neutrophils. Furthermore, this thesis demonstrated that AG120 was a highly effective and on-target IDH1R132H inhibitor for the treatment of IDH1R132H driven in vivo AML, and induced neutrophil-skewed differentiation with distinct kinetics throughout the hierarchy of leukaemic cells. Mechanisms of resistance to the hypomethylating agents AZA and GDAC were investigated utilising a genome-wide CRISPR/Cas9 screen in vitro. Members of the pyrimidine salvage pathway were implicated in the resistance to hypomethylating agents. Loss of DCK and UCK2 conferred resistance to GDAC and AZA, respectively, whilst SLC29A1 loss mediated resistance to both hypomethylating agents in both in vitro and in vivo models of haematological malignancies. This thesis demonstrated that DCK and UCK2 loss remained sensitive to AZA and GDAC, whilst cells with SLC29A1 loss were sensitive to inhibition of DHODH. This research provided comprehensive insight of the impact of IDH1R132H on distinct leukaemic cell subsets, transcriptional mechanisms underlying leukaemogenesis, and the transcriptional perturbations that may contribute to AG120 resistance. Furthermore, the results of this thesis suggest that AZA and GDAC treatment are potential therapeutic avenues for AML resistance driven by DCK or UCK2 depletion or loss, respectively. Moreover, the results described herein suggest that targeting the de novo pyrimidine synthesis pathway, through DHODH inhibition, is a therapeutic strategy to overcome resistance to hypomethylating agents mediated by the depletion of pyrimidine salvage pathway enzymes.
Deciphering tumour heterogeneity in acute myeloid leukaemia at the single cell level
The advent of next-generation sequencing (NGS) has allowed researchers to appreciate the enormous heterogeneity that exists between cells within a single tumour. This intratumour heterogeneity leads to diverse phenotypic outcomes, resulting in functionally distinct subpopulations of cancer cells. This functional heterogeneity fuels tumour evolution and therapeutic resistance and is thus a major barrier to producing cures in cancer. Acute myeloid leukaemia (AML) is an aggressive and heterogeneous malignancy with a high relapse rate. The prevailing paradigm to explain relapse in AML posits that genetic heterogeneity leads to pre-existing or acquired mutations that render certain cells refractory to therapy, resulting in the outgrowth of a resistant clone. Large-scale sequencing studies aimed at cataloguing genetic heterogeneity in AML have revealed several important observations. Firstly, AML has one of the lowest mutational burdens of any cancer. Secondly, a significant proportion of clinical relapse events cannot be attributed to an underlying genetic change. These important findings raise the possibility that mutations alone are insufficient to fully explain therapeutic resistance in AML. Indeed, we are now beginning to appreciate that both tumour evolution and clinical relapse can be driven by non-genetic processes. However, characterising the full extent of non-genetic heterogeneity and its relative contribution to both the evolutionary trajectory of the disease and therapeutic resistance requires innovative single cell methodologies. Single-cell RNA sequencing (scRNA-seq) has been instrumental in revealing the phenotypic heterogeneity of rare subpopulations of cells within a complex tumour. However, it is difficult to infer clonal relationships from scRNA-seq alone and this has hampered our ability to understand how individual malignant cells evolve over time. To overcome some of these challenges, we present a lentiviral method of tagging cells with unique heritable barcodes that are stably transcribed into RNA molecules in cells and therefore highly detected in microfluidic scRNA-seq workflows. This strategy, termed Single-cell Profiling and LINeage TRacking with expressed barcodes (SPLINTR), offers the ability to match the gene expression programmes of individual cells to their clonal lineage, in order to establish how initial transcriptional differences amongst heterogeneous malignant cells can shape thier future clonal behaviour during cancer progression. We apply our SPLINTR barcoding system to an in vivo model of clonal competition in order to determine the early transcriptional signatures that are associated with future clonal dominance in AML. We discover that clonal dominance is largely an intrinsic property amongst genetically identical clones. However, we find the deterministic nature of dominance is altered by the presence of other distinct competing mutational clones. Furthermore, SPLINTR enabled us to retrospectively identify a novel set of differentially expressed genes contained within certain clones prior to transplantation, which distinguished them from losing clones and was associated with their future dominance during disease progression. Finally, we find that resistance occurs to BET inhibitor therapy in the clinic in the absence of a clear genetic event. scRNA-seq of paired baseline and relapse AML patient bone marrow samples revealed than non-genetic resistance originates from either a population of pre-existing cells that phenotypically resemble LSCs, or through transcriptional adaptation as a result of therapeutic pressure. We then use SPLINTR coupled with scRNA-seq to interrogate our previously published in vitro model of non-genetic resistance to BET bromodomain inhibition. This provided further evidence that Lamarckian evolution in the form of gradual transcriptional adaptation drives non-genetic resistance. Future work aims to unravel the epigenetic states that mediate non-genetic transcriptional adaptation in a broader therapeutic context in AML. Collectively, the research presented in this thesis demonstrates the importance of applying novel single cell technologies to investigations of cellular diversity in cancer and highlights the underappreciated role of non-genetic heterogeneity in driving both disease evolution and therapeutic resistance in AML. These studies provide the molecular tools and rationale to further define the mechanisms by which non-genetic heterogeneity shapes cellular behaviour in cancer.
Enhancing CAR T cell therapy
Adoptive cell therapy using chimeric antigen receptor (CAR) T cells has shown remarkable efficacy in the treatment of haematological malignancies, with complete remission rates of 90% reported in early clinical trials. Following this success, two CD19 targeting CAR T cell products have been approved in Australia for the treatment of Acute Lymphoblastic Leukaemia (ALL) and non-Hodgkin lymphoma. However, acquired resistance to this therapy through CD19 antigen loss is an emerging problem. Moreover, such promising results have not been recapitulated against solid tumours. This is thought to be due in part to poor CAR T cell trafficking and infiltration, the immunosuppressive tumour microenvironment and antigen heterogeneity in solid tumours, as well as intrinsic and acquired tumour resistance. Therefore, it is necessary to uncover mechanisms of resistance to CAR T cell therapy in both the haematological and solid tumour settings, in order to improve patient outcomes. In this thesis, we sought to uncover the tumour-intrinsic mechanisms of resistance to CAR T cell therapy. Whole-genome loss-of-function CRISPR-Cas9 screening has recently emerged as a powerful tool to screen all protein-coding genes in the genome for conferring resistance to various immune and drug pressures. Herein, we utilised a genome-wide mouse pooled-sgRNA library to investigate genes that protect MC38 mouse adenocarcinoma cells from CAR T cell killing. Here, we identified that loss of cytokine response pathways, in particular, TNF and IFN-y signalling were critical for the effector function of CAR T cells. Additionally, we report for the first time that loss of the transcriptional co-binding partner Cbfb protects tumour cells against T cell killing. Investigation in to the mechanisms of Cbfb-mediated resistance uncovered that loss of tumour cell Cbfb protected tumour cells against T cell-derived TNF. Importantly, we report that mice bearing Cbfb-deficient MC38 tumours did not respond to anti-PD-1 in vivo, and analysis of clinical trial data suggests that in melanoma cohorts, low expression of CBFB or the transcriptional co-binding partner RUNX1 was correlated with a poorer prognosis following immune checkpoint blockade or adoptive cell therapy. Next, we sought to design a rational combination therapy to overcome some of the challenges associated with CAR T cell therapy against solid tumours and enhance CAR T cell therapy in this context. Based on data from the CRISPR screen which highlighted the role of TNF in CAR T cell-mediated cytotoxicity, we sought to enhance TNF-mediated killing using a small-molecule smac-mimetic. We demonstrated that antagonism of the Inhibitor of Apoptosis Proteins with the smac-mimetic birinapant, significantly enhanced CAR T cell killing in a TNF-dependent manner. Using a syngeneic HER2+ self-antigen model, we report that birinapant significantly enhanced CAR T cell-mediated tumour clearance in a combination therapy approach, and we demonstrated enhanced human CAR T cell killing in patient biopsy-derived tumouroids. Critically, we report that birinapant significantly enhanced TNF-mediated “bystander killing”, which occurred in the absence of target antigen expression, which may be a strategy to overcome the challenge of tumour antigen heterogeneity associated with solid tumours. Finally, we sought to uncover mechanisms by which B cells lose CD19 antigen expression following CAR T cell attack. By utilising a second genome-wide CRISPR screening approach, we applied a human pooled sgRNA library to the CD19+ B-ALL cell line MHH-CALL4, and sorted 3 times the cells with the lowest 20% CD19 surface expression by flow cytometry, in order to uncover genes or pathways of interest that are implicated in down-regulation of CD19 surface expression. Along with several known factors, such as CD81 trafficking and N-linked glycosylation, we also identified several novel processes including histone methylation, aryl hydrocarbon receptor processing and E3 ubiquitin ligase activity, which will be the focus of future investigations. In summary, insights gained from the work in this thesis elucidate some of the resistance pathways utilised by tumour cells in order to evade immune pressure from CAR T cells, and highlight the cytotoxic potential of TNF in CAR T cell therapy. In addition, we herein present a novel strategy to enhance CAR T cell efficacy in solid tumours, and demonstrate the potential for combination therapy approaches to overcome the challenges associated with CAR T cell therapy against solid tumours. Taken together, this work significantly addresses several of the existing limitation of CAR T cell therapy in both B cell leukaemias and against solid malignancies, and the novel combination therapy we describe is poised to be rapidly translated in to the clinic.
Profiling the immune and genomic landscape of anal squamous cell carcinoma and establishing preclinical models to explore new therapeutic options
Anal SCC is a rare disease that has increased significantly in both incidence and mortality over the last fourty years. Definitive chemoradiotherapy is the primary modality of treatment, offering a 5-year overall survival rate of 65%. For patients with locally persistent or recurrent disease, salvage surgery is an option with a 5-year overall survival of 50%. However, for those patients with un-resectable locoregional or metastatic disease, there are limited treatment options, and patients face a dismal outcome. Progress in identifying new treatment options for patients with anal cancer has been hampered by a deficiency in understanding the underpinnings of the disease and a lack of appropriate preclinical models. This thesis has focussed on addressing both of these deficiencies in addition to assessing the success of salvage surgery at a quaternary centre in Australia. Firstly, an attempt has been made to further our understanding of the biology of Anal SCC. This was undertaken by exploring the immune and genomic landscape of ASCC, to identify potential prognostic and therapeutic biomarkers. This has provided insight into the prognostic power of assessing the CD8+ immune infiltrate in Anal SCC. It has also identified PI3K aberrations as a frequent genomic event that may serve as a future therapeutic target. Secondly, it has led to the establishment of both human and mouse preclinical models of this disease. This includes the world’s first panel of human anal SCC cells lines and a syngeneic mouse model. Both of these pre-clinical models have been validated and characterised, with features closely resembling the human disease. These models can now act as a platform to further explore and facilitate investigation into potential new therapeutic options in this disease.
Harnessing senescence to control PI3K/AKT/mTORC pathway-driven cancer through the metabolic intervention
Oncogene-induced senescence (OIS) is a critical anti-tumour mechanism by arresting cell proliferation following oncogenic activation in normal cells. Cell cycle withdrawal along with senescence-associated secretory phenotype, deregulated metabolism and macromolecular changes are the hallmarks of cellular senescence. The PI3K/AKT/mTORC1 pathway is a key signalling pathway for homeostatic control of cell growth, proliferation, and survival. Molecular aberrations in the PI3K/AKT/mTORC1 signalling pathway have been identified in one third of solid tumours and more than 40 cancer types. Paradoxically, PI3K/AKT/mTORC1 hyperactivation causes a senescence-like phenotype in non-transformed cells. While most studies have focused on RAS/MAPK-induced senescence (RIS), little is known about the mechanisms regulating AKT-induced senescence (AIS) and its potential tumour-suppressive function. We hypothesise that understanding the molecular requirements for AIS and determining the essential regulators involved in AIS will identify therapeutic vulnerabilities for treatment of PI3K/AKT/mTORC1-driven cancer. To characterise the molecular landscape of AIS, we performed the RNA-seq and metabolomics analysis in BJ-TERT human skin fibroblasts overexpressing AKT1 or HRASG12V in Chapter 3. Compared to normal proliferating cells, AIS and RIS cells exhibited distinct metabolomes and transcriptomes, supporting metabolic and transcriptional reprogramming in OIS. The common molecular signatures in both AIS and RIS cells well represent the hallmarks of senescence and the molecular signatures that are unique for AIS or RIS are associated with the distinct molecular mechanisms underpinning these two types of OIS. The similar metabolic profile presented by the AIS and RIS cells with enhanced glycolysis, TCA cycle and oxidative phosphorylation to maximise energy production indicates a highly active metabolic status in OIS. To gain more insight of metabolic dependency of AIS, we utilised Cystathionine beta-Synthase (CBS), one of the top candidates in an RNAi screen we recently performed to identify key regulators of AIS and characterised its role in AIS maintenance in chapter 4. CBS acts as a hub for coordinating the transsulfuration and transmethylation pathways, regulating the biosynthesis of hydrogen sulfide (H2S) and glutathione (GSH) and modulating mitochondrial functions and cellular bioenergetics. We demonstrated that 1) the upregulation of transsulfuration pathway activity is an AIS-specific antioxidant response. 2) the metabolic reprogramming potentially couples to epigenetic alterations to maintain AIS. 3) CBS depletion releases oxidative stress through suppression of mitochondrial oxidative phosphorylation and causes AIS escape. The finding of loss of CBS resulting in the release of the senescence brake engaged by AKT hyperactivation promoted us to further evaluate the potential tumour suppressor role of CBS in gastric cancer. We provided in vitro and in vivo evidence to support that CBS depletion synergises with PI3K pathway activation to promote gastric cancer initiation and restoration of CBS inhibited gastric tumour growth via H2S. These findings provided an example of harnessing metabolic vulnerabilities therapeutically for treating cancer. Overall, our study provides novel mechanistic insights into AIS maintenance and more importantly, reveals the essential role of the metabolic reprogramming in AIS maintenance and escape. Further investigation of the molecular mechanisms underpinning PI3K/AKT/mTORC1-driven senescence and tumorigenesis will facilitate developing appropriate strategies to improve human health in aging, aging-related diseases, and cancer.
Study of the immunomodulatory effects of radiation therapy in solid cancers
Radiation therapy (RT) has evolved over more than a century into a well-established, highly sophisticated and major cancer treatment modality today. A paradigm shift that has occurred within the last two decades is the growing understanding that RT can induce host immune responses that contribute to tumour control, beyond direct radiation-induced cytotoxicity. Contemporaneously, the advent of modern cancer immunotherapy such as immune checkpoint inhibitors has revolutionised the field of oncology and highlighted the potential of harnessing the immune system to suppress and eradicate tumours. Inevitably, resistance to cancer immunotherapy has also brought into focus immunological barriers that preclude cancer immunity. In this context, an increasing body of pre-clinical and clinical studies substantiate the use of RT as a unique candidate to complement cancer immunotherapy in non-overlapping mechanisms to overcome such barriers. However, instruction on the optimal integration of RT and cancer immunotherapy is scarce. For the radiation oncologist, an outstanding gap in knowledge is how radiation dose-fractionation influences the immunomodulatory effects of RT and its synergy with cancer immunotherapy. In this PhD project, mouse models of solid cancer were used to systematically interrogate this question by employing a series of rationally selected radiation dose-fractionation regimens to dissect the immunological impact of dose per fraction (DPF) from that of total dose, as represented by biological effective dose (BED). In orthotopic AT3-OVA mammary carcinomas, radiation-induced CD8+ T cell responses were found to be regulated by radiation DPF, rather than BED. By contrast, radiation-induced natural killer (NK) cell responses in the same tumours were independent of radiation DPF but required a sufficient BED. Mechanistic investigations examining the cellular and transcriptional changes in AT3-OVA tumours evoked by radiation demonstrated that the differential regulation of anti-tumour immune responses by radiation DPF and BED was not primarily dictated by differences in tumour cell-intrinsic immunogenicity, but rather by the effector and suppressor dynamics in the tumour immune microenvironment, of which regulatory T cells played a central role. Furthermore, cross-examination of subcutaneously implanted MC38 colon carcinomas and publicly available transcriptomic data of human cancers pre- and post-RT suggested that radiation-induced immune responses are also significantly shaped by the tumour type. Lastly, the impact of radiation dose-fractionation on the anti-tumour activity of immune checkpoint inhibitors targeting the adaptive and innate immune arms was examined in AT3-OVA tumours, confirming the corollary that RT and immune checkpoint inhibitors do not universally synergise, but require selection of radiation regimens and checkpoint targets that are predicated on biological rationale. Overall, this PhD project represents a comprehensive side-by-side pre-clinical study of the effects of radiation dose-fractionation on host anti-tumour immune responses. Results presented herein contribute towards a clearer understanding of this complex and clinically urgent question. More broadly, insights from this project will help guide the refashioning of RT into an exciting key adjunct in the immuno-oncology era.
Molecular regulation of adipogenesis in secondary lymphoedema: a common complication of cancer therapies
Secondary lymphoedema is a common, chronic disease caused by inadequate drainage of interstitial tissue fluid from a limb due to damaged lymphatic vessels. It may develop after radiation therapy or cancer surgery involving lymph node dissection, in particularly for breast cancer, as well as a variety of other conditions. The resulting accumulation of interstitial fluid promotes pathological changes including oedema, expansion of fat and dermal fibrosis, which contribute to extensive chronic tissue swelling, typically in the upper or lower limbs. There is no curative treatment or molecular-based therapy for secondary lymphoedema, and current treatments for this condition have limited efficacy so it is an important unmet clinical need in medicine with an estimated 300,000 patients in Australia. A pharmacological intervention to restrict or reduce expansion of fat tissue and associated tissue swelling would be highly beneficial for patients however, the molecular mechanisms driving the development of fat in secondary lymphoedema are unknown. Here, a surgical mouse tail model of secondary lymphoedema is employed to investigate molecular pathways that drive expansion of fat tissue in this condition, and to explore a potential pharmacological approach for restricting this process. The mouse model of secondary lymphoedema employed exhibited key pathophysiological features of clinical secondary lymphoedema such as tissue swelling, subcutaneous oedema, dermal fibrosis and excess formation of fat. With the expansion of fat occurring during the early phase of lymphoedema, and elevated levels of fat persisting in the chronic phase, this mouse lymphoedema model was well suited for studying molecular mechanisms driving the initial expansion of fat as well as the persistence of excess fat tissue in the chronic setting. Whole-genome microarray analysis was used to study the mouse lymphoedema model which revealed that mRNA for insulin-like growth factor binding protein 5 (IGFBP5), an inhibitor of the insulin-like growth factor 1 receptor (IGF1R) adipogenic signalling pathway, was down-regulated in the model compared to controls. Further analyses by immunohistochemistry revealed the presence of IGF1 and IGF1R on adipocytes in the model and in a clinical sample of secondary lymphoedema, and activated IGF1R in the clinical sample. Moreover, the levels of IGF1 (an activating ligand for IGF1R) associated with adipocytes, were elevated in the mouse lymphoedema model. Thus, the IGF1R signalling axis could be active and promote expansion of fat tissue in secondary lymphoedema. Importantly, pharmacological targeting of this pathway in the mouse lymphoedema model with linsitinib, a small molecule tyrosine kinase inhibitor of IGF1R, led to significant reductions in tissue swelling and fat expansion which was associated with decreases in both the number and size of adipocytes. Furthermore, linsitinib also restricted the degree of dermal fibrosis in the mouse lymphoedema model. The work presented in this thesis demonstrated the role of the IGF1R signalling pathway in promoting adipogenesis in a mouse model of secondary lymphoedema. Hence, pharmacologically targeting IGF1R might be a viable therapeutic approach for restricting expansion of fat and tissue swelling in secondary lymphoedema patients.
Understanding the clinical implications of the evolution of breast cancers from primary to metastatic disease using next generation sequencing
Precision oncology refers to the use of sophisticated assays to tailor therapy to an individual patient. The feasibility of implementing such a program in a comprehensive cancer centre in Australia is unknown. The utility of precision oncology also depends on understanding how genomic profiles may evolve over time and differ between tumours in the same patient. A precision oncology program called SEGMENT was designed and implemented in a single centre. The program was popular, recruiting well over the study period. Timely acquisition of samples for sequencing was suboptimal from external pathology providers, and proved increasingly expensive during the study period. Delivery of results in a manner where they could be utilized by the patient was challenging in cases where patients were referred late in their natural history. A custom hybrid capture panel worked reliably. A total of 300 patients were recruited to the study, of which 288 had at least one sample received. Accounting for attrition, 214 patients or 71% went onto the main study. The spectrum of mutations and copy number alterations found in this study was similar to published cohorts. There were few differences between primary and metastatic lesions on average. Paired primary and metastatic samples however displayed discordance for both copy number and mutations. This was the case for actionable alterations in ESR1, ERBB2 and very rarely PIK3CA. Approximately 50% of patients had an actionable alteration. Of these 14% of the overall cohort received a therapy matched to their genomic profile. Five patients received matched therapy off trial and 26 received matched trial therapy. Three were zero responses in the off-trial group, and a response rate of 27% in the matched trial therapy group. To explore genomic heterogeneity in greater resolution, 4 patients with advanced breast cancer underwent rapid autopsies to collect large numbers of metastatic samples. Whole exome sequencing was performed on multiple lesions per patient which allowed inference of the subclonal structure. All patients displayed a monophyletic architecture, with truncal driver alterations giving rise to subclones with differing genomic profiles. One patient with a long disease free interval from primary to metastasis showed the acquisition of a new driver in the metastatic lesion. Driver alterations appeared to shape subsequent evolution.
Modelling the development of Barrett’s Oesophagus: Towards better treatment of Oesophageal Adenocarcinoma
Barrett’s oesophagus (BO) is a metaplastic condition in which the normal squamous epithelium of the oesophagus is replaced by a columnar gastro-intestinal like epithelium due to repeated gastro-intestinal reflux. BO is generally accepted to be a precursor condition with the potential to develop into oesophageal adenocarcinoma. Consensus for the cell of origin for Barrett’s oesophagus is still lacking. Different sources for the cell of origin have been proposed, one of which is the submucosal gland (SMG) and duct cells of the oesophagus. This hypothesis, however, has not been properly studied due to the lack of proper model systems. In this thesis, pig and human SMGs and ducts were characterised and compared to each other to assess the suitability of pig SMGs as a substitute for human SMGs using immunohistology and multiplex immunofluorescence staining. Pig epithelial SMG cells were also further characterised using single cell RNA sequencing. Furthermore, organoid culture systems were developed to functionally assess the potential of the progenitor cells found using histologic and transcriptomic characterisation. Pig and human SMGs show different distributions at the distal end of their respective oesophagi but are largely similar in cell type and progenitor cell marker expression as demonstrated by histologic characterisation. Both pig and human SMGs showed progenitor cell marker expression in their respective basal duct cells (CD49f and p75) and the myoepithelial cells (CD49f), suggesting that both the ductal and glandular compartments to contain their own respective pool of progenitor cells. The transcriptomic analysis of pig SMGs single cell RNA sequencing largely supports their role in maintaining oesophageal homeostasis, as previously is known for human. Furthermore, the pseudotime trajectory inference data support the notion of the basal duct cells to be progenitor which give rise luminal duct cells. Interestingly, the data suggest the myoepithelial cells to be progenitor cells that could give rise to both basal duct cells and the gland compartment cells. Sorting and organoid culturing of basal duct cells demonstrated their capacity to grow into squamous spheroids, supporting their role in contributing to normal oesophageal repair. This process could be inhibited by treatment with retinoic acid. Similarly, culture of gland compartment cells gave rise to spheroids with two distinct morphologies. Dense type spheroids showed a squamous morphology but also mucin production found in BO. The second type of spheroid showed a cystic morphology and similarly produces BO type mucin. Finally, viral induction of intestinalisation in submucosal basal duct cells, in the current culture system, did not show metaplastic changes similar to BO. In summary, pig SMGs show great similarity to human SMGs. The pig SMGs contain progenitor cells in their ductal and gland compartments. These progenitors can be purified and cultured in vitro. The current developed protocols could be used to test the hypothesis that the SMGs contain the cell of origin of BO.
Dual-specific Chimeric Antigen Receptor T Cells and an Indirect Vaccine against Pancreatic Cancer
Pancreatic cancer is one of the most aggressive malignancies with an overall 5-year survival rate of <7%. Pancreatic cancer is highly resistant to radiotherapy and chemotherapy, and surgery is not feasible in most patients. In this thesis, I developed a new form of treatment for pancreatic cancer, based on immunotherapy. Adoptive cell transfer (ACT) is a promising form of cancer immunotherapy, which involves the isolation and reinfusion of tumour specific T lymphocytes into patients. While ACT can eliminate substantial burdens of some leukaemia, the ultimate challenge remains the eradication of large solid tumours and metastases for most cancers, including pancreatic cancer. In this thesis, an enhanced ACT treatment strategy for pancreatic cancer was developed, which was termed ‘ACTIV: Adoptive Cell Transfer Incorporating Vaccination’. This treatment included dual-specific T cells that expressed a chimeric antigen receptor (CAR) specific for the tumour antigen Her2, and a TCR specific for the melanocyte protein (pMEL, gp100). These dual specific T cells were termed ‘CARaMEL T cells’. CARaMEL T cells were administered together with an injection of a recombinant vaccinia virus vaccine expressing gp100 (VV-gp100). We hypothesized that adoptively transferred CARaMEL T cells would proliferate mediated by their gp100 TCR, in response to the VV-gp100 vaccine, and kill Her2+ tumours through their anti-Her2 CAR. Functional assays performed in vitro indicated that murine CARaMEL T cells mediated antigen-specific cytokine secretion and killing abilities against pancreatic cancer cells, and demonstrated potent proliferative ability in response to gp100 antigen, confirming our hypothesis. In addition, I found that ACTIV therapy inhibited tumour growth and prolonged the survival of mice bearing Her2+ subcutaneous murine pancreatic tumour. However, tumours usually relapsed after ACTIV therapy administration. Therefore, I directed my study to augment the anti-tumour activity of ACTIV therapy by the administration of either a histone deacetylase inhibitor (Panobinostat) or an immune agonist monoclonal antibody specific for CD40. Panobinostat significantly suppressed the growth of pancreatic cancer cells in vitro through apoptosis and cell cycle arrest. Also, Panobinostat significantly increased the growth suppression of pancreatic cancer cells mediated by CARaMEL T cells. In addition, I found that the combination of ACTIV therapy and Panobinostat significantly reduced the tumour growth and prolonged the survival of mice bearing Her2+ subcutaneous murine pancreatic tumours. In addition, administration of an agonist CD40 monoclonal antibody with ACTIV therapy significantly reduced the tumour growth and prolonged survival of mice bearing subcutaneous Her2+ pancreatic tumours through a T-cell-dependent immune mechanism. Finally, I explored the clinical translational potential for ACTIV therapy through the generation of human CARaMEL T cells expressing both a Her2-specific CAR and a gp100-TCR. In vitro functional assays indicated that human CARaMEL T cells mediated powerful and antigen-specific killing and cytokine secretion against Her2, together with a strong proliferative ability in response to gp100 antigen. In addition, I found that the administration of both human CARaMEL T cells and an adenovirus vaccine expressing gp100 led to potent anti-tumour activity against subcutaneous human Her2+ pancreatic tumours in immunodeficient mice.
Targeting the tumour microenvironment to enhance immunotherapy against cancer
Cancer immunotherapies have shown astounding clinical results within the last decade, with complete eradication of advanced malignancies in certain cancer types, particularly melanoma and non-small cell lung cancer (NSCLC). These successes lead to clinical approval in multiple countries for checkpoint blockade and chimeric antigen receptor (CAR) T cell therapies, and the award of the 2018 Nobel Prize in Physiology or Medicine to James P. Allison and Tasuku Honjo for their research on checkpoint blockade molecules. Despite this, many patients receive minimal benefit and focus has shifted to understanding how to predict and enhance immunotherapy responses. It is now well established that the tumour microenvironment (TME) is a major limiting factor for immunotherapy efficacy. Studies in mice using genetically identical tumour lines implanted in different tissues have demonstrated that the location of tumour growth can directly impact the composition of the TME and response to anti-cancer therapies. Retrospective analysis of checkpoint blockade treated patients’ revealed tissue-specific patterns of response, where metastases in certain anatomical sites were more responsive than others. To date studies investigating the tissue-specific influence on immunotherapy responses in vivo have limited clinical relevance, and studies in patients are minimal. In this thesis, we investigated the influence of the tissue-specific TME on immunotherapy responses in vivo and assessed tissue-specific patterns in the TME of breast cancer metastases from patient samples. First, we investigated a murine breast cancer model comparing responses of primary tumours to tumours in the liver and lungs as common metastatic sites to two immunotherapies, anti-PD-1/anti-CTLA4 and trimAb (anti-4-1BB, anti-CD40, anti-DR5). We reported that the 67NR tumour line growing in the lungs was resistant to immunotherapy, whereas the same tumour line growing in the mammary fat pad (MFP, primary tumour site) or liver could be completely eradicated in a portion of mice. Our analysis revealed that the resistance of lung tumours was independent of the tumour cells, vasculature or drug delivery and that the immune TMEs of lung and MFP tumours were distinct. Specifically, we demonstrated that lung tumours had a more immunosuppressive TME, with increased myeloid derived suppressor cells (MDSCs), decreased T cells and decreased activation of T cells and natural killer (NK) cells. Furthermore, upon depletion of various immune subsets alongside therapeutic intervention we found that NK cell depletion had a significant impact on lung tumours, but not MFP tumours. Taken together our data suggests that tumours grown in different tissues sculpt different TMEs with varied levels of immunosuppression and require different immune cell subsets, and perhaps different immune stimulants, for optimal anti-tumour responses. Following on from this study, we next wanted to assess responses to immunotherapy in vivo in models where multiple tumours in different anatomical sites were present. The rationale of this model was to investigate a more accurate representation of advanced cancer, where tumours have metastasised to multiple locations throughout the body. We hypothesised that co-existing tumours in different sites with disparate TMEs could influence immunotherapy responses compared to tumours existing alone. Our results indicated that the presence of a concomitant MFP tumour enhanced responses of lung tumours to trimAb or anti-PD-1/anti-CTLA4 therapies compared with mice bearing only lung tumours. We observed a decrease in lung metastasis burden in mice with simultaneous MFP tumour growth even before therapy commencement, which likely contributed to enhanced therapy responses. Upon interrogation we found that CD8+ T cells were responsible for the decrease in lung tumour burden and that the lungs of mice with co-existing MPF tumours had more tumour reactive CD8+ T cells. From our results, we hypothesised that the presence of a tumour in a more immunogenic location, such as the MFP, promoted T cell priming within the tumour draining lymph node (TdLN) at this site and led to a systemic response against distal tumours, such as tumours within the lungs. Lastly, we aimed to identify tissue-specific patterns within metastases from human tumours. Herein, we utilised metastatic tumour samples collected as part of the cancer tissue collection after death (CASCADE) rapid autopsy program from three estrogen receptor positive (ER+) breast cancer patients and one triple negative breast cancer (TNBC) patient. We analysed the immune profiles of these samples by transcriptomic and immunohistochemical (IHC) analyses. Our data demonstrated that, although there were potential tissue-specific differences within the TME, the most significant trend delineated immunological differences between ER+ and TNBC patients. These results confirmed previous research describing a higher immune infiltrate in TNBC samples compared with ER+ samples. Our research highlights the potential of investigating metastatic tumour samples however, future studies with separation of disease subtypes and increased sample sizes are needed to truly investigate tissue-specific patterns within the TME. In summary, the data presented in this thesis highlights the importance in further defining tissue-specific response patterns and mechanisms in patients to optimise current and future immunotherapies. Our results indicate that an in depth understanding of the tissue-specific TME could reveal novel treatment options in tumours that are non-responsive to current immunotherapies.