Sir Peter MacCallum Department of Oncology - Theses
Permanent URI for this collection
Search Results
1 - 10 of 20
-
ItemFever and neutropenia in children with cancer: optimising clinical research and the delivery of care in Australia( 2017)Fever and neutropenia (FN) is the most common complication of childhood cancer treatment. Management traditionally involves hospital admission for antibiotics until resolution of fever and recovery of neutrophil count. However, children with FN are a heterogeneous group with varying risk of severe infection, and this approach over treats up to half of all episodes where risk of serious complications is low. Unlike FN management for adults, formal low-risk treatment strategies for children are not routinely employed. In Australia, little is known about the aetiology and management of FN in children and clinical decision rules (CDRs), designed to predict infection, have not been validated. These factors remain a critical barrier to implementing ambulatory-care programs for children with low-risk FN in this country. Such programs are safe, improve quality of life (QoL) and reduce healthcare expenditures. The overall aims of this thesis were to optimise clinical paediatric FN research; to advance our understanding of the assessment and management of FN in children with cancer in Australia; and to facilitate treatment that is tailored to the patient’s risk of infection. Each project addresses important evidence gaps, namely the absence of standardised paediatric FN research outcomes and definitions, the lack of a contemporary evaluation of FN management in Australia and the limited use of risk-based treatment algorithms. To optimise clinical paediatric FN research, Delphi survey methodology was used to achieve consensus on a set of core variables and outcomes that should be reported in all FN studies. This is the first time a paediatric-specific FN research framework has been developed and is the result of an international collaboration. Standardised FN research outcomes will reduce heterogeneity between studies, minimise reporting bias and enable research results to be compared, contrasted and combined. To advance our understanding of the management of FN in Australia, a national practice survey was conducted. There was clear evidence of heterogeneity in assessment, risk stratification and treatment of children with FN. The survey identified critical knowledge gaps and deviations from best practice, and the results will be used to inform guidelines, education and low-risk program implementation strategies. It also highlighted the necessity for validation studies to determine which CDRs are most appropriate for use in Australia. A series of studies were conducted to facilitate FN treatment that is tailored to the risk of infection. In a retrospective study, seven CDRs were validated, of which two exhibited the most clinically meaningful results. The accompanying economic evaluation highlights opportunities for substantial healthcare savings by reducing length of stay (LOS) for low-risk patients. The systematic review and meta-analysis of the predictive ability of novel biomarkers found that marked heterogeneity between studies limits firm conclusions, and further research is required. This thesis has addressed key evidence gaps and contributed new knowledge that will optimise clinical FN research and the delivery of FN care to children with cancer. It has informed the national Australian Predicting Infectious ComplicatioNs in Children with Cancer (PICNICC) project that will prospectively validate CDRs, identify novel biomarkers and evaluate the cost and QoL associated with standard FN treatment. It has also established a collaborative network that will ensure FN research continues in this country and results are translated into practice.
-
ItemAddressing the barriers to translating genetic sequencing into a clinical oncology setting( 2017)The molecular diagnostics of cancer are being transformed globally by the adoption of affordable high-throughput sequencing (HTS) to identify a patient’s genetic changes that can be matched to targeted therapies. However, many of the laboratories that have adopted these technologies do not have adequate capabilities in the key dimensions of clinical HTS diagnostics. Laboratories do not have the trained staff with the capabilities for analysis and do not have adequate software to tackle the bioinformatic pipelines, curation, and clinical informatics, which are essential for the maintenance of a robust, clinically defensible HTS assay. The clinicians who rely on results from laboratories that use HTS must be aware of the many limitations of these technologies, which can affect their patients’ outcomes profoundly. The pace of development of software in cancer diagnostics has not matched that of HTS hardware. This research addresses this software gap in the capabilities of HTS diagnostics. This is achieved by designing, developing and deploying a range of software that can be used in a HTS pathology laboratory. Additionally the software can be used by clinical trials and large research projects requiring volume HTS in a clinical setting. The software developed through this translational research covers three broad areas; 1) the bioinformatic pipelines that process the sequencing data, 2) the testing of these pipelines and 3) the analysis and reporting of processed sequencing data. This thesis describes three systems within these areas. Firstly, Canary [1] is an integrated single command pipeline that performs multiple tasks in a single Java executable file. Secondly, PipeCleaner is a pipeline testing framework incorporating the ability to generate synthetic data and write pipeline tests in a domain specific language. Finally, PathOS[2] is a web based application allowing laboratory scientists to filter, analyse, curate and report on patient variants in high volumes. Both PathOS and Canary have been deployed into a large cancer hospital pathology laboratory and are under active development. Additionally, PathOS, and the associated tools developed with it, are being actively used by the wider research community. The volume of molecular diagnostic testing in cancer has expanded from single exon and single gene assays into progressively larger panels of hundreds of genes and whole exome and genome assays. The introduction of ctDNA assays into clinical use, further allows a patient to be readily tested multiple times during their health care encounter. On the horizon, single cell sequencing may offer deeper diagnostic insights into a patient’s disease. To deal with these increasing volumes of assays and data, robust, high quality software will be needed to scale the laboratory capacity for the future.
-
ItemRadioprotection by combination of DNA binding antioxidants and aminothiol radical scavengers( 2017)The unavoidable irradiation of normal tissue during cancer radiotherapy can lead to serious dose limiting side-effects. For example, radiation-induced oral mucositis associated with head and neck radiotherapy is still a problem, even with modern dose delivery technology, and can lead to treatment interruptions. One strategy to ameliorate these side effects is the pharmacological radioprotection of “at risk” normal tissues. The first clinically approved radioprotector was amifostine, a prodrug of the aminothiol radical scavenger WR-1065. M2PB, an analogue of methylproamine, represents a new class of radioprotector that protects via a unique mechanism, involving electron transfer from the minor groove bound ligand to transient radiation induced oxidising species on the DNA. Importantly, in vitro clonogenic survival studies demonstrated near-additive radioprotection when these two drugs were used in combination (DMF=4.3), compared to 4 mM WR-1065 (DMF=2.5) and 40 μM M2PB (DMF=2.7) alone. Importantly, the drug concentrations were chosen as they correspond to the maximal achievable DMF. A major outcome of this PhD project is the demonstration that the additive radioprotection observed in vitro translates to studies with in vivo models, using M2PB and WR-1065 (or amifostine). The first in vivo model employed was the Withers mouse jejunum microcolony survival assay, which involves single fraction total body irradiation (TBI). In the Withers model, both M2PB (92 mg/kg SC) and amifostine (175 mg/kg IP) were found to protect the mouse small intestine from radiation damage with DMF values of 1.24 ± 0.01 and 1.26 ± 0.02 respectively. Importantly, when used in combination, M2PB and amifostine exhibit additive radioprotection with a DMF of 1.74 ± 0.04. Similar results were also obtained for the mouse tongue oral mucosa model, which involves the single fraction irradiation of the mouse oral mucosa and uses radiation induced ulceration as an endpoint. The observation of additive radioprotection in vitro and in vivo provides further insight into the mechanism of radioprotection, indicating different but complementary mechanisms of action. This complementarity of M2PB and WR-1065 radioprotection extends to radiation-induced mutagenesis in vitro. The frequency of radiation induced mutations at the HPRT locus in CHO-K1 cells, surviving 6 Gy gamma-radiation, was 65.4 mutants/106 cells. Protection of radiation-induced mutations was observed for WR-1065 (23.5 mutants/106 cells) and M2PB (24.5 mutants/106 cells). A further reduction was observed when the two radioprotectors were used in combination (10.0 mutants/106 cells). The results of the study provide the basis for contributing to the ultimate goal of developing effective protection of normal tissues during cancer radiotherapy. Such a decrease in treatment side-effects would be expected to result in fewer interruptions to radiotherapy treatments.
-
ItemPrediction and assessment of pathological complete response following neoadjuvant chemoradiotherapy for locally advanced rectal cancer( 2017)Introduction The management of patients with rectal cancer who develop a pathological complete response (pCR) following neoadjuvant chemoradiotherapy (nCRT) presents an ongoing challenge to clinicians. Some authors have suggested that the presence of a clinical complete response may allow patients to be spared the morbidity and potential risks of surgery through adoption of a ‘watch and wait’ policy with surgery only in the setting of clinical failure. However clinical response and pathological response are not always wellcorrelated and widespread adoption of this regimen is limited by accurate methods to assess and confirm the presence of a pCR without a surgical specimen. Method A systematic review of the literature has been performed to identify methods to predict and assess a pathological complete response to nCRT. The first part of the literature review focuses on factors predictive of a pCR, specifically clinical features, radiological features and histological and molecular markers. The second part of the literature review aims to determine methods to accurately assess the presence of a pCR following neoadjuvant treatment. Following this an institutional database was interrogated to determine clinical and radiological features associated with prediction and assessment of a pCR and a multimodal predictive model has been developed. Molecular analysis has been performed to identify genetic influences on pCR. The role of radiological imaging in the assessment of pCR will be explored and the prognostic and clinical significance of metabolic response assessment by 18F FDG PET CT has been investigated in detail. Results Assessment Histology and clinical assessment remain the most effective methods of assessment of pCR with histology considered the gold standard. Clinical assessment is limited to low rectal tumours and is open to significant inter-rater variability while histological examination requires a surgical specimen for accurate assessment. Radiological assessment of pCR demonstrates the greatest potential for assessment of pCR without the need for surgery with diffusion weighted MRI and 18F FDG PET CT providing the greatest accuracy. It is likely that improved accuracy will be achieved with multimodal assessment of response combining the benefits of clinical, serological, endoscopic and radiological methods of response assessment. Prediction Clear methods to predict pCR prior to the commencement of therapy have not been defined. Clinical and radiological features of the primary cancer have limited ability to predict response. Molecular signatures hold the greatest potential to predict response however adoption of this technology is limited by poor concordance of biomarkers between cohorts. Conclusion Accurate prediction and determination of a complete pathological response is paramount if a non-operative approach is to be undertaken with confidence in oncological outcomes. A variety of methods are available but currently they lack sufficient sensitivity and specificity to define management. Despite a large volume of research the ability to predict which patients are likely to sustain a pCR and accurately assess those patient who have a pCR remains elusive. Further research into models incorporating both prediction and assessment into decision-making is required.
-
ItemThe role of nature in cancer patients' experiences of health and recovery( 2017)This thesis explores the role of nature in cancer patients’ experiences of health and recovery. Using a 2-Phase mixed-method approach, the investigation aimed to generate new theoretical understanding about cancer patients’ use of nature and how they find nature engagement helpful or not when confronting cancer diagnosis. The project’s translational focus was to produce expert recommendations for nature-based care opportunities in oncology contexts based on patient-reported data. First, a systematic literature review and meta-synthesis was conducted to describe the existing qualitative research evidence base relating to nature experiences and nature-based interventions for cancer populations specifically. The aim was to describe current knowledge about the role of nature in cancer patients’ lives. From eleven eligible publications, seven inter-related core themes were identified as follows: connecting with what is valued; being elsewhere, seeing and feeling differently; exploration, inner and outer excursions; home and safe; symbolism, understanding and communicating differently; benefitting from old and new physical activities; and, enriching aesthetic experiences. Next, an in-depth investigation of cancer patients’ own experiences with nature used primary data to develop a new Grounded Theory describing the underlying and intrapsychic mechanisms of cancer patients’ phenomenal nature experiences. Based on qualitative data collected from semi-structured interviews with 20 cancer patients (9 female), the resulting theory model explores the unique role nature plays when diagnosed with cancer. It constitutes a core category and two inter-related themes, which explain a normalization process in which patients moved towards a state of 'new-normal' (Core Category). Nature functioned in this process as a support structure that repositioned patients and nurtured their inner and outer capacities to respond and connect more effectively (Theme A). Once enabled and comforted, participants could engage survival and reconstructive manoeuvres and explore the consequences of cancer in their present lives and possible futures (Theme B). A dynamic relationship was shown between moving away while, simultaneously, advancing towards the cancer reality in order for patients to incorporate their cancer experiences into a shifting normality. From a place of comfort and safety, patients felt supported to deal differently and more creatively with the threat and demands of cancer diagnosis, treatment, and outlook. The descriptively rich interview data provided further insights into patients’ own recommendations for nature engagement in the oncology context, which were extracted from the transcriptions using deductive content analysis and were consolidated into patient recommendations for nature-based care opportunities. These incorporated using nature for vital sensory stimulation and engagement, using nature for personal space and freedom to enable private and social exploration, using views of nature for distraction and comfort, and accessing nature for physical activity and movement. Three critical factors were determined to avoid adverse experiences: determining appropriate health-care expenditure and resourcing on nature-based interventions, selection of appropriate nature-based design materials, and exercising caution around demanding nature engagement and harsh weather conditions. A questionnaire survey study was conducted following an environmental intervention in an oncology waiting room to assess patient, visitor, and staff responses to design changes, which included the addition of artificial plant materials. Based on 143 returned survey questionnaires consisting of 73 cancer patients, 13 staff, 52 carers, and five ‘other’, it was found that the environmental intervention positively impacted patients, staff, and carers’ perceptions of the oncology waiting room environment. Patients, staff, and carers mostly accepted artificial plants as an alternative design solution to real plants. Comments included positive appraisals and occasional adverse reactions to artificial plants. No significant differences were found between patient, staff, and carer reactions. Insights gleaned from the initial, exploratory phase formed the basis for a second phase investigation comprising an international online Delphi study. The aim was to solicit knowledge from relevant experts drawn from a range of healthcare practitioners, management, designers, and researchers to determine feasible opportunities for, and barriers to, providing helpful nature engagement in oncology settings. Two hundred potential panellists were identified and sent an invitation to participate. Thirty-eight experts were recruited who represented 7 countries: Australia (19), USA (8), UK (3), New Zealand (2), Canada (2), Denmark (3), and Sweden (1). This study followed a structured, iterative feedback process that queried and synthesized expert opinion. Cancer patients’ own recommendations, extracted from phase 1, were used as a starting point for the Delphi panel to brainstorm and develop their own ideas about appropriate nature-based opportunities in oncology settings and the barriers to their provision. In total, 250 separate suggestions for opportunities and 205 suggestions for barriers were collected. Further analysis condensed these into 55 unique items (35 opportunities, 20 barriers). The Delphi panel’s list of recommendations included “Window views from clinical areas onto nature […]” as the highest rated opportunity, and “Building design and site constraints […]” as the highest rated barrier to providing nature-based supports for oncology care. Finally, a synthesis of findings from the overall investigation, which constitutes six publications, is provided to summarize and outline the salient findings and discern the study’s limitations in order to suggest pathways for future research. This synthesis produced a conceptual framework consolidating new theoretical understanding and empirical content from patient and expert-reported data about nature-based care opportunities in the oncology setting. The thesis findings provide evidence for multiple uses of nature as a supportive aid in the cancer care context. Concrete recommendations have resulted to guide the application of nature based concepts in future oncology setting design and may be considered when developing additional supportive care services. The findings may assist healthcare practitioners, designers, researchers, and patients themselves to creatively and practically participate in future oncology care practice and design.
-
ItemPrediction and prognosis in anal cancer: developing models to improve patient outcome( 2017)Anal squamous cell carcinoma is a human papilloma virus–related disease for which definitive treatment comprises chemoradiotherapy that has not changed substantially for forty years. Few advances in treatment have been made since then, especially for those patients who develop disease relapse and for whom no surgical options exist. Predicting responses in patients for whom conventional treatment will fail remains elusive and is a significant clinical problem. As anal cancer is reasonably described as a rare cancer, innovative approaches are required to address this pressing clinical issue as large clinical trials are exceptionally challenging and are unlikely to be undertaken. This thesis describes a range of research strategies to identify potential avenues to predict and improve patient responses to existing and novel therapies. It comprises a combination of clinical and translational research. Using our institutional database I have assessed the utility of post-treatment imaging with FDG-PET as it may serve as a means of early detection of poor response to treatment. I found that a complete metabolic response on post-treatment PET scan was predictive of overall survival and disease-free survival. The database, which spans a thirty year period, was also interrogated to explore patterns of treatment failure, subsequent salvage treatment and outcomes. I found that multiple treatment modalities have been utilised to treat patients with persistent or recurrent disease, with satisfactory survival benefit in carefully selected patients. I also evaluated the literature that investigated the molecular biology of anal cancer finding that no clinically valuable biomarkers have emerged. Some suggestions have been reported that regulators of apoptosis, including survivin, and agents targeting the PI3K/AKT pathway, might offer opportunities for targeted therapy. Additionally, antibody therapy targeting epidermal growth factor receptor may prove efficacious although the safety profile in combination with standard chemoradiotherapy has proven to be suboptimal. In the laboratory, next generation RNA sequencing was utilised in eleven anal SCC patient samples. Through stratification of the tumours into clinically relevant groups and Bioinformatic analysis, eight genes with differential expression were chosen for further validation. One of these genes was identified as a novel target which could ultimately lead to expanding therapeutic options in anal cancer management. Due to a lack of pre-clinical models, including cell lines and mouse models for testing new therapies, I developed a new anal cancer model based upon patient-derived tumour xenografts. I used this model in a pilot experiment to assess the novel drug target identified by RNA-seq. The outcomes were promising with stand-alone efficacy of the novel drug observed with statistical significance, while also validating the feasibility of using xenografts for anal SCC. This thesis builds upon the clinical experience of decades of management of patients with anal cancer identifying both clinical and laboratory approaches to advance assessment and identify novel treatment possibilities for this group of patients.
-
ItemInvestigating the role of polarity protein, SCRIB, in haematopoiesis( 2017)The evolutionarily conserved scaffolding protein, Scribble (SCRIB), acts as a tumour suppressor in multiple epithelial cancers. However, SCRIB’s role in haematopoiesis and haematopoietic malignancies is largely unknown. As SCRIB knockout mice are embryonically lethal, we utilised conditional knockout mouse models driven by the Mx1 or hScl promoter in conjunction with extensive phenotypic analyses to elucidate SCRIB’s role in haematopoiesis. Our comprehensive analyses revealed loss of SCRIB produced subtle but significant defects in lymphoid and myeloid progenitor fractions although the specific fraction affected varied between the conditional knockout mouse models. As these two models have different kinetics we utilised a reverse transplant assay, with similar kinetics to the Mx1-Cre model, to examine the cell intrinsic effect of SCRIB loss in haematopoiesis. Similar to our previous results, this assay revealed a significant role for SCRIB in early lymphoid and myeloid development. The subtlety of phenotype produced by deletion of SCRIB is most likely due to compensatory mechanisms so we induced stress haematopoiesis through irradiation to mitigate the effect of compensatory mechanisms. Lethally irradiated recipients receiving SCRIB knockout bone marrow revealed a more severe phenotype compared to previous assays with defects in erythropoiesis, myelopoiesis and lymphopoiesis. Taken together, our data reveals a role for SCRIB in multiple haematopoietic lineages during steady-state haematopoiesis thereby suggesting SCRIB may be important during leukemogenesis. Studies are currently underway to investigate how SCRIB impacts on the onset and severity of acute myeloid leukaemia and acute lymphocytic leukaemia.
-
ItemInvestigating prognostic and predictive tissue biomarkers in head and neck squamous cell carcinoma( 2017)Head and neck squamous cell carcinoma (HNSCC) comprises a diverse group of cancers that arise from a number of subsites of the upper aerodigestive tract, including the oropharynx, oral cavity, larynx and hypopharynx. These cancers have traditionally been linked to tobacco and alcohol use (Gillison et al 2008; Goon et al 2009) and despite improvements in treatment, the outcome for patients with locally advanced HNSCC remains poor. Current treatments are associated with significant acute and late toxicity which can have a significant long term negative impact on function and quality of life. Current tumor staging methods and biomarkers are limited in their capacity to consistently distinguish groups with different outcomes, and to permit tailoring of therapy based on prognosis and biological features. Furthermore, it is recognised that there are significant clinical differences between the different subsites of head and neck cancer, warranting molecular studies focused on individual sites. Thus, to improve patient outcomes, it is becoming increasing important to identify novel biomarkers of prognosis and response to therapy which will empower clinicians with better patient selection strategies for risk-adapted treatments and emerging molecular therapies. It is now accepted that a significant proportion of oropharyngeal cancers, are caused by infection with human papilloma virus (HPV) (Fakhry et al 2008; Ang et al 2010). HPV positive oropharyngeal cancer is known to differ from HPV negative epidemiologically, clinically and molecularly and most importantly, HPV positive status is associated with a greatly improved prognosis (Fakhry et al 2008; Ang et al 2010). The involvement and importance of HPV in non-oropharyngeal HNSCC sites however remains unclear. Other than HPV, which is often determined diagnostically via p16INK4A immunohistochemical staining (an accepted surrogate biomarker in oropharyngeal cancer), there are no clinically useful prognostic or predictive biomarkers in HNSCC. The identification of relevant biomarkers, which can be easily assayed in a diagnostic setting, utilising readily accessible formalin-fixed, paraffin-embedded (FFPE) tissue blocks, is critical to improving patient outcomes. Such assays include immunohistochemistry (IHC) to look at protein expression and fluorescence in situ hybridisation (FISH) for the study of gene copy number. This thesis comprises five published peer reviewed journal articles containing research investigating the role of HPV/p16INK4A in head and neck cancer; both establishing the role of HPV/p16INK4A in oropharyngeal cancer, as well as work demonstrating that the role of HPV in non-oropharyngeal HNSCC subsites including the oral tongue and larynx is not as clear or important as oropharyngeal cancer. Further, I present work investigating several putative predictive biomarkers and their potential roles in providing clinically useful information for potential patient stratification. All of these studies utilised several large unique HNSCC patient cohorts comprising FFPE tissue blocks and comprehensively annotated clinicopathological and outcome data.
-
ItemIdentification of cooperating oncogenic lesions in Myc-driven lymphoma( 2017)MYC is a potent oncogene that is deregulated in nearly 50% of all human malignancies and as such, is considered an attractive molecular target for inhibition. However, MYC is rarely mutated, has no enzymatic activity that can be pharmacologically exploited and is expressed by normal cells leading to the current view that MYC is “undruggable” and indeed, its pharmacological inhibition has proved elusive. Therefore, discovering genes and pathways that interact with oncogenic MYC signalling and identifying them as potential therapeutic targets in cancers with ectopic MYC expression is of high clinical importance. The Eμ-Myc mouse has been utilised extensively as a faithful model of MYC-driven B cell lymphomagenesis. The Eμ-Myc transgene mimics the t(8;14) translocation apparent in Burkitt’s lymphoma, where ectopic Myc expression is driven by the Eμ- (IGH) promoter elements. Despite being driven by a single oncogene, Eμ-Myc lymphomas demonstrate remarkable heterogeneity indicating that the pathway to frank clonal neoplasia relies on oncogenic Myc signalling and the acquisition of at least one other mutation that cooperates with Myc. Hence, the Eμ-Myc model is a powerful tool in identifying MYC-cooperative genes and pathways. However, to date there has only been partial characterisation of the secondary, tertiary and quaternary mutations that can cooperate with Myc in driving Eμ-Myc lymphomagenesis. To identify somatically acquired Myc-cooperative lesions, massive-parallel sequencing was applied to spontaneous Eμ-Myc B cell lymphomas. Whole genome sequencing was used to map three copies of the Eμ-Myc transgene to chromosome 19 in the germline corresponding with an adjacent chromosome 19 segmental copy number gain. The chromosome 19 amplicon is in a region syntenic to an oncogenic region frequently amplified in human B cell malignancies. The chromosome 19 amplicon was demonstrated to undergo additional somatic gain in 50% of Eμ-Myc lymphoma. In addition to the identification of mutations in genes already implicated in Eμ-Myc lymphoma (Trp53, Cdkn2a, Nras, Kras), whole exome sequencing identified high frequency protein truncating mutations in Bcl6-co-repressor (Bcor). Furthermore, co-occurring tertiary driver lesions involving Cdkn2a (p19ARF) deletion and either Bcor or Ras mutations were identified in clonal Eμ-Myc lymphomas. RNAi and CRISPR-Cas9 mediated knockdown/knockout of Bcor in Eμ-Myc foetal liver hematopoietic stem cells reconstituted into recipient mice demonstrated significantly reduced latency of disease onset, validating Bcor as a tumor suppressor gene in the Eμ-Myc model. Gene-expression profiling of these Eμ-Myc tumours with forced Bcor-loss identified a reliable signature of Bcor loss that was distinct to Trp53 mutation signatures and was redolent of Tgfβ-pathway activation signature. The Eμ-Myc model of lymphoma has been heavily utilised but never fully genomically characterised until now. By applying next generation sequencing technology to a first generation animal model of cancer, this thesis challenges several persisting assumptions made about Eμ-Myc lymphoma. Firstly, data herein suggests that both oncogenic Myc expression along with the chromosome 19 amplification is the initiating driver event in Eμ-Myc lymphoma. This has obvious implications for the conclusions drawn in many publications predicated on the assumption that ectopic Myc expression is the exclusive initiating oncogenic lesion in Eμ-Myc lymphoma. Secondly, the discovery that homozygous deletion of Cdkn2a does not totally attenuate selective pressure for the acquisition of tertiary driver mutations indicates the significance of Cdkn2a deletion in Eμ-Myc lymphoma is overestimated. Thus, deductions made about the cooperative mechanism between CDKN2A and how it opposes proliferative MYC-signalling in human neoplastic transformation may need to be revisited. Finally, the identification of biologically functional high frequency Bcor mutations in Eμ-Myc lymphoma has defined a novel pathway that is potentially capable of restraining oncogenic MYC activity. That Bcor inactivation is reminiscent of Tgfβ-pathway enhancement is suggestive of perturbation of the oncogene induced senescence pathway. If this is the pathway through which Bcor exerts its tumour suppressive activity then it is feasible that dissection of this pathway will lead to the identification of novel therapeutic targets that can be selectively exploited in human malignancies in which MYC is oncogenically deregulated.
-
ItemInvestigating therapeutic strategies targeting metabolism in NRAS-mutant melanoma( 2017)In human cancers, RAS mutations are among the most commonly identified mutations; however therapies targeting RAS remain elusive. Recent investigations have demonstrated that mutated RAS can reprogram metabolism in cancer cells. In the field of melanoma, the role of mutant BRAF in the regulation of metabolism has also been elucidated. However, surprisingly little is known about the importance of mutant NRAS in melanoma metabolism. Alongside this, there are limited targeted therapies available for the treatment of patients with NRAS-mutant melanoma. Consequently, this thesis aims to characterise the metabolic response of RAS-mutant cells to targeted therapies and to use this knowledge to develop novel therapeutic strategies targeting glucose metabolism in NRAS-mutant melanoma. Using human cancer cell lines, the studies in this thesis demonstrate that a number of similarities exist between NRAS and BRAF-mutant melanoma cells with respect to their metabolic responses to MAPK pathway inhibition. MEK inhibition, consistent with prior reports of the effects of BRAF inhibition in BRAF-mutant melanoma, resulted in suppression of glycolysis evidenced by decreased lactate production, glucose uptake and extra-cellular acidification rate. Importantly, known transcriptional regulators of glycolysis in BRAF-mutant melanoma (HIF1α, MYC and MondoA) also play a role in the response of NRAS-mutant melanoma cells to MEK inhibition. Furthermore, in the setting of MEK inhibition NRAS-mutant melanoma cells have increased oxidative metabolism, with increased PGC1α and MITF expression. This adaptation has previously been reported in BRAF-mutant melanoma. The studies in this thesis investigated the relative importance of the MAPK and PI3K effector pathways of RAS, demonstrating that MAPK pathway inhibition had the most consistent and significant effects on glucose metabolism in NRAS-mutant melanoma cells. Finally, a comparison of NRAS and KRAS-mutant cells revealed that NRAS-mutant cells are more sensitive to MEK inhibition, with a more pronounced reduction in parameters relating to glycolysis. Given these findings, it was hypothesised that combining a MEK inhibitor with an inhibitor of glucose metabolism would be an effective therapeutic strategy in NRAS-mutant melanoma. To this end, the MEK inhibitor trametinib was combined with the mitochondrial inhibitor PENAO. In vitro, PENAO enhanced the anti-proliferative activity of trametinib in NRAS-mutant melanoma cells, with additive effects on glycolysis and mitochondrial metabolism. In vivo, the combination was well tolerated, however the addition of PENAO did not enhance the effect of trametinib on tumour growth. These studies are important in demonstrating the feasibility of a combination targeting two key metabolic processes in vivo, particularly when one process is under the control of an oncogenic aberration. A chemical screen to identify combinations that enhance the suppression of glycolysis achieved by MEK inhibition has been commenced. In summary, this work has characterised important metabolic adaptations in the context of MEK inhibition in NRAS-mutant melanoma. Although an effective targeted therapy for RAS remains elusive, this research supports the ongoing exploration of strategies that target RAS effector pathways in combination with key metabolic processes, particular in the context of NRAS-mutant melanoma.