Surgery (St Vincent's) - Theses
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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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ItemEffect of melatonin and indomethacin on adipose-derived stem cells and fat graft survival and function( 2016)Background: Autologous fat grafting has emerged as a key technique in soft tissue reconstruction, and is currently utilized for various deformities secondary to burns, chronic wounds, trauma, irradiation injuries and post-oncological resections. Nevertheless, a significant proportion of the transplanted fat fails to survive within the host environment, which may partly be attributed to cell death of its constituents. A process known as cell-assisted transfer is now used clinically to further enhance fat graft retention by adding stromal vascular fraction (SVF), an adipose-derived stem cell (ASC) rich content to lipoaspirate. However, there are certain concerns about cell-assisted lipotransfer especially regarding its oncologic safety and feasibility. Methods: The basis of this project stems from the need for reproducible and predictable clinical outcomes following transplantation of adipose tissue. To this end, the properties of two safe, commercially available and FDA-approved drugs, melatonin and indomethacin were studied. The in-vitro effects of melatonin on human ASCs were investigated through functional and survival assays including oxidative stress and cell-death assays, MTT Assay, monolayer scratch assay, cell membrane integrity assay, a human cytokine array, and a specific TNF-alpha ELISA kit. The in-vitro effects of indomethacin and melatonin on human ASC adipogenesis were investigated through a lipid (Oil Red O) staining kit as well as reverse transcription polymerase chain reaction (RT-PCR) to determine RNA expression of key adipogenic genes. The effects of melatonin-treated autologous fat grafts injected into the dorsums were tested in-vivo with a murine model, and its weights and volumes were assessed at three time points of two, four and twelve weeks. Hematoxylin and eosin staining, perilipin and CD31 immunostaining were performed with morphometric analysis of adipose tissue and vascularization. Results: The in-vitro results indicate that melatonin significantly protects human ASCs from hydrogen peroxide induced cell-death in a dose-dependent fashion. Addition of melatonin to ASCs improved cell-viability, promoted cell migration and preserved membrane integrity as compared to controls. In addition, it induced a potent anti-inflammatory response by down-regulating acute inflammatory cytokines particularly TNF-alpha but melatonin itself did not exert an adipogenic effect on human ASCs. Indomethacin stimulates adipogenesis of human ASCs in 2 weeks cell culture and upregulates several key adipogenic genes including PPAR-gamma, lipoprotein lipase and fatty acid binding protein 4. For the first time, this work demonstrated in-vivo that melatonin enhances fat graft volume retention at 4 weeks by increasing the percentage of adipose volume within fat grafts with comparable volumes to that of cell-assisted lipotransfer. Melatonin treatment, however, had no effect on angiogenesis. Based on these findings, melatonin and indomethacin may potentially be useful pharmacological adjuncts in clinical fat grafting based on their respective cytoprotective and adipogenic properties.
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ItemBiocellular aspects of high mammographic density as a risk factor for breast cancer( 2016)High mammographic density (MD) is one of the strongest risk factors for breast cancer after high-risk mutations, with a 4-6 fold increased risk comparing the highest to lowest MD quartiles. MD is of great clinical relevance, given that the attributable risk of breast cancer (BC) due to high MD in the population may be as high as 30%, and the already widespread use of mammography for breast assessment. However, the biological basis for high MD and its associated cancer risk is poorly understood. A validated xenograft model where the dynamic effects of drug interventions and gene perturbations on human MD tissue can be investigated in the preclinical setting will be valuable. Prior to commencement of the PhD thesis, there were no animal models of human MD that could maintain the MD differential of tumour-free breast tissues. In the first published study, we developed a xenograft model of human MD, where matched high and low MD human tissues were precisely-sampled under stereotactic-guidance from fresh mastectomy tissues and maintained in separate vascularized biochambers in SCID mice. This study demonstrated that the high and low MD biochamber tissues retained their differential radiographic density and histologic features of the original human tissue. The high compared to low MD biochamber tissues were composed of increased stromal and decreased adipose percentage areas, reflecting the MD phenotype of the original human breast samples. The MD xenograft model was then extended to examine the changes in radiographic density and histology that occurred during murine pregnancy, lactation and postpartum involution states, and after exposure to exogenous endocrine treatment. These studies demonstrated the dynamic nature of the MD xenograft model, with decreased stromal and increased adipose tissue percentage areas observed in high MD biochamber tissues during murine lactation and postpartum involution, and also in Tamoxifen-treated compared to placebo-treated mice. High and low MD biochamber tissues had increased radiographic density with postpartum involution, and increased duration of implant. The radiographic density decreased in high MD biochamber tissues of Tamoxifen-treated compared to oestrogen-treated mice. As increased Cox-2 levels have been observed in breast tumours and stromal regions of high MD tissues, we investigated the expression of Cox-2 in the epithelial and stromal cells of matched high and low MD breast tissues. We demonstrated increased staining in both epithelial and stromal cells of high MD breast tissues. We then showed that the differential Cox-2 expression in high and low MD human breast tissues was maintained in murine biochambers, and was sensitive to hormonal supplementation. Collectively, this thesis indicates that the MD model will be valuable for investigating the mechanisms of how modifying factors, such as lifestyle behaviours and endocrine treatments like Tamoxifen, can reduce MD in cancer-free human breast tissues, and would be useful in research aiming to develop preventative therapies to reduce MD-related risk, such as Cox-2 inhibition.
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ItemThe contribution of genetic variations in the region of the parathyroid hormone-like hormone, PTHLH, gene to breast cancer susceptibility( 2016)Aims: • Analyse the evidence for PTHLH and PTHrP, its protein product, playing a role in breast cancer and update the empirical definition of the gene. • Describe the 3-Dimensional structure of the PTHLH region and determine its system of regulatory interactions, including remote regulatory elements affecting PTHLH. • Integrate existing tools in addition to the novel perspectives generated above to enable a comprehensive annotation and analysis of genetic variants identified through molecular epidemiological techniques including Genome-Wide Association Studies (GWAS) and somatic DNA sequencing of tumour tissue to derive putative molecular mechanisms for the region’s involvement in breast cancer susceptibility. Methodology: • Review published studies and databases, integrating findings from diverse sources. • Acquire and analyse DNA and RNA sequencing, regulatory, expression, algorithm-inferred, and proximity-ligation data from multiple public data sources including ENCODE, ROADMAP, dbGAP, COSMIC and other to update the definition of PTHLH, and advance concepts of structure and regulatory function in the region. • Acquire and analyse primary GWAS data, performing imputation with multiple algorithms and references, and annotating associated variants with a suite of tools. • Use genome browsers including UCSC, WUSTL, and Golden Helix SVS, and their associated databases and tools, to analyse and visually integrate findings. Results: • PTHrP has multiple discretely functional segments active throughout the cell. It likely plays a bivalent and context-dependent role in cancer biology. Analysis of somatic variation in cancer suggests PTHrP may have a tumourigenic role within the nucleus. • PTHLH sits within a 1.3Mb TAD featuring multiple sub-structures that are integrated with the region’s regulatory function. There are activated chromatin hubs (ACHs) at protein-coding genes with evidence of extensive interaction between them. This is facilitated by the TAD’s structure, collocating them at the neck of the TAD. • The ACHs at MRPS35, KLHL42, and CCDC91 each monopolise a subordinate regulatory sub-net with a hierarchical structure. They each appear to act as important remote regulatory elements that integrate regulatory signals generated within their respective sub-nets, transferring them to PTHLH, and other genes, via ACH-ACH interactions. • There appear to be multiple discrete GWAS breast cancer association signals in the PTHLH region. Annotation of the associated variants suggests three particular regulatory elements may be its key drivers. In the context of the regulatory concepts developed in this thesis, the variants may affect a particular regulatory signal at multiple points in its assembly. PTHLH is the likely downstream target of this signal. • There are multiple poorly-describe coding, and non-coding, genes in the region that are also potential actors in breast cancer and should be investigated. Conclusion: • The PTHLH region is likely involved in the pathogenesis of breast cancer through the modification of PTHLH expression. There are likely to be other mechanisms in parallel that are yet to be fully described.