Surgery (St Vincent's) - Theses
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ItemGene expression profiling of the ictal hippocampus(University of Melbourne, 2009)
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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.
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ItemDeveloping liver specific vasculature to support the growth of liver progenitor cells for liver tissue engineering( 2015)Tissue engineering is the combination of organ/tissue specific cells in matrices or scaffolds to grow new tissues. Tissue engineering and the related area of cell therapy hold much promise for the repair, replacement and regeneration of organs and tissues damaged by disease and trauma. To date, liver tissue replacement research has focused on cell therapies – particularly hepatocyte transplantation into the diseased liver which specifically aims to reduce the need for liver transplantation, and aims to treat the myriad of end stage liver diseases and metabolic disorders. Despite the technological advances, a major limitation to the clinical success of hepatocyte cell therapy and other liver tissue engineering strategies is the inability to generate a vascular supply capable of supporting the engineering of large, three-dimensional (3D) tissues and organs. The liver sinusoidal endothelial cell (LSEC) makes up the liver specific microvascular network (sinusoids) and plays an integral role in liver development, liver homeostasis and liver regeneration. Vascularisation itself is rarely addressed in liver tissue engineering; let alone the incorporation of LSECs in liver constructs. In this study murine LSECs were used to construct liver specific blood vessels and were identified by their surface markers (LYVE1+/ CD31-), as opposed to capillary endothelial cells (LYVE1-/ CD31+), and lymphatics (LYVE1+/ CD31+). LSECs were tested in vitro and in vivo with and without the addition of murine liver progenitor cells (LPCs). Liver progenitor cells are a native liver progenitor cell, capable of differentiating into both hepatocytes and cholangiocytes. Whilst hepatocytes are the gold standard for liver tissue engineering, LPCs offer an alternative, rarely investigated source of hepatocytes for tissue engineering. LPCs are responsible for liver regeneration during end stage liver disease, when hepatocyte proliferation has been impaired. As LSECs and LPCs play important roles in liver regeneration, both cell types were investigated for their possible applications in liver tissue engineering. LSECs and LPCs were cultured as 3-D multicellular spheroids of one cell type-termed homospheres, or co-cultured together as heterospheres in vitro, for subsequent in vivo implantation in a vascularized tissue engineering chamber. This thesis demonstrates that LSECs and LPCs are capable of forming homogeneous homospheres, and heterogeneous spheroids of co-cultured heterospheres. Furthermore, LSECs form vascular structures in vitro when cultured as homospheres heterospheres. The ability to generate vascular structures through co-culturing with endothelial cells in vitro is termed pre-vascularization, and is currently at the forefront of vascular tissue engineering. The thesis also demonstrates that LSECs were capable of integrating into native vasculature when implanted in vivo to form a liver specific vasculature at an ectopic site in healthy SCID mice. The liver specific vasculature was identifiable as LYVE1+/ CD31- and through the detection of DiI labeled LSECs. Implantation of LSECs also increased the generation of native neo-vasculature (LYVE1-/CD31+)- this was significant when the matrix Matrigel was used in the tissue engineering chamber. However, the generation of liver specific vasculature and increased native vasculature did not support significant survival of LPCs. Whilst the results indicate that heterospheres of LSECs and LPCs improve the survival and spontaneous hepatocyte differentiation of LPCs, the overall survival was inconsistent throughout the in vivo experimental groups in SCID mice. Finally, LPC spheroids were implanted in a mouse model of the metabolic disorder methylmalonic aciduria (MMA). LSECs were not used in the final study; as LPC survival was inconsistent and low regardless of the presence/absence of LSECs. Instead, the total number of LPCs implanted was increased 4 fold. Again, survival of LPCs was low and appeared to elicit an immune reaction in MMA mice. Ultimately, the final study did not demonstrate any functional disease reversal, due to poor LPC survival. This is the first liver tissue engineering study that has used LSECs with LPCs. Isolated LSECs demonstrated significant promise in their ability to generate a liver-specific vasculature in vivo for liver tissue engineering, however the use of LPCs presented a number of issues, which require further investigation – the most significant of which was their inconsistent and generally poor survival in vivo.
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ItemThromboembolism and neoadjuvant chemoradiation for rectal cancer( 2015)Thromboembolism (TE) is one of the leading causes of morbidity and mortality in cancer, is an independent predictor of reduced survival, and the overall rate of TE in cancer patients is increasing. Assessing the TE risk of an individual patient at a given time point and the benefit of thromboprophylaxis (TP) can be complex, involving widely variable TE rates according to tumour related factors (such as cancer subtype and disease stage), as well as patient and treatment related factors. Some, such as surgery and hospitalisation are well recognised and appropriately targeted with mechanical and pharmacological thromboprophylaxis (TP) strategies backed by Level I evidence. There is evidence that TP after hospital discharge following surgery (extended TP) is also beneficial. Recently, subgroups such as those with metastases receiving palliative chemotherapy have been shown to be at equally high risk, and also benefit from primary TP. Both chemotherapy and central venous access devices (CVAD) have been shown to be independently associated with development of TE. In addition, early studies examining neoadjuvant radiotherapy (nRT) in rectal cancer reported higher rates of TE however this finding was not repeated in subsequent studies using modern radiotherapy techniques. Extrapolating these findings raises the question of TE risk and the potential benefit of TP during neoadjuvant radiotherapy (nRT) or chemoradiotherapy (nCRT) for rectal cancer. This thesis explores the current evidence and contemporary guidelines concerning TE risk during nCRT, demonstrating considerable uncertainty and a lack of robust data. Randomised trials examining nCRT in rectal cancer are systematically reviewed to determine rates of TE, demonstrating a failure to capture TE events due to inadequate complication reporting frameworks. Existing attitudes and prescribing practices of specialist rectal cancer surgeons are surveyed, as well as barriers to TP prescribing. Issues of equipoise, ownership and logistical problems with outpatient TP prescribing were identified. The historical TE rate and epidemiology over a prolonged follow-up period in rectal cancer patients, as well as the relationship to nCRT is examined at both Peter MacCallum Cancer Centre in Australia and the Cleveland Clinic in the United States. These studies demonstrated that most TE events occurred in patients with metastatic disease receiving ambulatory palliative chemotherapy, and that the overall rate of TE in patients treated with nCRT was not elevated over those patients who had surgery alone. Finally, a prospective study of thrombogenic biomarkers in patients with rectal cancer was undertaken demonstrating significant coagulation abnormalities in colorectal cancer patients at baseline, but no major alteration during neoadjuvant chemoradiation. Marked and prolonged procoagluant abnormalities were demonstrated in the post-operative phase. Thus the current literature, attitudes of treating clinicians, historical rates, as well as candidate biomarkers for prospective TE risk were examined in nCRT for rectal cancer for the first time, identifying future potential research questions aimed at reducing this common complication of cancer care.
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ItemMicropapillary pattern in lung adenocarcinoma: the molecular and clinicopathological characterization of an emerging cancer phenotype( 2015)Adenocarcinoma of the lung is a tumour of mixed morphological appearance under light microscopy with haematoxylin and eosin staining. Last decade, the recognition of a micropapillary pattern by academic pathologists led to its introduction as a subtype in the multidisciplinary pathological classification of a combined taskforce from the International Society for the Study of Lung Cancer, American Thoracic Society and European Respiratory Society. However, its inclusion was controversial, and supported only by a handful of peer-reviewed publications. Little work has been done to further define this pattern beyond its original morphologic description in 2002. Using a top-down approach combining genomic, transcriptomic, immuno-histochemical and clinico-pathological features of a bank of clinically annotated tumours, I sought to define the phenotype and genotype of the micropapillary pattern in lung adenocarcinoma. Additionally I tested the feasibility of next generation RNA sequencing of formalin fixed paraffin embedded (FFPE) tumour tissue. The architecture of micropapillary pattern was confirmed as several variations on the original description of papillary structures lacking fibrovascular cores or small rosettes of cells floating in alveolar spaces, glandular and lymphovascular spaces. The cytology of cells in the micropapillary pattern was one of high-grade nuclear atypia. Using immunohistochemical techniques all micropapillary tumours studied stained for the transcription factor TTF-1. I demonstrated circumferential cytoplasmic staining with MUC1 (a trans-membrane glycoprotein) in micropapillary pattern, as opposed to luminal only or poor staining for other subtypes and variants of adenocarcinoma. The combination of these two stains allowed confirmation of the presence of “spread through alveolar spaces” as another form of micropapillary pattern where small clumps of cells are identified in alveolar spaces of normal lung well away from the main tumour. Intratumoral heterogeneity has been increasingly recognized as a phenomenon in tumours that may be responsible for variable response to therapies and development of resistance. I have demonstrated that individual subtypes within tumours, and especially micropapillary pattern, may harbour mutations not present in the remaining tumour. Additionally, micropapillary tumours had a higher incidence of EGFR and BRAF mutation than adenocarcinomas generally. The differential gene expression profiles of separately sampled subtypes of lung adenocarcinoma were determined from RNA extracted from FFPE tumours using the Nanostring platform. Further evidence of both intratumoral heterogeneity and of a discrete micropapillary phenotype was elicited by the fact that the gene expression signatures of micropapillary pattern generally clustered closer with micropapillary sampled from different adenocarcinomas than they did with other patterns within the same adenocarcinoma. The clinical phenotype of micropapillary predominant adenocarcinoma (MPA) was investigated based on classification by comprehensive histologic subtyping. This involved a quantitative estimate of all subtype patterns in a tumour present in greater than 5% of the tumour volume. Tumours were classified according to the predominant pattern. MPA was found to have the highest rate of lymph node metastasis relative to its presence in primary tumours, and to share the invasive and metastatic potential of solid with mucin predominant adenocarcinoma. These findings give weight to predominant subtype pattern being useful as a form of grading in lung adenocarcinoma, with MPA and solid predominant tumours graded as high grade. Micropapillary pattern of adenocarcinoma appears to be a discrete phenotype with particular genomic aberrations and up-regulation of embryonic stem cell and fibroblast growth factor pathways. Its clinical behaviour is typified by high rate of nodal metastases, spread through alveolar spaces and early relapse and death after adequate surgery.