Sir Peter MacCallum Department of Oncology - Theses

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http://hdl.handle.net/11343/38322

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Management of Regional Disease in Prostate Cancer

Doughton, Jacki Anne ( 2021)

The aim of this project is to critically review evidence for and against placing node positive disease in the “curable” category and to suggest ways to improve its management. In the last decade, there have been improvements in both the detection and treatment of lymph node positive disease. The work presented examines the role of more aggressive treatment for those men with node positive prostate cancer but no evidence of distant metastasis. Surgery and radiation therapy are both options for treatment of known (N1) lymph node disease. Androgen deprivation therapy and radiation therapy are commonly considered for adjuvant treatment, as per surgeon and patient preference. A systematic review of the literature updating the role of radical prostatectomy, ADT and radiation therapy in this space is presented. GRADE guidelines are used to assess the quality of the studies. Given the dearth of data from strong randomised control trials, a multivariate analysis of a large cancer database with health records was undertaken and reported with the aim of providing another key in predicting the effectiveness of aggressive up-front treatment for improving cancer specific and overall survival. An in vivo investigation of anatomical pathways for cancer spread also contributes to the hypothesis testing. A cohort study of men underwent sentinel lymph node imaging using a novel tracer and PET/CT imaging. Method and results are presented. The images are an intriguing call-back to historical studies and reveal the opportunities for targeted treatment in regional disease. The last chapter of this work summarises my findings and provides an outline of areas that hold the most potential for pragmatic implementation. A guideline for staging and management of potentially node positive prostate cancer is proposed. Carefully targeted treatment to high risk areas is now possible and casting aside early use of ADT in these patients would in itself be a worthwhile goal.
Discovery and validation of novel ovarian carcinoma predisposition genes

Subramanian, Deepak Naga ( 2021)

Epithelial ovarian carcinoma (EOC) has a significant hereditary component, over half of which cannot be explained by known hereditary breast and ovarian cancer (HBOC) genes (e.g. BRCA1 and BRCA2). Gene discovery studies to date have generally been limited to a small number of candidate genes in relatively few families and have failed to consistently identify any compelling new genes that may account for this missing heritability. The underlying hypothesis of this thesis is that the remaining unexplained EOC families are due to individually rare deleterious variants in numerous genes, each explaining a small proportion of families. To overcome the limitations of earlier targeted panel sequencing efforts, germline whole exome sequencing (WES) was performed on 516 likely familial high-grade serous ovarian cancer (HGSOC) patients with no pathogenic variants in BRCA1 or BRCA2 to discover novel predisposition genes. Forty-three candidate genes enriched for rare loss-of-function (LoF) variants were identified, along with LoF variants in several proposed EOC predisposition genes (e.g. ATM, PALB2). A high degree of genetic heterogeneity was observed, with no single gene harbouring LoF variants in more than 1% of cases. These candidate genes represent diverse functional pathways, with relatively few involved in DNA repair and only a small enrichment for genes involved in homologous recombination (HR) repair. This suggests that many of the remaining HGSOC families are explained by genes in pathways that have been previously under-explored. Since candidate gene variants were individually very rare, orthogonal approaches of tumour sequencing and segregation analysis were undertaken to validate these genes. WES and/or Sanger sequencing was performed on tumour DNA from 105 germline variant carriers, along with bisulphite sequencing of promoter CpG islands for selected genes, to identify evidence of biallelic inactivation and mutational signatures that might support a causative role for that gene. Two genes previously implicated as HGSOC predisposition genes, PALB2 and ATM, displayed biallelic inactivation in nearly every germline variant carrier tumour, associated with characteristic mutational signatures defined principally by the presence or absence of HR repair deficiency, respectively. Of the candidate genes, 19 out of 38 demonstrated biallelic inactivation in at least one tumour from affected carriers, but only three- LLGL2, SCYL3 and MIPOL1- displayed this result consistently in multiple samples, with the others showing loss of the variant allele or returning inconclusive results. Distinctive mutational signatures were found in the LLGL2 and SCYL3 tumours, similar to those for ATM and PALB2, respectively. In the limited number of segregation studies performed amongst six families, none of the tested germline variants consistently segregated with disease. In conclusion, these studies provided data supporting PALB2 and ATM as likely moderate-risk HGSOC predisposition genes, demonstrating the utility of this approach for validating novel familial cancer genes. Several candidate genes showed evidence to indicate a potential predisposing role, but the extreme genetic heterogeneity of unexplained familial HGSOC will necessitate larger studies to confirm these findings.
Developing novel methods of infection surveillance in haematology-oncology patients and implications for health policy

Valentine, Jake Christopher ( 2021)

Healthcare-associated and opportunistic infections are a leading cause of mortality, morbidity, and increased healthcare costs in haematology-oncology patients. Surveillance is recognised as the cornerstone of infection prevention to guide clinical decision making and to monitor quality improvement. The utility of current case ascertainment methods is poorly delineated in patients with underlying malignancy. Administrative data comprise a standardised ontology to classify disease with the potential to support large scale infection surveillance programmes; however, it is unclear if administrative data can support surveillance activities in high-risk settings. The overall aims of this thesis were to: (i) present and argue the case for novel case ascertainment methods; (ii) develop and apply a methodology using administrative data to identify infection in haematology-oncology patients, and determine the classification performance and healthcare funding implications of these data in line with current health policy; and (iii) establish and evaluate a hospital-wide linked dataset integrating multiple data sources, together with administrative data, to achieve maximal performance for automated surveillance in patients with haematological malignancies. Methods include a systematic review to describe the scope of existing surveillance methods among haematology-oncology units, analysis of continuous statewide surveillance datasets and hospital-level administrative data extracts, performance evaluation of administrative data to classify infection and simulation of pay-for-performance funding methodology in a cancer casemix, and development of a hospital-wide linked dataset to identify discrete data combination yielding highest performance for automated infection surveillance. The thesis findings demonstrate significant heterogeneity in existing infection monitoring methods among haematology-oncology patients. Estimates of the burden of disease in a predefined haematology-oncology population relative to a statewide cohort were determined, together with longitudinal trends in incidence over time. Administrative data show to be a feasible alternative to current surveillance data to enable standardised comparison of intra- and interhospital infection epidemiology in patients with underlying malignancy, however, at the expense of poor-to-moderate classification performance associated with significant shortfalls in hospital remuneration. Linkage of administrative data with microbiology, histopathology, and antimicrobial-dispensing data according to specific data combinations demonstrated improvements in classification performance for discrete opportunistic and healthcare-associated infections in patients with haematological malignancy. It was demonstrated that although administrative data enable standardised comparison of infection epidemiology, these data are an unreliable proxy for infection surveillance in Australian haematology-oncology units. Refinements to current pay-for-performance funding specifications are necessary before administrative data can reliably be used as quality improvement measures in a cancer casemix. This thesis posits data linkage as an efficient means to optimise the utility of administrative data, together with hospital-level datasets, to support an automated surveillance strategy in haematology-oncology patients. Future research agenda are outlined regarding the evaluation of electronic medical record data and other codified nomenclature to support electronic surveillance and quality improvement monitoring.
Overcoming resistance to PI3K inhibitors in colorectal cancer

de las Heras Vila, Francesc d'Assis ( 2021)

Colorectal cancer (CRC) is responsible for the second highest number of cancer deaths worldwide, with stage IV patients having a 5-year survival rate of only 14%. One of the treatments in development involves a targeted therapy directed at PI3K alpha, a protein mutated in 18% of CRC patients. Particularly, PI3K alpha is involved in several cancer hallmarks including survival, metabolism and migration. The introduction of PI3K inhibitors as targeted therapy in clinical trials caused an increase in overall survival and a decrease in disease progression. However, ultimately, treatment failure and tumour progression still occur due to development of drug resistance. Therefore, the aim of this project was to identify mechanisms of resistance to PI3K targeted therapy that can be exploited to overcome treatment failure. Specifically, the focus in this study is on BYL719, a PI3K alpha specific inhibitor. Firstly, a novel subcutaneous syngeneic mouse model of CRC was generated by the syngeneic transplant of Pik3ca mutated and Apc deleted gastrointestinal tumours from a mouse model of CRC. Tumour growth in this model was reduced upon treatment with BYL719. Mice were then chronically treated with BYL719 to induce resistance. Although the development of resistance was not confirmed within the time-frame of this thesis, this model proved to be a useful tool for pharmacological studies. Secondly, in silico analyses were performed, correlating the sensitivity of solid cancer cell lines to PI3K inhibition with CRISPR KO/mRNA/protein/metabolite data from online pharmacogenomic datasets. These analyses identified multiple pathways potentially involved with sensitivity to PI3K inhibition, including proteins involved in the regulation of the cytoskeleton, the PI3K/MAPK pathway, the endomembrane system and lipid and glutamine metabolism. Finally, an in vitro approach was performed using genome-wide CRISPR KO screen techniques, to identify additional functional pathways involved with sensitivity to PI3K inhibition. The results identified potential involvement of PI3K/Akt/MAPK signaling, mTOR/protein synthesis pathways, the Wnt pathway, TGF-beta pathway and metabolism, including lipids, glycolysis and the mitochondrial respiratory chain. Ultimately, these findings should enable the rational design of novel combination treatments with PI3K inhibitors to prevent or overcome resistance. Moreover, the findings might also be used to screen patients to predict their response to PI3K inhibitors.
Investigating the role of Mucosal-Associated Invariant T (MAIT) cells in cancer

Petley, Emma Victoria ( 2021)

The success of immunotherapy in patients has highlighted the importance of the anti-tumour role of the immune system. The function of conventional T cells within the tumour microenvironment (TME) have been intensively studied, while the role of mucosal-associated invariant T (MAIT) cells is yet to be determined. MAIT cells are abundant in humans and enriched in mucosal tissues, such as the colon and lung, and have been found within primary and metastatic tumours. Upon activation, MAIT cells exert rapid effector functions and can secrete both the anti-tumour cytokines (IFNg and TNF) and pro-tumour cytokines (IL-17 and IL-22). MAIT cells also produce granzyme B and perforin, suggesting they are capable of killing target cells. Although direct evidence of MAIT cells precise function in cancer is limited, some studies show that increased numbers of MAIT cells within tumours are correlated with a good prognosis, whilst other studies have indicated MAIT cells are associated with a poorer prognosis. These divergent results have made it difficult to interpret whether MAIT cells have an anti-tumour or pro-tumour role. Therefore, this thesis investigated the role of MAIT cells in cancer and the potential for MAIT cells to be exploited for adoptive cellular therapy. The first results chapter of this thesis explores the anti-tumour role of MAIT cells in both murine and ex vivo human models. It was observed that at steady-state, MAIT cells negatively regulate NK cell maturation and anti-tumour activity. Conversely, activating MAIT cells through either pulsing of tumour targets or intranasal administration of free MAIT cell antigen, led to robust protection against the development of lung metastases. Upon further investigation, it was discovered that activated MAIT cells enhance NK cell maturation and anti-tumour activity in an IFNg-dependent manner. This chapter proposes the existence of a MAIT-NK cell axis that can control NK cell mediated anti-tumour efficacy. The second results chapter aims to further improve the anti-tumour efficacy of activated MAIT cells, by combining this therapy with additional immunotherapies. The additional immunotherapies tested in combination with MAIT cell activation were selected on the basis of their ability to activate MAIT cells and/or NK cells. Notably, additional therapies that increased both MAIT cell and NK cell activity were most promising. This chapter also found that intravenous administration of MAIT cell antigen led to systemic expansion of MAIT cells and an increase in MAIT cells within the tumour tissue, broadening the application of activating MAIT cells in the clinic. The third results chapter aims to investigate the potential of MAIT cells in the context of Chimeric antigen receptor (CAR) T cell therapy. CAR T cell therapy is currently ineffective in solid tumours, due to the immunosuppressive TME, antigen heterogeneity, poor trafficking to solid tumours and decreased persistence. Furthermore, this therapy requires autologous generation of CAR cells in order to avoid graft versus host disease (GVHD). Excitingly, MAIT cells represent an allogeneic source of CAR cells as they are not restricted to conventional MHC. Chapter 5 demonstrates that MAIT cells are able to be efficiently transduced with CAR and upon target recognition CAR MAIT cells produce cytokines and directly kill tumour cells. Collectively, this data illustrates the potential anti-tumour activity of MAIT cells through a MAIT-NK cell axis. Furthermore, this thesis demonstrates the potential for MAIT cells to be used in adoptive cellular therapy, namely as CAR MAIT cells.
Therapeutically targeting RNA Polymerase II transcription in leukaemia

Todorovski, Izabela ( 2021)

RNA Polymerase II (RNAPII) transcription is a discontinuous and unidirectional process, control of which is necessary for normal gene expression and cellular functioning. Regulation occurs at discrete checkpoints throughout transcription and is in part mediated by Cyclin Dependent Kinases (CDKs). This includes CDK7, where it phosphorylates RNAPII among other transcriptional substrates to enable transcriptional initiation and coordination of co-transcriptional processes, such as splicing. However, the genome-wide role of CDK7 in nascent RNA synthesis and splicing, in addition to whether its function is negatively regulated, has not been robustly established. The loss of RNAPII transcription control invariably leads to disease, including cancer. Mutations in cancer can affect several proteins involved in regulating RNAPII and engenders a reliance on sustained functioning of the core RNAPII machinery for continued malignant cell survival and proliferation. This dependence is termed ‘transcription addiction’ and is the basis for targeting RNAPII core components for therapeutic benefit. Small molecule perturbation of transcription in these cancers results in selective gene expression changes attributed to the target gene’s specific chromatin context, which includes the occupancy of oncogenic transcription factors (TFs) and association with genomic elements such as super enhancers (SEs). Whether selective transcriptional changes are also influenced by RNA stability however, is largely unexplored. In this thesis, we demonstrated that RNA decay rates largely determined gene expression changes to clinically relevant transcriptional and epigenetic compounds. Transcripts that were the most down-regulated on the total RNA level had both short half-lives and high production rates and often encoded TFs, including the proto-oncogene c- MYC. Moreover, genetically engineering the three prime untranslated region (3’UTR) of c-MYC to prolong its RNA half-life rendered c-MYC transcripts resistant to transcriptional targeting on the total RNA level. Therefore, these finding highlight an under-appreciated role of RNA stability in gene expression changes to therapeutic RNAPII perturbation in cancer. Among the most promising therapeutic transcriptional targets is CDK7, and inhibitors targeting this protein are currently clinical trials and have been expedited for Food and Drug Administration (FDA) approval. Proteomic and transcriptomic characterization of the CDK7 inhibitor, YKL-5-124, in the THP-1 Acute Myeloid Leukaemia (AML) cell line revealed that it perturbed the phosphorylation of several proteins involved in cell cycling, transcription and co-transcriptional processes, namely splicing. This was paralleled with a global decrease in de novo RNA synthesis, most prominently towards the 5’ ends of genes, and total RNA levels. In addition, CDK7 inhibition increased alternative isoform expression, most strikingly related to exon skipping and intron retention. On a phenotypic level, these changes were also associated with a decrease in THP-1 cell proliferation. Taken together, these findings improve our understanding of the significance of CDK7 kinase activity in RNAPII transcription and splicing. To identify factors that functionally antagonize CDK7 and may confer resistance to sustained CDK7 inhibition, CRISPR-Cas9 knockout positive enrichment screening in THP-1 cells was performed using YKL-5-124 treatment as a selective pressure. The screen showed that knockout of histone acetyltransferase (HAT) and core structural components of the Spt-Ada-Gcn5 acetyltransferase (SAGA) complex mediated resistance to CDK7 inhibition. Further investigation into the resistance mechanism revealed that cells knocked out for the SAGA HAT subunit, TADA2B, were less sensitive to nascent RNA down-regulation with YKL-5-124. Moreover, this was concomitant with maintained H2BK120 mono-ubiquitin levels across gene bodies genome-wide. Therefore, these data highlight SAGA complex components as novel negative regulators of CDK7 and how cancers might overcome the effect CDK7 inhibitors in the clinic. Overall, the results presented herein improve our understanding of CDK7 in RNAPII transcription and the importance of RNA decay in defining the anti-cancer effects of therapeutic perturbation of the core RNAPII machinery.
Targeting Protein Tyrosine Phosphatases (PTPs) to enhance T cell immunotherapy

Du, Xin ( 2021)

The advancement of cancer immunotherapy has revolutionized cancer treatment. In particular, the success of immune checkpoint blockade, including anti-PD-1/L1 and anti-CTLA-4 in the clinic has changed guidelines for treating multiple solid tumours such as melanoma, non-small-cell-lung cancer (NSCLC), renal cell carcinoma and head and neck squamous cell cancers. Adoptive T cell therapy against haematological malignancies has also achieved remarkable success. Two anti-CD19 CAR T cell products have been approved by Food and Drug Administration (FDA) for the treatment of B cell Acute Lymphoblastic Leukaemia (ALL) and non-Hodgkin lymphoma. However, the efficacy of adoptive CAR T cell therapy against solid tumours has been less impressive. Even though numerous preclinical or clinical trials have been conducted to identify novel neoantigens or to improve CAR T cell anti-tumour efficacy against solid tumours, all attempts have so far not matched the extraordinary clinical responses observed in patients with haematological malignancies. There is an urgent need to 1) understand CAR signalling in T cells, 2) identify the key mechanisms intracellularly and extracellularly limiting CAR T cell function, and 3) to develop strategies to overcome the immunosuppressive tumour microenvironment to improve the efficacy of CAR T cells in the context of solid tumours. Recent studies have highlighted the potential of targeting negative regulators of T cell signalling to enhance the efficacy and extend the utility of CAR T cells to solid tumours. Protein tyrosine phosphatases (PTPs) including PTPN2 and PTPN22 are key regulators of T cell signalling and contribute to the maintenance of immune tolerance. PTPN22 is known as a critical autoimmunity-associated PTP that suppresses T cell receptor (TCR) signalling by dephosphorylating substrates such as the Src family kinase LCK and the Syk family kinase ZAP70 in effector/memory T cells. Loss of function SNPs in PTPN2 have also been associated with the development of autoimmunity. Studies from our group have shown that PTPN2 plays pivotal role in negatively regulating T cell receptor (TCR) signalling by dephosphorylating and inactivating the Src family protein tyrosine kinase LCK to limit T cell proliferation and function. PTPN2 also attenuates cytokine signalling by dephosphorylating JAK-1, JAK-3 and their target substrates STAT-1, -3 and -5 in a cell context-dependent manner. Beyond PTPN22 and PTPN2, early studies established that PTP1B could directly dephosphorylate and inactivate JAK-2 and Tyk-2 to attenuate cytokine-induced JAK/STAT signalling. There is also evidence that PTP1B can dephosphorylate STAT family members. In this thesis, we found that basal STAT-5 Y694 phosphorylation and IL-2/IL-7/IL-15 induced STAT-5 Y694 phosphorylation were enhanced by the deletion of PTP1B in T cells. Since CARs signal via LCK, and cytokines can affect CAR T cell function, differentiation and memory, we postulated that inhibiting PTPN2, PTP1B and PTPN22 individually or combinatorically might bolster the anti-tumour activity of CAR T cells. Work in this thesis characterized the role of PTPN22, PTPN2 and PTP1B in adoptive T cell therapy and demonstrated that the deletion or inhibition of PTPN2 or PTP1B resulted in enhanced T cell signalling and promoted CAR T cell anti-tumour efficacy in vitro and in vivo. In addition, we developed a CRISPR/Cas9 RNP gene editing based method to target PTPs in CAR T cells for translational purposes.
Investigating mechanisms for Elotuzumab and Lenalidomide therapy in Multiple Myeloma

Richardson, Kelden James ( 2021)

Multiple myeloma (MM) is a haematological malignancy of plasma cells with disease present in the bone marrow. Despite the success of new therapies in extending life expectancy, MM is still considered incurable and requires further investigation into new treatments. In the context of refractory/relapsed MM (RRMM), the combination of the two immunotherapies Elotuzumab (Elo, anti-SLAMF7 mAb) and Lenalidomide (Len, IMiD) has resulted in an 82% objective response rate and 16% complete remission in patients. This success led to FDA approval of Elo plus Len treatment for patients with RRMM and has warranted further investigations into the exact mechanism of action. Elo targets SLAMF7 antigen which is highly expressed on MM tumour cells and activates NK cells for antibody dependent cell-mediated cytotoxicity (ADCC) resulting in MM tumour cell death. Len is an immunomodulatory agent commonly used to treat a wide range of haematological cancers. Len has been shown to activate various cells of the immune system as well as directly suppress tumour progression. This thesis aims to identify how the mechanisms of Len intersect with, and enhance, Elo activated NK cell control of tumours. The work from this thesis revealed that to induce ADCC, Elo required both expression of SLAMF7 on MM target cells and NK cell CD16 engagement. Elo-activated NK cells had increased expression of CD107a and CD69, as well as loss of CD16 expression which was a result of ADAM17 induced cleavage. Elo activation of NK cells also secreted increased levels of cytokines and chemokines associated with recruitment of effector immune cells. In both in-vitro and in-vivo studies, Len significantly enhanced Elo-induced healthy donor NK cell killing of MM tumour cells. In contrast, only 4/12 RRMM patient (responders) NK cells induced increased MM cell killing in the context of Elo plus Len. Important for this increased cytotoxicity was the expression of CD54 on NK cells that allowed differentiation between these responder and non-responder patients. CD54 was also more highly expressed on NK cells activated by Elo plus Len compared to Elo activation alone. However, further phenotypic studies and RNA sequencing did not reveal any further mechanisms in NK cells by which Len enhances Elo activation. Further studies revealed that MM tumour (myeloma) cells specifically upregulated CD54 and CD11a expression in response to Elo plus Len activation of NK cells and this did not occur with Elo treatment alone. This increase in CD54/CD11a expression on myeloma cells was also dependent on the presence of CD14+ monocytes. RNA sequencing also revealed enrichment of cytokine and chemokine signalling/secretion pathways in MM cells specifically in the context of Elo and Len activated NK cells and monocytes. In conclusion, these data indicate that a complex network of direct and indirect mechanisms between NK cells, monocytes and myeloma cells contribute to the Elo plus Len treatment induced response in RRMM patients. Elo alone activates NK cells and monocytes, whereas the Len effect is largely on myeloma cells. Collectively the two treatments led to robust myeloma antibody dependent cellular cytotoxicity (ADCC). There was a strong correlation between increased myeloma cell CD54/CD11a expression and myeloma cell ADCC suggesting that stabilisation of the immune synapse with strong adhesion may be a key factor for this improved myeloma cell killing. Finally, this study revealed that upregulation of MM cell CD54 expression on MM cells may be a useful predictive biomarker to stratify RRMM patients for Elo plus Len therapy.
Molecular and functional analysis of Mucinous Ovarian Carcinoma

Salazar Ruales, Carolina Alejandra ( 2021)

Mucinous ovarian carcinoma (MOC) is a rare subtype of ovarian cancer (OC) distinct from all other subtypes. MOC represents 3% of OC cases; however, it responds very poorly to conventional chemotherapies contributing to the poor outcome of OC. Moreover, there is a lack of effective therapies for MOC since treatments with platinum-based chemotherapy or PARP-inhibitors are unlikely to be effective. The recent description of the molecular landscape of MOC, including contributions by work in this thesis, allowed the identification of novel therapies that target the most recurrent genetic features of the disease. Nevertheless, there is still the need to develop accurate pre-clinical models in order to test the efficacy of these new therapies. This thesis evaluates the transcription factor ELF3 as a new MOC candidate driver gene. We performed a comprehensive analysis of 210 human mucinous ovarian tumours and identified that ELF3 was mutated in 8.2% of our sample cohort. To assess whether ELF3 acts as a tumour suppressor or oncogene in MOC and how its mutated function is expressed in cancer, we evaluated the ability of MOC cells to proliferate, migrate and invade, by introducing truncating mutations using CRISPR-Cas9 technology. It was found that ELF3 could possibly act as a tumour suppressor in MOC; however, its role might depend on coexisting mutations in the cells. This thesis also describes a novel 3D patient-derived therapeutic approach to pre-test the effectiveness of drugs, as monotherapy or in combination, before exposing patients to ineffective therapies or side effects. A mucinous ovarian tumour 3D platform that enables the correlation of patient genetic data with drug testing was established in the first result chapter of this thesis. MOC organoid development was based on the MOC genetic profile and characteristics as well as optimization that adapted human colorectal and pancreatic organoid culture systems. It was found that Wnt-3a is essential for mucinous ovarian tumour organoid long-term derivation. Moreover, MOC organoids faithfully recapitulate MOC primary tumours at the genomic level and retain histological characteristics of their corresponding primary tumours. The development of MOC organoids and 3D spheroids from cell lines together with the description of the genetic profile of MOC allowed the development and testing of a panel of novel therapies that targets the most frequent mutations found in MOC tumours, including a mutant TP53 activator, a Wnt inhibitor, RAF/EGFR/RAS/CDK4 and CDK6 inhibitors, and two antitumour compounds for HER2-overexpressing tumour cells (APR-246, OMP-18R5, BGB-283, BKM120, PD-0332991, PLX4032, Trastuzumab and Lapatinib). The organoids and spheroids showed different responses to the individual therapies tested, depending on the individual genetic features of the models. Spheroids showed sensitivity to PD-0332991, Trastuzumab, PLX4032 , and BKM-120 . MOC organoids also showed different responses: MOC organoid MOCOR-16 showed sensitivity to APR-246, and resistance to Trastuzumab; however, MOCOR-38 was sensitive to Trastuzumab but resistant to APR-246. This chapter also shows the first attempt of drug synergy scoring using automatic robots to create 3D MOC models. Synergistic combinations were identified for one of the spheroid lines when APR-246 and BKM-120 were combined. Interestingly, the spheroids were resistant to APR-246 in monotherapy. If validated independently, these results could potentially represent a more effective treatment for MOC. Antagonistic interactions were also found, with the strongest antagonism occurring between PLX4032 and BGB-283. The findings of this study substantially increase our understanding of mucinous ovarian tumours together with potential personalized therapeutic targets to improve the survival outcomes for women with this deadly disease.
Applications of massively parallel sequencing technology in the evaluation of haematological malignancies

Yannakou, Costas Kleanthes ( 2021)

Massively parallel sequencing (MPS) technology has revolutionised the genomic exploration of human disease. This is especially true in the case of cancer, which is primarily driven by the development of acquired genomic aberrations. The body of work described within this thesis represents a broad yet in-depth array of novel applications of MPS technology in the evaluation of haematological malignancies. This field is currently surging in relevance and clinical utility as the ongoing movement of MPS technology from the research to the routine diagnostic setting continues to facilitate the development of increasingly personalised medicine. High impact contributions have been made in a number of areas encompassing myeloid and lymphoid malignancies as well as haematological malignancies as a collective. Key achievements include: quantifying the risk of incidentally detecting germline variants of potential clinical significance during unpaired MPS testing of cancer samples; definitively proving that ASXL1 NM_015338.5:c.1934dup;p.Gly646Trpfs*12 is a true somatic alteration and developing an accurate and sensitive assay for its detection; exploring the pathogenesis of and mechanisms of resistance to histone deacetylase inhibitors in cutaneous T-cell lymphomas as well as defining the clinical features, outcomes and genomic landscape of transformed marginal zone lymphoma. This thesis represents a diverse portfolio of novel research with a strong translational focus. Despite the wide scope of the individual lines of inquiry described herein there is a common thread that binds the narrative together: the pursuit of innovative yet practical ways of utilising the powerful technology now available to improve the genomic characterisation of haematological malignancies and ultimately the lives of the patients and families they affect.

Sir Peter MacCallum Department of Oncology - Theses

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