Medicine (St Vincent's) - Theses
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ItemDifferentiation of dermal papilla cells into a myogenic lineage for the treatment of Duchenne muscular dystrophy( 2016)Duchenne muscular dystrophy (DMD) is the most common form of muscular dystrophy in children. It is due to an X-linked recessive mutation in the dystrophin gene resulting in the lack of, or production of non-functional, dystrophin. Dystrophin is a pivotal part of the dystophin glycoprotein complex, which transfers mechanical force from the extracellular matrix to the cytoskeleton and stabilizes the myofibre structure. DMD is characterized by significant muscle weakness leading to loss of ambulation and respiratory/cardiac failure and death. No specific treatment has been established so far, thus new therapeutic strategies are being sought. The application of stem cells for therapy in DMD is one of the more promising approaches. Multipotent stem cells residing in the hair follicle dermal papilla are highly plastic and are reprogrammable to bone, cartilage, fat, haematopoietic and myogenic differentiation. Dermal papilla cells (DPC) from the hair follicles of skin were specifically the focus of this study. DPC derived from mouse whisker pad were shown to undergo myogenic differentiation when co-cultured with different types of myoblasts, including normal and dystrophic human myoblasts. Lamin A/C staining was used to distinguish DPC- and myoblast-derived myonuclei inside myotubes to enable measuring the contributions of DPC to differentiated myotubes in cocultures. DPC incorporated into myotubes and up-regulated the muscle marker myogenin in coculture with human myoblasts, suggesting that DPC fully underwent myogenic differentiation in these co-cultures. This study also defines new methods for quantifying myogenic reprogramming in vitro. The data obtained with this method suggested that DPC incorporation efficiency was low in all co-cultures and differed significantly between various types of myoblast; however, no significant difference was observed between normal and dystrophic human myoblasts. These encouraging findings suggested that the altered properties of dystrophic myoblasts did not compromise the myogenic differentiation of DPC in vitro, supporting their in vivo application and possible therapeutic potential. This dissertation also presents a comprehensive evaluation of the in vitro effects on DPC myogenic differentiation of different bioactive molecules, including galectin-1, reversine and activation of the Shh signalling pathway via recombinant Shh and purmorphamine. None of the bioactive molecules could induce or increase myogenic differentiation in DPC, but the data generated in this study suggested that the activation of Shh signalling might function in a dose-dependent manner. Weaker Shh signalling promotes differentiation and stronger signalling promotes proliferation. In summary, this dissertation reports that DPC are a readily accessible source of stem cells that can undergo myogenic differentiation in vitro. Thus DPC remain a candidate stem cell type for DMD treatment; however, their differentiation efficiency requires improvement in order to make them suitable for therapeutic applications in muscle wasting disorders. Furthermore, these newly defined methods to quantify myogenic reprogramming in vitro could be used to identify strategies to enhance the myogenic differentiation of DPC and other stem cell types. This dissertation provides an impetus for future studies investigating stem cell treatments for myogenic differentiation.
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ItemThe role of Hepatitis B surface antigen in the development of severe liver disease and hepatocellular carcinoma( 2016)Hepatitis B virus (HBV) infection continues to be a major public health issue worldwide, affecting an estimated 2 billion people globally. In spite of safe vaccination and effective therapy, there is still a large burden of disease due to chronic hepatitis B (CHB), affecting an estimated 240 million people, which may lead to cirrhosis and or the development of hepatocellular carcinoma (HCC). Liver cancer is rapidly emerging as the single greatest challenge in hepatology and liver transplantation and the burden of disease is set to explode in the next 20-30 years. Viral hepatitis is ranked as the seventh leading cause of death worldwide, resulting in at least 1.2 million deaths annually from liver cirrhosis and HCC. HCC is currently the sixth most common cancer and the second leading cause of cancer-related deaths globally. Case-control studies have shown that chronic HBV carriers have more than 100-fold increased risk of HCC compared with non-infected individuals. Approximately 80% of all HCC cases can be attributed to viral hepatitis, more than half of that due to HBV, which is the second most potent carcinogen after tobacco (World Health Organization), and this is despite remarkable improvement in therapy with the advent of nucleos(t)ide analogues (NA). The global reservoir of HBV infection serves as the basis for the generation of HBV variants via recombination and a high frequency of mutation in the HBV genome. Due to the inherent molecular biology of this virus which replicates its DNA genome via a low fidelity viral reverse transcriptase (rt)/polymerase, a population of closely related genetic variants known as a quasispecies is produced. The last two decades have seen a significant increase in the emergence of mutants as the virus responds to selective pressures, such as vaccination and antiviral therapy. Surveillance for clinically significant HBV mutations and an improved understanding of the impact of these emerging variants on the natural history of the disease and its diagnosis, control and management will pose a challenge to global health care in the foreseeable future. This is because these mutants have the potential to alter current diagnostic and treatment algorithms. NA therapy was approved in 1998, which efficiently lowers the HBV DNA viral load in HBV-infected patients. Given that the HCC risk is particularly high in the presence of cirrhosis and/or persistent high HBV DNA replication, NA should be a rational treatment to prevent liver disease progression including liver cancer in such patients. However since the introduction of antiviral therapy, the HCC incidence has continued to rise. Registration for the liver transplant waiting list due to HBV-related HCC in the U.S. has increased in the NA era, which may in part be due to patients no longer dying of liver disease. There is clearly a need for a greater understanding of the role that NA therapy might play in the development of HCC. Numerous risk factors for HBV-related HCC development have been identified. However the relationships are complex. Older patient age, liver cirrhosis, DNA viral load, hepatitis e antigen (HBeAg) status, HBV genotype, gender and family history have all been found to be important risk factors for HCC in most studies. The exact mechanism of HBV-related carcinogenesis is not fully elucidated, however it is likely a multi-factorial process reliant on a combination of mechanisms which include: ongoing inflammation, cycles of damage and regeneration of hepatocytes, increased chromosomal instability through multiple HBV DNA integrations, oxidative stress as a consequence of NA driven alterations to the HBV life cycle, and finally any direct effects of the virus or viral proteins themselves. The aim of this study was to determine the role that the hepatitis B surface antigen (HBsAg) might play in patients with CHB in the development of severe liver disease and HCC in the era of hepatitis B virus (HBV)-specific NA therapy. The hypothesis tested was that the development of HCC may be associated with altered expression of phenotype and abnormal cellular distribution of HBsAg, and that NA-induced drug resistance can influence this process. This is an important question given the rising incidences of HCC. This study will investigate the effects of NA treatment on HBV replication and any subsequent variation in oncogenic potential from common NA resistant variants. This study will also use various novel techniques such as HBV splicing and next generation sequencing to explore new approaches for the molecular pathogenesis of CHB. The findings are important as virological factors may have prognostic importance in patients developing HCC, which may impact existing CHB treatment strategies and professional practice guidelines.
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ItemTherapeutic effects of phosphodiesterase 4 inhibitor roflumilast in chronic kidney disease( 2016)Progressive chronic kidney disease (CKD) is a major health problem in Australia, and its prevalence is increasing due to the growing incidence of diabetes and hypertension. However, current clinical management of CKD, particularly the use of antihypertensive drugs, and glycaemic control for diabetic patients, is only partially effective against preserving renal function and in delaying end stage kidney disease (ESKD). Therefore, there is a need for new approaches to slow or halt the progression of CKD to ESKD. As chronic inflammation and consequently progressive fibrosis are important features of CKD, this thesis explored the therapeutic potential of phosphodiesterase 4 (PDE4) inhibitor, roflumilast (RFL), a drug has been found to broadly inhibit inflammation, in progressive CKD. Firstly, the efficacy of RFL to inhibit TGF-β1 stimulated effects, where TGF-β1 is a major pro-fibrotic cytokine, was tested in vitro in renal cell cultures. RFL exhibited both anti inflammatory and anti-fibrotic properties, diminishing the expression of pro inflammatory cytokine MCP-1 and weakening TGF-β1 induced pro-fibrotic responses. RFL also normalised cell proliferation changes triggered by TGF-β1 stimulation. However, the mechanism of how PDE4 inhibition by RFL led to the attenuation of these TGF-β1 stimulated effects remains inconclusive, and was at least not solely due to the changes in intracellular cAMP levels. The efficacy of RFL was then investigated in an animal model of progressive CKD, the subtotal nephrectomised rat. RFL treatment attenuated renal decline in this rat model by preserving glomerular filtration rate, reducing proteinuria, and attenuating structural deterioration, such as glomerulosclerosis and tubulointerstitial fibrosis. Moreover, RFL indirectly downregulated TGF-β1 mediated response, implying that the therapeutic effects of RFL may be through a simultaneous inhibition of inflammation and fibrosis than merely through the suppression of inflammation and subsequent attenuation of fibrosis. Finally, the localisation of PDE4 isoforms was investigated in the renal cortex. PDE4 isozymes were mainly localised in the cells of the distal tubules of the tubulointerstitium and the glomeruli. In addition, the expression of PDE4A, PDE4B and PDE4C isozymes in the glomerulus were elevated in the diseased kidney, and colocalised with podocytes. RFL treatment was found to reduce the elevated PDE4 expression, which correlated with the preservation of podocyte number, suggesting that PDE4 inhibition may be relevant in preserving podocyte integrity. In conclusion, it was clearly demonstrated in this thesis that RFL treatment was able to reduce inflammatory and fibrotic processes, in vitro and in an animal model of progressive CKD, and preserve podocyte survival, mechanisms which were independent of hemodynamic changes. Instead, these effects presented by RFL might be through the inhibition of inflammation and TGF-β1 stimulated responses, although the exact mechanism of this remains inconclusive in this thesis. Nonetheless, there is strong basis for use of RFL as a novel therapy to delay the progression of CKD to ESKD. Further investigation on PDE4 inhibition in combination with antihypertensive drugs is necessary to determine potential clinical utility of PDE4 inhibitors in treating CKD.
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ItemInduction of antigen-specific tolerance and development of autoreactive T cells in an experimental model of autoimmune diabetes( 2016)Immune responses to proinsulin initiate anti-islet autoimmunity in non-obese diabetic (NOD) mice and possibly in humans. This results in autoimmune destruction of insulin secreting beta cells leading to type 1 diabetes (T1D). Therapies that bolster immune tolerance to islet antigens are highly desirable, however such approaches have failed to prevent clinical T1D. The major aim of this thesis was to determine a stage of life when antigen-specific tolerance is most effective in preventing anti-islet immune responses. Chapter 2 describes generation and validation of transgenic NOD mice engineered to express islet antigens proinsulin (TIP mice) and IGRP (TII mice) in the antigen presenting cells (APCs) in a tetracycline dependent manner. MHC class II IEα promoter in combination with tet-OFF transactivator induced robust, doxycycline dependent and APC specific expression of proinsulin and IGRP in TIP and TII mice respectively. TIP mice expressing proinsulin did not develop insulitis and were protected from cyclophosphamide-induced diabetes, suggesting that proinsulin expression in TIP mice was sufficient to induce functional antigen-specific tolerance. In chapter 3, we examined the impact of antigen-specific tolerance on the development of autoreactive T cells and spontaneous diabetes by expressing islet antigens proinsulin and IGRP in the APCs during defined periods of life in TIP and TII mice. Our results indicate that tolerance to proinsulin in early life until the weaning period is sufficient to prevent diabetes development in TIP mice. The protection from diabetes was not due to dominant tolerance, but mainly due to a significant reduction in the insulin reactive T cells. Although insulin reactive T cells were not completely absent, the residual autoreactive T cells lacked pathogenic potential. By tracking IGRP reactive T cells in TII mice we demonstrate that IGRP T cells also emerge during early life. These data suggest that early life is a vulnerable period for escape of islet reactive T cells, and that boosting immune tolerance to islet antigens during this time imparts durable protection from islet autoimmunity. Immune tolerance to proinsulin-2 imparts robust protection from autoimmune diabetes in the NOD mice. Whether dampening immune responses to proinsulin-1 would influence diabetes development in NOD mice remains to be investigated. Chapter 4 describes the generation of transgenic NOD mice that express proinsulin-1 in the APCs (TIP-1 mice) in a tetracycline dependent manner. TIP-1 mice displayed a significantly reduced incidence of spontaneous diabetes, which was associated with reduced severity of insulitis and insulin autoantibody development. Antigen experienced proinsulin specific T cells were significantly reduced in number in TIP-1 mice indicating immune tolerance. Although immune response to downstream antigen IGRP was reduced in TIP-1 mice, tolerance to proinsulin-1 was unable to prevent diabetes in NOD 8.3 mice with a pre-existing repertoire of IGRP reactive T cells. Thus, despite being highly conserved to proinsulin-2, tolerance to proinsulin-1 only partially prevents islet-autoimmunity in NOD mice, which suggests an ongoing residual immune response to proinsulin-2 epitopes in TIP-1 mice.
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ItemHair follicle transplantation: implications for cell migration and wound healing( 2016)Chronic wounds are a major problem in the health care system. The increasing incidence of obesity and diabetes and longer life expectancy are adding to the burden. Treatment options are currently limited indicating needs for new treatment modalities. Impaired innate healing response is the main characteristic of chronic wounds. In recent years, there has been a focus on stem cell therapy and tissue engineering as an alternative treatment for chronic wounds to replace the damaged tissue with cells that possess a normal injury response. The hair follicle’s continuous cycling and their response to trauma stimulation, indicate a strong regenerative potential. It has been shown that hair follicles participate in wound re-epithelialization and respond to skin injury by promoting skin innervation and vascularization. This dissertation explores the potential of hair follicle transplantation to enhance wound healing. It was hypothesized that hair follicle transplantation can promote healing in skin wounds. Whole hair follicle were isolated and re-established in the wound site where they preserved their structure and function. This approach exploits the normal endogenous function of hair follicle cells to enhance skin wound healing. First, a vibrissae follicle transplantation technique was established and optimised in nude mice. Using donor follicles from GFP transgenic mice made it possible to investigate cell trafficking from transplanted follicles during epidermal and dermal repair. Transplanted vibrissae follicles integrated normally into the host epithelium and ninety percent of grafted vibrissae produced a hair shaft. The GFP labelled cells were confined to the transplanted hair follicles and were not found in the interfollicular epidermis. It was then possible to identify migrating hair follicle cells from recipient inter-follicular epidermis in lineage tracing wound experiments. Healing responses were then compared in excisional wounds with and without adjacent transplanted follicles. My findings re-confirmed that hair follicle cells participated in wound re-epithelialisation. Neighbouring transplanted follicles accelerated wound closure. Wounds adjacent to transplanted follicles also showed four times higher nerve density in the granulation tissue than the wounds without adjacent follicular transplants. There was no significant difference in capillary density and macrophage density in wounds with and without hair transplants. I also conducted a preliminary investigation of incisional wounds perpendicular or parallel to transplanted follicles, to compare hair follicle cell migration from damaged and intact hair follicles. In these experiments, intact hair follicles participated only in the epidermal component of wound healing and cells migrated from transplanted hair follicles to the healing dermis only when the hair follicles were cross-sectioned. The results of this dissertation indicated that re-epithelialization and promotion of innervation were possible mechanisms by which transplanted follicles contributed to wound healing. These findings lay the foundations for using follicular transplantation to promote healing in chronic or neuropathic wounds and peripheral nerve injuries. In addition, damage to hair follicles may be a starting signal for migration and participation of hair follicle cells in the repair of dermis, which may subsequently influence the healing outcome.
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ItemInvestigating the natural history of Barrett’s oesophagus and its progression to oesophageal adenocarcinoma in the setting of improved assessment techniques and endoscopic therapy( 2016)Barrett’s Oesophagus (BE) is a condition whereby the columnar cells that normally line the oesophagus are replaced by an intestinal metaplastic cell type. Over time, these new cells can become dysplastic and ultimately can develop into oesophageal adenocarcinoma (EAC). Dysplasia is generally graded by pathologists as either low or high grade dysplasia, though previously the terms mild, moderate and severe dysplasia, as part of a three tier system, were used. The risk of malignant progression increases with worsening dysplasia. At present, we know that for high grade dysplasia (HGD) the risk of progression is very high and these patients must be treated to prevent the development of cancer. What is not clear from the current literature is how we should be managing low grade dysplasia (LGD) in BE, as malignant progression rates are variably reported and it is unclear whether we should be treating these patients aggressively or whether, in fact, surveillance only is appropriate. Additionally, over the last five to ten years there have been significant advancements in the therapies used to treat and eradicate dysplastic BE endoscopically, most notably with the use of endoscopic resection of early neoplastic lesions and radio frequency ablation of dysplastic BE. While these techniques have been shown to be safe and effective in trial populations, there are limited data on effectiveness in real-world cohorts. Furthermore, data have been lacking regarding the durability of outcomes following treatment. This thesis involved a range of clinical studies that aimed to address the above issues. I studied a cohort of patients who were diagnosed over ten years ago with LGD within BE, and assessed the percentage of those that went on to develop high grade and cancerous lesions without intervention/treatment. The original histology slides from this group were reviewed by two expert gastroenterology pathologists to 1) confirm the original diagnosis of low grade dysplasia and, 2) also grade the dysplasia according to the three tier system. I have shown that while a consensus diagnosis of LGD does appear to confer an increased risk of malignant potential, it is primarily a diagnosis of moderate LGD that accounts for this risk increase. This finding suggests that in addition to BE patients with LGD undergoing expert histological review, consideration should be given to further risk stratifying this group as having mild or moderate dysplasia, as this may influence a clinician’s decision to heighten surveillance or commence endoscopic therapy. In the second study I evaluated whether a Specialised Barrett’s Unit was better able to detect and stage dysplastic Barrett’s Oesophagus when compared with community endoscopists. I found that assessment of these patients at a Barrett’s unit resulted in improved detection of mucosal abnormalities and cancers, that were often missed by community endoscopists. Endoscopic mucosal resection (EMR) of early cancers was a critical step in determining a patient’s appropriateness for endoscopic therapy. These results highlight the importance of adequate assessment by endoscopists with the appropriate training in Barrett’s assessment and technical ability to perform EMR of suspected cancers, prior to embarking on a definitive management course. In a third clinical study, I assessed the safety, effectiveness and durability of combined endoscopic therapy (radiofrequency ablation and endoscopic mucosal resection) in one of Australia’s largest cohorts of patients with dysplastic Barrett’s oesophagus. This group had predominantly advanced histopathology and often complex Barrett’s segments. I found that in this real world cohort, the effectiveness and safety profile are similar to published data but that there was significant risk of recurrence of both Barrett’s and dysplasia. I demonstrated that recurrence appeared to occur more commonly in patients with advanced pre-treatment histology, and frequently at the gastroesophageal junction. This finding has important clinical implications and we advocate careful ongoing surveillance of this region, particularly in patients with prior advanced dysplasia or cancer.
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ItemInvestigating the effects of downstream parathyroid hormone (PTH) targets on bone strength and quality( 2016)The only currently approved anabolic therapy that increases bone mass and reduces fracture risk in patients with osteoporosis is parathyroid hormone (PTH). To investigate how PTH increases bone mass and improves bone strength, I have investigated the function of two gene products regulated by PTH in osteoblasts on bone structure and strength (gp130 and ephrinB2). Gp130 deletion in late osteoblasts/osteocytes resulted in greater femoral dimensions indicating a role for osteocytic gp130 in maintaining bone width. Although the bending load required to fracture these bones was not altered, they showed a significant reduction in material strength which was associated with a greater proportion of disorganised woven bone suggesting that the increased bone width may have been a compensatory mechanism for poor bone quality. This indicated that gp130 in late osteoblasts/osteocytes maintains the material strength of the cortical bone matrix collagen production and the deposition of organised lamellar bone. The role of another PTH downstream target, ephrinB2, in controlling bone strength and quality was investigated in both osteoblasts and osteocytes. Work conducted prior to my PhD showed that specific deletion of ephrinB2 within the entire osteoblast lineage in 12-week old female mice caused osteoblast apoptosis and delayed initiation of bone mineralisation, indicating a role for ephrinB2 in osteoblasts that promotes mineralisation by preventing apoptosis. During my PhD, I found that mice lacking ephrinB2 in the osteoblast lineage had slender bones which were more compliant when assessed by mechanical testing. Despite their greater bending ability, their bones were more fragile but they exhibited no change in bone composition measured by synchrotron-based Fourier transform infrared microscopy (sFTIRM). This suggested that the previously observed delay in the initiation of mineralisation caused by ephrinB2-deficient osteoblasts may have resulted in the fragile bone phenotype. Since ephrinB2 is expressed throughout the entire osteoblast differentiation pathway, I next sought to determine the role of ephrinB2 in late osteoblasts/osteocytes in 12-week old female mice. Mice lacking ephrinB2 in osteocytes showed an opposing strength phenotype compared to the earlier deletion of ephrinB2 mentioned above. Their bones were more brittle due to greater mineral deposition and parallel stretching/compression of the collagen fibres detected by sFTIRM, highlighting stage-specific roles of ephrinB2 during osteoblast differentiation that regulate bone size and material composition. Correlation analysis revealed that the relationships between collagen fibre alignment and bending strength and between mineral composition and material toughness that normally exist in control mice were lost in bones from mice lacking ephrinB2 in late osteoblasts/osteocytes. This demonstrated that ephrinB2 within late osteoblasts/osteocytes restrains mineral deposition to maintain bone strength. I next investigated the trabecular bone structure in these mice and found that deletion of ephrinB2 in late osteoblasts/osteocytes resulted in greater trabecular bone containing enlarged osteoclast size observed in vivo suggesting impaired osteoclast function. This may also relate to greater carbonate content within the bone mineral which requires further investigation. Finally, I assessed whether osteocytic ephrinB2 is required for the anabolic action of PTH. In contrast to impaired anabolic effect of PTH in mice lacking ephrinB2 within the entire osteoblast lineage, PTH treatment in mice lacking ephrinB2 in late osteoblasts/osteocytes did not significantly impair the anabolic action of PTH on trabecular and cortical bone formation. This led me to use the sFTIRM technique to analyse bone composition in vehicle and PTH-treated bones from control mice. This showed that bone deposited during PTH treatment undergoes a normal process of collagen maturation and mineral accrual. In conclusion, downstream targets of PTH, gp130 and ephrinB2 play distinct roles in regulating bone size, strength and quality. This highlights that osteoblasts utilise these proteins during the formation of bone matrix and the initiation of its mineralisation. In contrast, osteocytes maintain the quality and composition of bone matrix and mineral by regulating mineral accrual and collagen arrangement. Understanding how specific genes expressed in these cells can control bone strength, coupled with the sFTIRM technique developed during my PhD, can provide a new way for investigating different causes of bone fragility. Current diagnostic tests cannot fully predict risk of fracture and treatment options are not patient specific and are limited in their use. In the long term, this study may allow specific targeting of cell types to increase bone mass and improve bone quality which could lead to the development of different personalised treatments for bone fragility.
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ItemSelection and management of men for active surveillance in low risk prostate cancer( 2016)Aims: To investigate: 1. Selection of men for active surveillance of prostate cancer a. Validation of risk calculators b. Suitability for inclusion of Gleason 3+4 disease. 2. Performance of prostate biopsy during AS a. Differences in quality of diagnostic biopsy between academic and referral centres. b. Optimization of biopsy templates c. Examination of prognostic indicators for disease progression Methods: Data were obtained from several difference sources: • Men suitable for AS on prostate biopsy but undergoing upfront radical prostatectomy were pooled from 3 international academic institutions in Cambridge (UK), Toronto (Canada) and Melbourne (Australia). • Prospectively maintained AS prostate cancer database at Princess Margaret Cancer Centre (PMCC) (1997-2012). Analyses performed: • Four risk calculators were assessed for their ability to predict different definitions of insignificant prostate cancer by area under the curve (AUC) of receiver operating characteristic curves and Brier scores for discrimination, calibration curves and decision curve analysis. • Men with biopsy Gleason 3+4 disease, suitable according to modified Royal Marsden, Sunnybrook Toronto and PRIAS selection criteria, were assessed for presence of adverse pathology at upfront radical prostatectomy. • Patients on AS at a tertiary referral centre (PMCC) were dichotomized depending on where their diagnostic biopsy was performed (interval versus external). Multivariate logistic regression was performed to examine for predictors of re-classification at the second, or confirmatory, biopsy. • Mapping of all patients with pathological progression at PMCC for location of disease progression enabled comparison of hypothetical biopsy templates (sextant and standard extended) to the institutional template used. • Men on AS at PMCC were evaluated for presence of disease progression at serial biopsy in the prostate transition zone (TZ). Multivariate Cox proportional hazards regression evaluated predictors of TZ progression. • At PMCC, men were dichotomized based on presence of cancer at their confirmatory biopsy. Pathological progression was investigated using a Cox proportional hazards regression model. Results: • All 4 models predicting presence of insignificant prostate cancer had weak discrimination at best (AUC 0.618-0.664). • Presence of Gleason 3+4 at biopsy, compared to 3+3 disease, increases risk of adverse pathology at radical prostatectomy if modified Sunnybrook Toronto criteria are used (19% versus 33%, p≤0.001). Using a stricter protocol such as PRIAS, there was no statistical difference between the groups. • External biopsy predicted both grade related re-classification (OR 4.14, C.I. 2.01-8.54, p<0.001) and volume related re-classification (OR 3.43, C.I. 1.87-6.25, p<0.001). • Sextant and standard extended biopsy templates were inferior to the institutional biopsy template in detecting presence of cancer (84% and 99% versus 100%), and pathological progression (47.9% and 81.9% versus 100%). • At each subsequent biopsy during AS, 2.7-6.7% of men had disease progression only in the TZ which would not have been detected if TZ biopsy was not performed. Predictors of TZ progression were maximum % single core (HR 1.99, C.I. 1.30-3.04, p=0.002), and MRI reporting cancer (HR 3.19, C.I. 1.23-8.27, p=0.02). • Men with no cancer at confirmatory biopsy were less likely to have pathological progression (HR 0.47, CI 0.29-0.77, p=0.003). Sub-analysis showed this was predictive of volume-related progression (HR=0.36, CI 0.20-0.62, p=0.0006) and not grade-related progression. Conclusions: • Utilization of models predicting suitability for AS should be used with caution as external validation in our cohort was weak. • If considering biopsy Gleason 3+4 disease for AS, a stricter protocol such as PRIAS must be utilized. • At PMCC, patients who had their initial diagnostic prostate biopsy for AS done externally, were more likely to have worse pathological features and re-classify on the second biopsy. • For men on AS, sextant and standard extended biopsy are less likely to detect prostate cancer or disease progression than the template used at PMCC. • TZ biopsy should be considered for all men having serial biopsy on AS, in particular those with high % core involvement or positive MRI findings. • Absence of cancer on B2 is associated with a significantly decreased risk of volume-related but not grade-related progression.
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ItemAdvanced characterisation of pulmonary hypertension: Assessment of right ventricular diastolic function and pulmonary artery wave reflection( 2016)Pulmonary hypertension is the net haemodynamic consequence of a wide variety of underlying pathologies. As disease progresses, right ventricular systolic dysfunction may develop. However, by the time this occurs, prognosis is poor. Like the situation in the left ventricle, chronically increased right ventricular afterload first leads to right ventricular hypertrophy and hypothetically, diastolic dysfunction. Although there is some evidence from animal models for this, human data is limited. Theoretically, the identification of right ventricular diastolic dysfunction may assist in the earlier diagnosis of pulmonary hypertension. This thesis provides evidence that right ventricular diastolic dysfunction does exist in the setting of pulmonary hypertension, that it occurs earlier than systolic dysfunction, and that it can be identified by invasive pressure measurement in the right ventricular cavity. Although echocardiography provides a useful way to assess left ventricular diastolic function, data presented here will show that currently available echocardiographic measurement of right ventricular diastolic function may not be sensitive enough to detect abnormal function. The secondary hypothesis tested is that a pressure/time analysis of pulmonary wave reflection can provide additional information in the assessment of patients with pulmonary hypertension. Data suggests that a metric of wave reflection, the pulmonary augmentation index, is closely associated with standard measures of right ventricular afterload, and therefore may not add value. However, the time to wave reflection is related to the site of obstruction in the pulmonary circulation and could theoretically assist in identifying disease aetiology.
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ItemBreast cancer risk assessment and risk management:: Development of a personalised, web- based, decision support tool (iPrevent®)( 2016)Breast cancer risk is a complex interaction of environmental factors, inheritance and genetics. Awareness of her personal breast cancer risk can allow a woman to make informed decisions regarding the management of her risk, through screening or risk reduction measures such as surgery or medication. The effect of risk factors, both inherited and modifiable, on breast cancer risk is complex but mathematical models exist to estimate an individual’s risk. These models use epidemiological data to quantify the risk for an individual, based on a range of their risk factors. As these models were developed primarily for research purposes, they are not designed for ease-of-use by a range of clinicians, nor designed to be used by those unfamiliar with estimating breast cancer risk. Once estimated, a woman’s breast cancer risk must then be explained in a way that is comprehensible to the woman, together with ways to manage that risk in a similar format. If this were achieved for all women, it may allow them to make informed choices and potentially even prevent breast cancer or reduce its impact greatly. The ultimate aim of this research was to develop a user-friendly computerised, web-based breast cancer risk assessment and risk management support tool. This tool, called iPrevent©, uses the existing mathematical models to estimate individualised breast cancer risk, but using a user friendly interface. It then goes further, and provides Cancer Australia guideline-based recommendations, based on that risk, for each individual woman. It presents the risks and befits of each evidence-based intervention in a similar manner to the risk so that women can make an informed choice regarding their breast cancer prevention strategy. Before developing iPrevent©, I first examined the other possible effects of being a carrier of a mutation in breast cancer predisposition genes, as it was hypothesised that other factors such as fertility effects, could have a large bearing on any future decisions women may make, including risk reducing surgery. I then explored current behaviours to reduce risk among women at highest risk, in an attempt to understand the magnitude of the possible benefits of iPrevent© in this highest risk group. Through focus group studies, I examined the information needs of clinicians to facilitate breast cancer risk discussions. Understanding the needs of end-user clinicians of iPrevent© ensures it could meet their needs. This usability may increase uptake and use, of both the tool and breast cancer risk management strategies where appropriate. This tool, iPrevent©, is currently undergoing clinical validation studies, outside the scope of this thesis, but will shortly become freely available with the aim of increasing individual awareness of each woman’s own breast cancer risk, enabling her to manage that risk according to the evidence, Cancer Australia guidelines and her own preferences.