Medicine (Austin & Northern Health) - Theses
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The effect of sodium on sympathetic nervous system activity and endothelial function in people with type 2 diabetes
Lowering dietary sodium intake is recommended by public health bodies to lower blood pressure with the ultimate aim of reducing cardiovascular-related disease. However, lower sodium intake has been demonstrated in observational studies to be associated with adverse health outcomes such as higher cardiovascular-related morbidity and mortality in people with type 2 diabetes. They may be due to pleiotropic effects of sodium lowering, such as activation of the renin-angiotensin-aldosterone system (RAAS) and increases in circulating adrenaline and noradrenaline. Dedicated intervention trials are lacking but greatly needed to investigate the complex role of sodium on cardiovascular outcomes further. In an effort to underpin the mechanistic links behind the associations between low sodium intake and poorer cardiovascular outcomes in people with type 2 diabetes, the studies presented in this thesis aimed to examine the effect of sodium on cardiovascular health by measuring the primary endpoints of sympathetic nervous system activity, as assessed by microneurography, and endothelial function, as assessed by endoPAT and endothelial microparticle measurements, and the secondary endpoints of cardiac baroreflex, serum aldosterone, as markers of RAAS activity, and simultaneous measures of 24h blood pressure and heart rate variability. We demonstrated, that compared to other groups along the glucose continuum, individuals with treated type 2 diabetes who had low sodium intake presented with worse endothelial and baroreflex function. This occurred even though these individuals were appropriately managed for their cardio-metabolic risk factors and importantly, adhered to recommended low sodium intake guidelines. This study provided the rationale to conduct an interventional study where, for the first time, we assessed the effect of salt supplementation on cardiovascular endpoints of sympathetic activity and endothelial function as assessed by endoPAT, in people with type 2 diabetes. Salt supplementation was associated with a small increase in sympathetic activity. However, this did not reach levels associated with pathological disease states. Importantly, there were no detrimental effects on endothelial function or alterations in blood pressure. Interestingly improvements in baroreflex function and RAAS activity were seen. When participants were categorized based on salt-sensitivity, salt-resistant individuals demonstrated a trend towards improved endothelial function after salt supplementation. To explore the endothelial response further, we examined the association between habitual sodium intake, RAAS blockade, and endothelial function as assessed by circulating microparticles. Despite expecting higher endothelial microparticles being associated with lower sodium intake, we did not demonstrate any significant association between sodium intake and endothelial microparticles. However, we observed a trend towards higher erythrocyte-derived microparticle levels being associated with lower sodium excretion and higher platelet-derived microparticles being associated with the lowest tertile of 24hUNa excretion. Additionally, RAAS blockade was not associated with lower microparticles counts in the setting of a low sodium intake, questioning whether the therapeutic benefits of RAAS blockade may be attenuated during lower sodium intake. Despite public health bodies advocating for lowering dietary sodium, we demonstrated that these guidelines are simply not being met and sodium intake is unlikely to change over time. Furthermore, we compared the two most common methodologies used to estimate sodium intake in cardiovascular outcome trials which have largely formulated the basis of dietary sodium guidelines. We demonstrated poor agreement between 24h dietary recall and 24h urine sodium excretions, likely due to underestimation and underreporting of sodium intake via 24h dietary recall. Taken together, these findings begin to provide mechanistic insight that sodium lowering in people with type 2 diabetes may not provide the benefit on the sympathetic and endothelial system as intended. Therefore, the necessity for stringent sodium lowering is questioned in this population. Our studies additionally demonstrate the need for future studies to use sound methodological approaches to determine the ideal yet feasible dietary sodium intake targets in people with type 2 diabetes.
The Association Between Mitral Valve Prolapse With Ventricular Arrhythmias and Sudden Cardiac Death
The mitral valve is one of four heart valves, and is situated between the left atrium and left ventricle. Its role is to facilitate the unidirectional flow of blood through the left atrium and ventricle by opening in diastole and closing in systole. Mitral valve prolapse (MVP) involves the atrial displacement of the mitral valve during ventricular systole. While MVP has been linked to the development of sudden cardiac death (SCD) through the development of malignant ventricular arrhythmias (VAs), this association remains controversial. This thesis examines the relationship between MVP, VAs and SCD. Chapter 1 reviews the published literature surrounding MVP, VAs and SCD. A historical context and contemporary link between MVP and SCD are discussed. Various histopathological, cardiac imaging and electrophysiological findings in MVP and VAs or SCD are explored. The current pathophysiological understanding of SCD in MVP is highlighted, and provides a premise for subsequent chapters. Chapter 2 systematically reviews all cases of MVP and SCD reported in the literature. The cases in the literature describe a predominantly young, female population with frequent premature ventricular complexes (PVCs) and prolapse involving both mitral valve leaflets. Cardiac arrest usually occurs as a result of ventricular fibrillation. Leaflet redundancy (defined as thickness greater than or equal to 5mm) is the only independent predictor of SCD. Chapter 3 describes histopathological findings and cardiac arrest rhythm in individuals with isolated MVP (iMVP, whereby other potential causes of death are excluded) and SCD. Individuals with iMVP-SCD have increased cardiac mass, mitral annulus size and left ventricular fibrosis compared to a control group matched for age, sex, height and weight. In those with iMVP and witnessed cardiac arrest, ventricular fibrillation is the predominant cardiac arrest rhythm. These findings suggest that histopathological changes in MVP may provide the substrate necessary for the development of VAs leading to SCD. Chapter 4 comprehensively and systematically quantifies left and right ventricular fibrosis in individuals with iMVP-SCD compared to a matched control group. Individuals with iMVP-SCD have more left ventricular and interventricular septum fibrosis but similar degree of right ventricular fibrosis compared to the control group. In those with iMVP-SCD, there is more fibrosis in the lateral and posterior walls of the left ventricle with an endocardial-epicardial gradient of fibrosis, which is similar to other conditions that cause cardiac remodelling. These findings indicate that non-uniform left ventricular remodelling with both localised and generalised fibrotic changes are important in the pathogenesis of SCD in MVP. Chapter 5 compares the incidence of VAs detected with continuous cardiac rhythm monitoring in patients with MVP and controls, and examines whether certain factors predicted for the development of VAs within the MVP group. Over a 24-month follow-up period, those with MVP had a significantly higher overall incidence of VAs compared to control groups. Within the MVP group, the presence of late gadolinium enhancement on cardiac magnetic resonance imaging was predictive for the development of VAs. Chapter 6 evaluates the role of mitral regurgitation and mitral valve surgery with regards to VAs in patients with redundant leaflet MVP. In patients with redundant leaflet MVP, severity of mitral regurgitation does not affect PVC burden while mitral valve surgery does not reduce PVC burden in unselected patients with MVP. Based on the significant change in PVC burden in two patients, the role of mitral valve surgery in selected patients with MVP warrants further investigation.
The effects of lung volume recruitment therapy on respiratory function and quality of life in people with neuromuscular disease
Respiratory muscle weakness results in substantial discomfort, disability and ultimately death in many neuromuscular diseases (NMDs). Respiratory compromise manifests as some or all of, shallow breathing, poor cough and associated difficulty clearing mucus, respiratory tract infections, hypoventilation, sleep-disordered breathing and chronic ventilatory failure. As survival outcomes improve for many NMDs, there is a shift towards more proactive and preventative chronic disease multi-disciplinary care models that manage symptoms, improve morbidity and reduce mortality. Unfortunately, clinical care guidelines for chronic NMD care are based largely on clinical rationale and consensus opinion rather than level A evidence. These guidelines typically recommend therapies to enhance lung inflation and cough effectiveness, however there is minimal evidence that performing techniques regularly is beneficial. Lung volume recruitment (LVR) is one such therapy. Simple, inexpensive and widely-accessible, it delivers air via a manual resuscitation bag to augment lung inflation above a person’s own deepest breath. Given the absence of prospective controlled research, this thesis aimed to investigate the effect of regular LVR in people with NMD. Firstly, a cross-sectional cohort study of 80 community-dwelling adults with NMD and respiratory system impairment identified that participants with slowly-progressive forms of NMD have smaller lung volumes and respiratory system compliance (Crs) than participants with rapidly-progressive motor neurone disease, despite having a similar degree of respiratory muscle weakness. Stiffness was associated with smaller lung volume in long-standing NMD, supporting the hypothesis that maintaining lung volume and Crs may ameliorate respiratory decline. The second component of this thesis confirmed the feasibility of LVR; 95% of participants naïve to the therapy could successfully augment their lung insufflation capacity (LIC, the maximum inflation capacity obtained by assisting inflation). Moreover, LIC and Crs increased following a single-session of LVR therapy. These immediate effects were only evident when naïve; when assessed three-months later there was no change in respiratory function following a single-session of LVR. The third and primary component of this work, a randomised controlled trial of twice-daily LVR or an active control treatment for three-months, found a statistically significant difference in LIC between groups favouring LVR. No demonstrable change in lung volumes, respiratory muscle strength, symptoms or quality of life was found, suggesting a learning effect or acclimatisation to higher inflation pressures may be responsible for the increase. However, an improvement in Crs predominantly in the LVR group means a beneficial effect on underlying respiratory mechanics cannot be excluded, especially if conducted for a longer duration. Notwithstanding the need for further longitudinal studies, the observed improvement in the primary outcome of LIC in the absence of apparent harm or burden, provides robust preliminary data supporting clinical recommendations and practice that regular LVR be performed by people living with NMD. The clinical significance of a higher LIC is still to be fully realised, but this thesis has demonstrated an effect that is compatible with the clinical and biologically-plausible rationale for this therapy.
Predictors of Relapse in Polymyalgia Rheumatica Patients Treated with Low-Dose Glucocorticoid Therapy
Polymyalgia rheumatica (PMR) is a chronic inflammatory condition characterised by subacute onset shoulder and pelvic girdle pain, and early morning stiffness. Oral glucocorticoids represent the treatment mainstay and whilst cessation of therapy is the goal, a subset of patients with relapsing disease remains poorly delineated. This thesis aimed to characterise the demographic, clinical, laboratory and imaging phenotype of patients that relapse by undertaking a prospective longitudinal cohort study of newly diagnosed, steroid naive PMR cases (2012 EULAR/ACR classification criteria) treated with a weaning schedule of prednisolone (BSR guideline). Disease relapse, as defined by the PMR activity score (PMR-AS), represented the primary outcome measure and was observed in 25/32 participants (78.1%) during the 46-week follow-up period. Older age at diagnosis (HR 1.07) and female sex (HR 2.38) were subsequently associated with an increased risk of relapse, whilst a volar pattern of 18F-FDG uptake on whole body PET/CT conferred a distinct prognostic advantage (HR 0.25). Half of the study participants also met criteria for the secondary endpoint of glucocorticoid-resistant disease. Baseline predictors for this outcome again included older age and female sex, along with high BMI and high neutrophil to lymphocyte ratio (NLR). Results from this work additionally informed a proof-of-concept PET/MRI fusion study identifying hamstring peritendonitis as the anatomic correlate of abnormalities observed adjacent to the ischial tuberosities. A nested case-control subsequently achieved a sensitivity of 90.9% and specificity of 92.4% for diagnosing PMR on whole body PET/CT based upon a novel scoring algorithm that combined this finding with other key sites of extra-capsular pathology. Finally, data was utilised for preliminary validation of candidate instruments being considered for inclusion in a PMR core outcome measurement set to be endorsed by the international body, OMERACT. Overall, the findings presented in this thesis confirm the hypothesis that discrete phenotypic differences exist among PMR patients with relapsing disease and make a substantive novel contribution to the literature concerning the pathology, diagnosis, monitoring and management of this rheumatic disease. The low incidence of sustained clinical remission observed in this study following a standardised wean of glucocorticoid therapy should however serve as a timely reminder of the ongoing need for an alternate treatment paradigm in PMR.
Diabetic Kidney Disease with a focus on Renal Hyperfiltration
Currently, it is not known if the process of renal hyperfiltration is adaptive as a compensatory effect akin to pregnancy or is maladaptive and can result in non-reversible renal damage and the development of diabetic kidney disease. The overall aim of my study was to investigate renal hyperfiltration in diabetes, by comparing this to renal hyperfiltration in pregnancy, assessing renal biomarkers and utilizing functional magnetic resonance imaging. Estimated glomerular filtration rate based on The Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) formula was used as the main surrogate measurement of renal function in this thesis. Where possible, measured glomerular filtration rate using renal Diethylenetriamine Penta-acetic Acid (DTPA) and plasma inulin clearance were employed. The threshold of renal hyperfiltration in this thesis is set at an estimated or measured glomerular filtration of >120ml/min/1.73m2. Candidate inflammatory and oxidative stress serum and urinary renal biomarkers were selected based on current evidence in diabetic kidney disease. 3-Tesla magnetic resonance imaging was used to acquire images on renal tract, with data providing information on renal hypoxia and microstructural changes. The main findings from assessing women with type 1 and type 2 diabetes during pregnancy were that women with type 1 diabetes may have an attenuated ability to hyperfiltrate in pregnancy, which is possibly related to pre-existing impaired renal function. However, in general, women with type 1 and type 2 diabetes, including those with mild to moderate diabetic kidney disease are not at risk of accelerated renal function decline from pregnancy. Both type 1 and type 2 diabetes carry a similar high risk for poorer maternal and fetal outcomes compared to healthy pregnancies. For these outcomes, a lower estimated glomerular filtration rate was associated with pre-term birth and neonatal intensive care admission independent from albuminuria. Understanding how pregnancies in type 1 and type 2 diabetes can affect maternal renal function; and on the other hand, how maternal renal function can affect pregnancy outcomes are important in guiding clinicians to provide optimal care this high-risk antenatal cohort. Secondly, with regards to functional magnetic resonance imaging, we demonstrated that there is no evidence that renal hypoxia or micro-structural changes occur in those with renal hyperfiltration and type 1 diabetes. Magnetic resonance imaging parameters also do not reflect level of renal function determined with a direct measurement of glomerular filtration rate, nor do they strongly correlate with selected inflammatory and oxidative stress markers in type 1 diabetes. Finally, we did not observe any difference in the concentration of renal biomarkers in those who were hyperfiltering, compared to those who were not hyperfiltering in type1 diabetes. Elevation of renal biomarkers during peak hyperfiltration in the second trimester of pregnancy were comparable in women with and without diabetes. Our findings of a lack in rise of inflammatory and oxidative stress biomarkers do not support the hypothesis that inflammation is present in renal hyperfiltration in diabetes. In conclusion, findings from this thesis suggest that hyperfiltration in diabetes may not necessarily reflect a maladaptive stage of diabetic kidney disease. Due to the exploratory nature of the studies, along with the limitation of estimated glomerular filtration rate, future work involving larger cohort of participants and measured glomerular filtration rate may provide more in-depth understanding of renal hyperfiltration in diabetes. Understanding the significance of renal hyperfiltration is important, particularly as this process occurs in the early stages of diabetes and would be an avenue of targeted therapy to prevent not only the progression, but development of diabetic kidney disease.
Development, prevalence and treatment of blood pressure abnormalities in spinal cord injury
Disorders of blood pressure control arise from disruption of the autonomic nervous system and result in symptomatic orthostatic hypotension and large fluctuations in blood pressure. Ambulatory blood pressure monitoring is used in the general population for assessment of blood pressure control and to detect episodes of hypotension. In spinal cord injury (SCI), impaired control of the sympathetic nervous system leads to orthostatic intolerance and autonomic dysreflexia. Smaller studies in restricted populations have examined ambulatory pressures in SCI and observed abnormalities in diurnal blood pressure variation in complete cervical SCI. Altered diurnal blood pressure is associated with abnormalities in diurnal urine production and orthostatic intolerance in autonomic failure. This triad may also occur in SCI to explain the orthostatic intolerance. A retrospective examination of ambulatory pressures of patients with SCI referred for clinically significant blood pressure disorders revealed a high prevalence of abnormalities in diurnal blood pressure and urine production in acute and chronic tetraplegia and in acute paraplegia. To characterise the course of diurnal blood pressure, urine production and orthostatic symptoms in SCI, two prospective studies were performed. First, consecutive patients admitted with acute SCI were screened for recruitment, and consenting volunteers were compared with immobilised and mobilising controls. In the second study, people with chronic SCI (>1 year) living in the community were compared with mobilising controls. Compared with mobilising and immobilised controls, there was a high prevalence of abnormal diurnal blood pressure variation in SCI. The abnormalities were most prevalent and marked in higher and earlier SCI. Abnormalities persisted over time in both complete and incomplete cervical SCI. Abnormalities in high paraplegia were no different to that of cervical SCI in early SCI, but were no different to that of controls in chronic SCI. Nocturnal hypertension occurred in the absence of day hypertension in SCI, thus clinic pressures may miss elevation in blood pressures and ambulatory monitoring may be beneficial in assessment of blood pressure in SCI. Abnormalities in diurnal urine production were present and persisted over time in cervical SCI, but appeared to improve over time with thoracic SCI, mirroring changes in diurnal blood pressure. Orthostatic hypotension and orthostatic intolerance were more prevalent in SCI than in controls. Mild orthostatic intolerance is common in chronic SCI and a small proportion had more severe symptoms. A single centre study to investigate a drug treatment for orthostatic intolerance in the setting of a loss of diurnal blood pressure variation was unsuccessful due to low recruitment rates. As life expectancy increases in the SCI population, there is evidence of increased rates of cardiovascular disease. Similar to the general population, the loss of diurnal blood pressure variation and elevated nocturnal pressures may be a contributor to the increased cardiovascular disease in SCI, and thus a potential therapeutic target. Ambulatory blood pressure monitoring may be useful to detect these changes and examination of a larger SCI population with a longer duration of follow up for cardiovascular disease may help to determine this.
Mitochondrial Stress and Metabolic Dysfunction in the Liver: A Role for The Unfolded Protein Response
Liver dysfunction and fat accumulation in liver correlates with metabolic syndrome, also increases the risk of development of type 2 diabetes, advanced liver disease and several other complications. There are no simple or widely effective medical solutions to these conditions and further studies are required for clarification of mechanisms involved. Mitochondria are the powerhouses of the cell, responsible for cellular energy production and help maintain the cellular environment through complex procedures. Thus, it is fundamental for the mitochondria to function properly. Mitochondrial dysfunction is known to be associated with metabolic diseases including NASH and Type 2 diabetes where it has been implicated in hepatic fat accumulation and hepatic insulin resistance. Various enzymes and proteins are required to make mitochondria function properly. Mitochondrial dysfunction can be caused by build-up of reactive-oxygen-species which leads to the activation of a stress response known as the mitochondrial-unfolded-protein-response (UPRmt). Failure of these responses to adapt to or repair damage from stress results in mitochondrial dysfunction and may lead to apoptosis and eventually death. Ubiquitin-like-5 (UBL5) is a protein that was first discovered in the obese Israeli sand rat, associated with weight gain. Studies in C. elegans have shown that UBL5 is involved in the activation of the mitochondrial unfolded protein response (UPRmt) to ensure that chaperone proteins are transcribed to relieve the ensuing stress. This pathway upregulates mitochondrial proteases and chaperone proteins to alleviate proteostatic stress. To further explore how mitochondrial stress through the mitochondrial unfolded protein response lead to liver dysfunction, we generated a tamoxifen-inducible, liver-specific UBL5 knockout mouse. In earlier studies, we performed systematic assessments of the role of UBL5 in mitochondrial function and energy metabolism. We found that mice with complete deletion of UBL5 in liver are glucose intolerant and present with features of liver failure (gross steatosis, apoptosis and significantly increased hepatic enzymes). Furthermore, these mice present with oxidative stress reduced mitochondrial respiration, increased ROS levels and decreased UPRmt gene expression (CHOP, ClpP, ClpX, Lonp1, HSP10, HSP70, ATF-5) while mtHSP70 was upregulated. PGC1a mitochondrial biogenesis gene was also downregulated. Our heterozygous mice (50% UBL5 reduction), however, did not present with any defects when on a chow diet. Therefore, our aim was to determine whether exposing the heterozygous mice to high-fat diet rich in cholesterol (HFD) would make them more susceptible to liver stress. We found that following 2 months of HFD feeding, the heterozygous mice were healthier than the wildtype controls. They displayed better glucose tolerance, liver enzyme levels and histology. Furthermore, gene expression of UPRmt were significantly higher in the HFD heterozygous mice compared to the HFD fed wildtype controls. The UBL5 KO mice were then treated with pioglitazone, or an ACE2-containing virus to determine if such treatments provide benefit. Liver enzymes and fat accumulation showed significant improvement after treatment with both pioglitazone & ACE2. Which had UPRmt upregulated in pioglitazone treated group and normalized in ACE2 treated UBL5 KO mice. The current dissertation demonstrated that a liver-specific deletion of the mitochondrial stress response gene UBL5 in adult mice leads to reduced UPRmt, mitochondrial dysfunction / hepatic failure and early death. We have generated a model that develops severe fatty liver disease and have shown that both genetic & pharmacological therapies may diminish this disease process. This study provides the first evidence for a critical role for UBL5 and UPRmt in hepatic function. However, the main reason of liver failure remains unknown and further study is required. In lower species the homeostatic nature of the UPRmt it is well-known. If this pathway is sustained in mammals, controlling this stress response might help to establish a therapeutic strategy to cure disease characterized by mitochondrial disruption.
Optimising Clinical Outcomes in Patients with Acute Coronary Syndromes
Ischaemic heart disease remains the leading cause of death worldwide despite significant advances in diagnosis and treatment. Over the past two decades, interventional and medical therapies have been tested through rigorous large scale randomized controlled trials. These data form the backbone of national and international guidelines. However, there are still numerous controversial issues in the short- and long-term management of patients with acute coronary syndromes (ACS) and there are limitations in translation of scientific evidence into clinical practice. Using a well-established multi-centre interventional cardiology registry, this thesis focuses on contentious issues in the management of ST-elevation myocardial infarction (STEMI) and non-ST-elevation acute coronary syndromes (NSTEACS). The topics addressed range from the safety of pretreatment with dual antiplatelet therapy in STEMI and NSTEACS, to the impact of door-to-balloon time in high-risk patients and the long-term prognostic significance of optimal medical therapy and smoking cessation. In addition, single-centre studies were used to explore in detail the management of patients older than 85 years with acute coronary syndromes and the impact of periprocedural myocardial infarction on long-term mortality. Finally, through a randomized controlled trial of a smartphone based cardiac rehabilitation program, this thesis underscores the value of an innovative model to optimize secondary prevention of coronary artery disease.
Spontaneous versus spontaneous timed mode of assisted ventilation in extrathoracic restrictive lung disease
Restrictive lung disease can be divided into intrathoracic restriction due to parenchymal causes and extrathoracic restriction due to chest wall disease or neuromuscular disease. Patients with extrathoracic lung restriction suffer from shortness of breath, disturbed sleep and eventually type two respiratory failure when their condition progresses. Type two respiratory failure is characterised by hypoxaemia and hypercapnia. Effective treatment of this type of respiratory failure involves ventilatory support in the form of assisted mechanical ventilation. Non-invasive positive pressure ventilation has been a major advance in delivering mechanical ventilation since the late 1980’s. This can be in the form of volume-cycled ventilation or pressure-cycled ventilation. Pressure-cycled ventilation is more commonly practised in Australia and around the world; particularly Europe; according to large multicentre surveys. This can be delivered in spontaneous mode (S) or spontaneous timed mode (ST). There is no current evidence that one mode is superior to the other, and the two modes are roughly equally practised around the world and in Australia according to the available surveys. The aim of this research was to compare S mode to ST mode in a double-blind cross over design over six weeks in 20 patients with stable extrathoracic lung restriction from chest wall and neuromuscular disease. The study aimed to objectively assess adequacy of ventilation, quality of sleep and impact on the patient in psychomotor function, sleepiness as well as patient preference. This was assessed by performing blood gas analysis, sleep studies, psychomotor vigilance testing (PVT) as well as patient questionnaires. We also performed a physiological arm of the study in a subset of patients, assessing the participants for ineffective efforts and poor triggering by inserting an oesophageal balloon and monitoring for markers of effort not followed by a ventilator breath. The oesophageal balloon-catheter system was calibrated for frequency response under different conditions prior to starting this arm of the study and for adequacy of signal acquisition before each sleep study. Due to difficulty in recruitment of participants and some patients withdrawing or failing the safety criteria for the study, a total of 13 patients completed the two arms of the study. The results of this research demonstrated that ST mode was associated with improved nocturnal oxygenation, sleep quality and fewer respiratory events than S mode in patients with stable chest wall and neuromuscular disease. There was no significant difference in daytime hypoventilation as assessed by carbon dioxide measurements. The physiological arm of the study showed that ST mode resulted in fewer ineffective efforts than S mode, the former contributing to better sleep efficiency and correlating well with fewer respiratory events. On the other hand, the mode of ventilation had no significant effect on poor triggering of the ventilator. The participants appeared to be less sleepy on ST than S mode; however, neither mode was associated with pathological sleepiness. There was no significant difference in patient preference between the two modes, nor was there a significant difference in psychomotor function between the modes as assessed by PVT. In conclusion, ST mode of non-invasive positive pressure ventilation was superior to S mode in many aspects of ventilation, sleep quality and impact on the patient. Physiologically there were less ineffective efforts on ST mode, which was related to improved sleep efficiency and the frequency of respiratory events while on ventilation. The participants were less sleepy on ST than S mode, however there was no significant difference between the two modes of non-invasive ventilation on on psychomotor function and patients did not prefer one mode over the other. The sample size of this study was small and less than the original target, due to difficulty in recruitment and the need for participants to present to hospital on three different occasions, a considerable expectation in this relatively frail cohort of patients. The concept of measuring oesophageal pressure in awake patients was also a limiting factor in failure to achieve the target number of participants in the sub-study. This would have impacted the power of the study and the overall significance of some of the results. Based on the results of this study, ST mode of non-invasive ventilation should be utilised rather than S mode in patients with extrathoracic lung restriction due to neuromuscular and chest wall disease. Centre specific preferences and cost need to be taken into account when implementing this recommendation. Acknowledging the difficulties with recruitment, larger (possibly multi-centre) studies of longer duration are required to determine long term health benefits such as hospital admissions and mortality in this population.
Characterisation of the epidermal growth factor receptor, vascular and stromal biomarkers in mesothelioma for oncogenic targeted therapies
Malignant Mesothelioma (MM) is an aggressive malignancy of the pleura and other serosal surfaces with limited treatment options. The mainstay of medical treatment is the combination of cisplatin and pemetrexed chemotherapy. Despite initial responses to chemotherapy, nearly all patients will progress. Only recently, the results of the Mesothelioma Avastin Cisplatin Pemetrexed Study (MAPS) demonstrated a statistically The tumour microenvironment represents an important target in mesothelioma given the absence of oncogenic drivers within the tumour itself. Towards this, I have investigated an anti-EGFR monoclonal antibody [ABT-806 (mAb806)] developed by co-supervisor Scott and now licenced to Abbott (AbbVie), which selectively binds an epitope of wild type (wtEGFR) only found in the overexpressed or amplified EGFR, or its oncogenic truncation mutant EGFRvIII, on cancer cell surfaces. The presence of overexpressed wtEGFR that binds the ABT-806 antibody was also found to be present in a number of MM samples. In this thesis, I aim to characterise specific biomarkers in MM, with particular focus on vascular markers and EGFR expression in MM (Chapter 3). The tumour microenvironment that comprises the interface between the tumour and stroma is of particular interest. This thesis focused on EGFR and angiogenesis given the access to novel anti-EGFR antibody drug conjugates. Angiogenesis, assessed by microvessel density (MVD) has previously been reported by several groups to be a poor prognostic factor in MM. The results of an angiogenic and stromal biomarkers analysis in a large mesothelioma patient cohort are consistent with the literature. I identified high Chalkley count of CD31 immunohistochemistry staining (more than or equal to 5) and increased PDGF-CC expression are associated with a poorer prognosis in 326 MM patients. CD31 was found to be an independent prognostic marker in the multivariate analysis. This project seeks to build on previous work establishing these antibodies as novel therapies for cancer and determine their potential in MM, a disease where we have previously established ligand expression. Using our large repository of MM tissues, I have investigated the expression of the selected targets overexpressed/ amplified EGFR to establish the potential for use of these targets in mesothelioma (Chapter 4). I have also created a MM patient derived xenograft (PDX) library via patient samples derived from the institute and our collaborators from Toronto, Princess Margaret Hospital. Via the library of MM PDXs generated during the course of this PhD, I have evaluated EGFR and mAb806 immunohistochemistry to help select specific models to investigate the validity and feasibility of targeting EGFR in MM using novel mAb806 based antibody drug conjugates (ADCs) (Chapter 5). These novel anti-EGFR ADCs were explored in mesothelioma cell line xenografts, then taken to a panel of novel PDX models to further characterise these mAb806 based ADCs to determine if there are any signals of clinical efficacy (Chapter 6). I have demonstrated the efficacy and selectivity of these anti-EGFR compounds with MM cell line MSTO 211H, as well as 2 other PDX models. The cell line and the PDX model which are mAb806 IHC positive had demonstrated significant tumour suppression and therapeutic efficacy with these mAb806 drug conjugates. These findings contribute towards the understanding and development of potential prognostic biomarkers of interest in MM. The data presented in this thesis also provides novel insight into anti-EGFR antibody drug conjugates in mesothelioma and reveal potential targets for development of targeted therapies in this disease where none was thought feasible.
Understanding the molecular basis for response and resistance to targeted therapies in colorectal cancer
Active treatment options for patients with metastatic colorectal cancer (CRC) have evolved significantly over the last 20 years. During this time, the median overall survival of these patients has approximately doubled. However, the five-year survival of patients with metastatic CRC is still poor, and further research to optimise treatment is required. The use of targeted therapies has contributed to improvements in clinical outcomes in these patients. However, not all patients respond to these drugs, and they are expensive. The central aim of this thesis was to explore ways to optimise the targeted treatment of metastatic CRC. Specifically, mechanisms of response and resistance to two targeted therapies - the BRAF inhibitor vemurafenib and the anti-angiogenic drug bevacizumab, were investigated. Whilst treatment with single-agent BRAF inhibitors has been associated with significant improvements in response and survival outcomes in patients with advanced BRAF mutant malignant melanoma, results of clinical trials of BRAF inhibitors for BRAF mutant metastatic CRC have been unexpectedly disappointing. First, by screening a panel of BRAFV600E CRC cell lines, cell lines which were sensitive and resistant to BRAF inhibition were identified, and these models were used to explore mechanisms of resistance to these drugs. High phosphorylated-epidermal growth factor receptor (p-EGFR) protein expression was identified to be a potential basal biomarker of sensitivity to vemurafenib. In resistant BRAF V600E CRC cell lines, BRAF inhibitor treatment was demonstrated to induce rapid p-ERK reactivation which was not associated with increased p-EGFR expression, suggesting the presence of EGFR-independent mechanisms of inherent resistance. Next, ways to overcome resistance of BRAFV600E CRC to BRAF inhibitors were explored. BRAF mutant CRC cell lines which were relatively resistant to BRAF inhibitors were found to be broadly similarly resistant to other MAPK inhibitors, and this was also characterised by p-EGFR independent reactivation of p-ERK. This suggests that in BRAF mutant CRC, resistance to MAPK inhibitors is tumour- rather than drug-specific, and using combinations of multiple MAPK inhibitors is unlikely to overcome resistance. BRAF inhibitor treatment did not induce preferential RAS activation, increased p-CRAF expression, nor increased p-MEK expression in resistant BRAF V600E CRC cell lines, suggesting that alternative mechanisms of resistance originate downstream of MEK. These findings provide the basis for further exploration of non-EGFR mediated mechanisms of resistance, which ultimately may enable the development of new rational targeted drug combination strategies for BRAF mutant CRC. Bevacizumab, an anti-human VEGF-A monoclonal antibody, is currently approved for the treatment of metastatic CRC in combination with chemotherapy. Discovery of a predictive biomarker for response to bevacizumab would enable us to identify patients who are unlikely to respond, commence them on alternative therapies sooner, and avoid unnecessary side effects. Identification of a predictive biomarker may also improve the cost effectiveness of bevacizumab use. We explored whether single nucleotide polymorphisms (SNPs) in VEGF-A, VEGFR1 and VEGFR2 are predictive biomarkers for bevacizumab response, using archived tissue samples and clinical outcome data from participants in the MAX Phase III trial of capecitabine, bevacizumab and mitomycin in the first-line treatment of metastatic CRC. VEGF-A SNPs rs25648 and rs699947 were found to be prognostic, but not predictive biomarkers of survival outcomes.
Clinical Studies in HFE haemochromatosis
HFE haemochromatosis is the most common iron overload disease. Since the discovery of the HFE gene is 1996, it is readily diagnosed using a genetic test rather than using liver biopsies. It is an autosomal recessive disease and the most common form of HFE haemochromatosis is homozygosity for p.C282Y. Homozygosity for this substitution accounts for more than 90% of haemochromatosis in Australia. Excessive iron accumulates due to malfunction of the HFE protein that leads to excess iron absorption. As a result, excess iron builds up in various organs including liver, joints, heart, pancreas, pituitary gland and skin, that may cause end-organ damage including liver cirrhosis, cardiac failure, diabetes mellitus, hypopituitarism and skin pigmentation. Symptomatically, fatigue and arthralgia are the major complaints reported by patients with haemochromatosis. This disease is easily treatable, as the blood contains a significant proportion of the body’s iron, so excess iron can be removed via the blood through phlebotomy. Multiple studies have found that individuals with high total body iron, defined by serum ferritin of more than 1000ug/L, have the highest risk of developing complications including liver cirrhosis. In the last decade, some have suggested that patients with elevated body iron but with serum ferritin less than 1000ug/L, here defined as moderate iron overload, might not need treatment, as they might not have symptomatic manifestations of the disease. However, there have been no randomised controlled trials to examine the treatment benefits for individuals with moderately elevated iron. To answer this question, a randomised controlled trial (Mi-Iron) was conducted which was the major aim of my PhD to examine if removing excess iron would have an impact on patient-reported outcomes, particularly fatigue, as well as liver fibrosis and oxidative stress. This demonstrated that with treatment, there was an improvement in fatigue and its cognitive subcomponent, and the affect component of the arthritis score. There was also an improvement in the liver fibrosis marker, Hepascore, and oxidative stress marker plasma F2-isoprostane, by removing excess iron in this cohort when compared to the control group who did not have iron reduction. These results, therefore, support current guidelines that all patients with haemochromatosis with elevated serum ferritin should have phlebotomy to return body iron levels to normal levels. In the second clinical study, the relationship of serum ferritin with non-invasive markers of liver fibrosis including transient elastography, Hepascore, aspartate aminotransaminase (AST) to platelet ratio index (APRI) and Fibrosis-4 (FIB-4) to assess for liver fibrosis and cirrhosis in HFE p.C282Y homozygotes was examined. This study was conceived due to the shift from using liver biopsy to the increasing use of non-invasive techniques to assess liver fibrosis and cirrhosis. This showed that there was a linear relationship of serum ferritin with Hepascore, indicating that higher body iron is associated with more advanced liver fibrosis and cirrhosis. This relationship was also found for APRI and FIB-4 scores. These findings are important as they provide extra information in utilising these scores to assess liver fibrosis and cirrhosis in haemochromatosis. In the third study, the hand joint arthritis in people with haemochromatosis was examined. Arthralgia is one of the major complaints of individuals with haemochromatosis and is often one of the earliest symptoms of haemochromatosis. It is often difficult to differentiate between haemochromatosis arthropathy and osteoarthritis in the hands. The second and third metacarpophalangeal joints are described to be more commonly affected in individuals with haemochromatosis. By examining the data from HealthIron, a haemochromatosis cohort extracted from a population study that assessed the burden of disease due to iron overload, I found that there was an increase in first metacarpophalangeal joint abnormalities in those with HFE p.C282Y homozygosity, comparable to the frequency of involvement of second and third metacarpophalangeal joints.