Medicine (St Vincent's) - Theses
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ItemThe relationship between occupational sunlight exposure and non-melanoma skin cancer( 2015)Aims and Background: Australia has the highest incidence of skin cancer in the world and ultraviolet (UV) radiation exposure is the dominant environmental determinant of all major forms of skin cancer. Two recent systematic reviews and meta-analysis of the available epidemiological evidence clearly indicate that occupational ultraviolet radiation (UVR) exposure is a substantial risk factor for the development of non- melanoma skin cancer. This study is an initial attempt to investigate the role of occupational sunlight exposure in the development of non-melanoma skin cancer in men in Australia. Methods: A case-control study was conducted. One hundred cases were recruited from the Skin and Cancer Foundation Inc. Controls included 14 age- and gender-matched subjects nominated by the cases. A questionnaire was administered through face-to- face interviews to collect information on pigmentary and genetic characteristics, occupational and recreational exposure assessment, and time of first diagnosis of non- melanoma skin cancer, confirmed with histopathological diagnosis. Conditional logistic regression models were implemented. Subsequently, a one-way analysis of variance (ANOVA) was used to investigate the relationship of each risk factor with occupational sun exposure. Results: There was insufficient evidence to conclude that there was a relationship between occupational sun exposure and non-melanoma skin cancer, because of the small sample size of matched case-control pairs. As the results for the small sample size of the matched case-control group were not statistically significant, a one-way analysis of variance (ANOVA) was performed to compare the risk factors. Fitzpatrick Skin Type was the only risk factor that showed a statistically significantly different occupational exposure profile (with a P-value of 0.035) but even then the differences were minor and only between skin phototype 2 and 3. There were no significant relationships between occupation exposure and the other risk factors. Conclusion: Unfortunately, analysis of the small number of matched case-control pairs was not statistically significant and it was not possible to draw any conclusions regarding the role of occupational sunlight exposure in the development of non- melanoma skin cancer. We experienced unexpectedly high levels of reluctance from case subjects to propose control subjects to participate in the study, which impacted on data collection and subsequently the study results. It has been well established that solar UVR is a risk factor for skin cancer. Outdoor workers are potentially exposed to high levels of UVR. More epidemiological studies are needed to improve our understanding and awareness of skin cancer as well as aid the development of prevention strategies at workplaces to reduce outdoor workers’ exposure to UVR.
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Item“A tale of two cities”: Studies on host control of chronic hepatitis B infection & Comparative evaluation of methods for quantifying cytokine production( 2016)Globally, an estimated 240 million people are living with chronic hepatitis B (CHB) [1], and the number is approximately 218000 in Australia [2]. Inactive hepatitis B e antigen (HBeAg)-negative patients are the most prevalent group, who are defined by with sustained virological response (SVR). This state is associated with favorable prognosis and low risk of liver complications. Through monitoring the serological markers and immune markers by flow cytometry, as well as advanced techniques such as transcriptome mapping, progress has been made on unveiling the mechanism of host immunological control of the CHB infection. Furthermore, it is of great clinical significance to accurately identify relapse of patients (Chapter 1). Few studies have focused on effectively identifying the treatment-naïve inactive carriers (IC), and we are particularly interested in predicting sustained inactivation through monitoring the serum HBV DNA level and HBsAg titer. Based on a retrospective/longitudinal study, we report that HBV DNA is a better predictor over HBsAg, and it is not beneficial to add HBsAg into the prediction model. The cutoff value of HBV DNA>2.5log IU/mL is effective for identifying IC with the NPV 93.6% for 12 months and 92.3% for 24 months. In another words, IC with baseline HBV DNA load<2.5 log IU/mL is likely to maintain the sustained inactivation for another 1 and 2 years, with a probability over 90% (Chapter 2). We also developed a protocol for effectively selecting peripheral blood mononuclear cells (PBMCs) subpopulations- including monocytes, Natural Killer (NK) cells, and rest of the PBMCs from inactive HBeAg negative patients undergoing clinical relapse after NA therapy cessation. Corresponding selection kits (from Miltenyi Biotec or Stemcell Technologies) were compared, and sample quality and the purification of the sample was analysed. Extracted mRNA samples will undergo expression profiling in the future, expected to pinpoint the biomarkers and unveil the mechanism of the carrier relapse (Chapter 3). For host immunity studies, accurate assessment of cytokine profile is an important perspective. We performed the comparative evaluation of two most commonly used methods for quantifying cytokines, flow cytometry and ELISA, based on the model of TLR stimulated human monocytes. Fundamentally different biological patterns were ascertained regarding cytokines from the two assays, and were mainly evidenced by the more comprehensive information that was available by flow cytometry (Chapter 4). For applying this optimized methodology, which is presenting results from flow cytometry and ELISA in parallel, to CHB studies, we adopted a human hepatocyte cell line, HepG2 (Chapter 5).
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ItemNo Preview AvailableBioimaging in colorectal cancer - prediction of response to neoadjuvant treatment( 2015)Over the last decade the management of colorectal cancer has changed significantly with the benefits of neoadjuvant therapies and new adjuvant treatments becoming apparent. Surgical strategies have also evolved with initial evidence that some patients can be successfully managed with local excision or omission of any surgery at all, resulting in a shift towards the individualisation of cancer management. The management of rectal cancer is based on primary staging assessment which relies on imaging techniques such as CT, MRI and ERUS. Recent advances in technology have improved the accuracy and widened the applications of these techniques. With the progress in medical and surgical treatments for rectal cancer, the optimal management of rectal cancer has become more complex. The evolving ability to tailor optimal treatment to the individual has created new roles for imaging such as prediction of response to treatment, restaging with assessment of response to treatment and prediction of prognosis. Consequently, prediction of response will become an important component of modern pre-operative assessment of rectal cancer to optimise individualisation of medical and surgical treatment. Beyond the established role of primary staging of malignancies, the role of conventional imaging techniques in re-staging following neoadjuvant treatment may be of increasing importance. Novel functional imaging techniques such as FDG-PET, DW (diffusion weighted) MRI are also emerging, the roles of which are yet to be determined. This thesis will examine the current status of bio-imaging and explore new imaging techniques in rectal cancer. At the Peter MacCallum Cancer Centre, we have been routinely performing staging and restaging imaging with CT, MRI and PET for the last 5 years which has resulted in a cohort of patients in whom these imaging techniques can be evaluated. This thesis also aims to evaluate a recent and evolving functional imaging technique- DW-MRI, in the prediction of response of rectal cancer to chemo-radiation.
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ItemIncreasing the hair inductive potential of human dermal papilla cells: stimulating and characterising cell aggregation( 2015)Dermal papilla cells (DPCs) are able to induce hair follicles. DPC tend to aggregate both in vitro and in vivo. This tendency is associated with their ability to induce hair growth. The use of DPCs to treat alopecia is limited because human DPCs lose their hair-inducing activity in culture, whereas ovine DPC do not. The aims of this study were to characterise the molecular phenotype of ovine DPC aggregates, and to determine whether aggregating ovine DPCs secrete factors affecting the aggregative behaviour or inductive potential of human DPCs. Expression of papilla markers in cultured ovine DPCs was characterised. The effects of ovine factors, different culture substrates and medium compositions on aggregative behaviour of human DPCs were determined. Co-cultures of ovine and human papilla cells, separated by a permeable membrane were observed to determine whether the ovine cells secrete soluble factors that affect human cell aggregation. Ovine DPC aggregates expressed 16 papilla markers, showing they have a similar phenotype to papillae in vivo. In co-culture experiments, well-formed aggregates were produced in human:ovine DPC mixtures. In contrast, unmixed human DPCs remained in a monolayer state, indicating that ovine cells are required to initiate aggregation but the human cells are then able to incorporate into aggregates. Both human and ovine DPCs had a higher tendency to aggregate in medium containing 20% (v/v) lamb serum compared to 10% (v/v) foetal calf serum. The effect of co-culturing human with ovine DPCs separated by a permeable membrane gave positive additional effects to human aggregation. In summary, ovine biomolecules show potential for increasing the aggregative behaviour of human DPCs in culture. These biomolecules might eventually be used to treat androgenetic alopecia.
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ItemExamining non-melanoma skin cancer trends in Australia using data from Medicare Australia( 2014)Background: Non-melanoma skin cancers (NMSC) are the most common cancer in Australia, costing the Australian Government $511 million in 2010. NMSC incidence studies have typically utilised face-to-face interview methods that rely on self-reporting of skin cancers. Studies overseas have employed national medical system billing data to examine trends in NMSC. The work in this thesis has adopted this novel approach for evaluating skin cancer trends. Objectives: 1. Determine the completeness of Medicare Australia (MA) in capturing skin cancer cases. 2. Examine the up-to-date incidence rates of NMSC to 2012. 3. Examine rates of recurrent and incompletely excised (residual) NMSC in Australia. Methods: Data from MA, Medicare Benefits Online and Victorian Cancer Registry (VCR) from 2004-2008 were extracted for use in this thesis. MA and VCR melanoma cases were compared. Regression and a paired-samples t-test was performed. NMSC incidence was estimated by determining the number of separate patients treated for a NMSC. The proportion of recurrences and residuals were calculated. Results: Medicare Australia data were found to correlate with the VCR data set. Incidence rates for NMSC were 856 per 105 people in 2012. This incidence was forecast to increase to 1636 per 105 in 2015. 1.54% of NMSC required treatment for a recurrence and 1.26% of NMSC required retreatment for incomplete excision. Conclusion: This thesis has contributed the following: 1. Demonstration of a correlation between MA and VCR melanoma data, suggesting that MA could potentially be used for examining trends in other skin cancers including NMSC. 2. An up-to-date incidence of NMSC in Australia to 2012. 3. Proportion of NMSC that required treatment for residual lesions. 4. Proportion of NMSC that required treatment for recurrent lesions.
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ItemFactors influencing cryopreserved allograft heart valve degeneration( 2006)Heart valve replacement is becoming more commonplace in developed nations. Despite this the ideal valve prosthesis has not been found. The allograft valve has been used for over 40 years and remains an important prosthesis with many advantages. However, like other biological valve prosthesis, they have a finite durability. The causes of allograft valve degeneration are still unknown. The study aims to identify factors associated with cryopreserved allograft valve degeneration. Knowledge of such factors will improve our understanding of the potential causes and mechanisms of allograft heart valve degeneration. (For complete abstract open document)
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ItemPatterns of pain in patients with advanced cancer( 2000)Extensive literature detailing pain in cancer has been published in the past 15 years with point prevalence studies for populations, effects of interventions and general pain management approaches receiving most attention. The experience of pain over time for individual patients has not been studied. There is value in identifying patterns that pain may follow in order to predict future pain levels for a particular patient. Aims: This prospective longitudinal study of patients with cancer, for whom treatment is palliative, seeks to determine if pain reported by patients follows characteristic patterns. The study took place in the context of a larger epidemiological study of symptoms, quality of life, health service utilization and the quality of information regarding cancer and treatment for patients and their carers. Within this epidemiological study, an observational study of pain intensity, characteristics and analgesic levels was undertaken over a 6 month period. The investigators enrolled 202 participants, with 165 participants completing data for evaluation. Method: Data series were examined as a whole group, then individual participants' time series data were examined for visual and mathematical patterns. A series of mathematical models were fitted to successive pain scores based on the assumption that current pain scores are not independent of previous pain scores. Results: As a group, mean pain levels did not alter over the 6 months, but differences between individuals within the group were marked. Considered as a whole, 20.50/0 reported using paracetamol at assessment, and just 16.3% used slow release morphine. Visual examination of pain levels over time revealed three broad groups of patients: those for whom pain scores were always high, those for whom pain scores were always low, and those who had fluctuating pain scores. Those with consistently high pain scores were more likely to have bone metastases (p <0.05), arthritis (p <0.005), to describe pain as burning (p <0.003), stabbing (p <0.001), present with light touch (p <0.001), than the fluctuating or consistently low pain scores groups. In addition these patients used more paracetamol, codeine, slow release morphine and morphine mixture (p < 0.001), though not nonsteroidal anti-inflammatory drugs. There was no significant difference in gender, treatments planned or survival between the groups. A series of mathematical models were fitted to each individuals’ time series data in an attempt to simply describe patterns within the data. The model that most followed the plotted data points with the least variation of points from that model was a 'cubic' pattern for 95% of patients. This pattern is best described as fluctuating, typically with an initial increase of pain early in the study, followed by some lessening of pain, to again increase more steeply towards the end of the study. The degree to which this model 'fitted' or the mean residual square value for all time series ranged from r2=0.02 - 1.0 (median r2=0.38, mode r2=1.0). For 22 subjects the mean residual square value for cubic modeling was r2=0.8 or greater indicating an excellent correlation between the data and the model. These patients were significantly more likely to be treated with radiotherapy (p <0.01), codeine (p <0.05) and morphine mixture (p <0.05), and had a poorer ECOG performance status (p <0.001) and shorter survival (p <0.05) than the other patients. Therefore a cubic model may assist to predict future pain levels for patients with poor performance status. Clinical Implications: The results of this study have implications for the clinician caring for patients with advanced cancer, enabling forecasting of likely future pain levels for a particular patient. This information will assist when planning analgesic interventions, frequency of consultations and resource allocation for a patient, as well as interpreting information gleaned from analgesic trials. Conclusion: Mean pain scores for a whole population with advanced cancer do not change considerably with time, but for individuals marked fluctuations are apparent. Patients with a poor performance status, arthritis, bone metastases and describing certain pain descriptors are more likely to have consistently high pain levels. For 95% of patients a cubic function model best fitted their time series data, and this model is likely to be most reliable in forecasting pain levels when patients have a poor performance status.
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ItemBreast cancer stem cell properties in the PMC42 breast cancer cell line system( 2010)Epithelial to mesenchymal transition, EMT, is a developmental process that may be adopted by tumour cells to facilitate metastasis. EMT changes resulting in a more mesenchymal phenotype have been associated with a “migrating stem cell” pattern in breast and other carcinoma cells. PMC42 human breast cancer cells can be induced by epidermal growth factor (EGF) to a more mesenchymal state and adopt cancer stem cell characteristics. This study investigated the cancer stem cell properties in EGF-induced EMT in the PMC42 system, which consists of the predominantly mesenchymal PMC42-ET cell line and its epithelial-like subline, PMC42-LA. Quantitative reverse transcriptase polymerase chain reaction, qRT-PCR, was used to measure relative gene expression levels following EGF-induced EMT ofPMC42 cells in two- and three-dimensional cell cultures. Stem cell properties were evaluated through immunocytochemistry and two-channel fluorescence-activated cell sorting (FACS). CD44+/CD24- expression, a marker of breast cancer stem cells (BCSC), was measured after EGF-induced EMT. Ten ng/mL EGF treatment increased the number of cells expressing CD44+/CD24- in PMC42-ET but not in PMC42-LA cells as shown by FACS analysis. A mammosphere formation assay, which measures the ability of cells to form complex structures, was used to assess the functionality of the stem like properties exhibited by the different cell populations. This evidence of stemness was found in the BCSC-like population (CD44+/CD24-) of PMC42-ET cells. Expression of embryonic stem cell markers such as ABCG2 and ITGA6, EMT-BCSC related genes generated from EMT and BCSC bioinformatic comparison, and CD24-correlated genes (SerpinE1, EMP1, EMP3, AXL and VIM) were examined in the PMC42 system. EGF-induced EMT increased those gene expressions in the PMC42 system. PLP2 and VIL2, components of the Invasiveness Gene Signature derived from BCSC (Liu et al., 2007), showed no change in expression during EGF-induced EMT of PMC42. In conclusion, transcriptional changes demonstrating an increased stem-like or BCSC-like characteristic was observed in the PMC42 system following an EGF induced EMT. These results suggest the existence of a subpopulation of PMC42 cells which inherently resemble BCSC and provide new insights into the potential role of these subpopulations in EMT-associated metastasis.
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ItemOver-expression of human CD39 in mouse liver protects against ischemia reperfusion injury in a model of liver transplantation( 2009)Primary graft non-function is one of the major limitations of organ transplantation increasing the risk of rejection and early graft failure. A major cause of primary non-function is ischemia reperfusion injury (IRI), an obligatory insult in transplantation. During procurement, the donor is subjected to a period of ischemia inducing the release of tissue-damaging factors such as nitric oxide and reactive oxygen species. Upon engraftment and reperfusion with the recipient blood, these ischemia-induced factors cause rapid cell death and amplification of the inflammatory response leading to further tissue damage. CD39 is an integral vascular and immune ectonucleotidase. CD39 hydrolyses extracellular nucleotides ATP and ADP into AMP, which is then hydrolysed into adenosine by CD73. Extracellular adenosine produced by the concerted action of CD39 and CD73 has potent anti-inflammatory and anti-coagulation effects acting principally via the purinergic adenosine receptor A2a. NKT cells have only recently been recognised and constitute an important subset of T lymphocytes that display both effector and suppressive functions. NKT cells are found in high proportion in the liver of mice and are implicated by depletion studies in protection against hepatic IRI. We have generated mice transgenic for human CD39 (hCD39) and have shown they have an anti-coagulant phenotype. As CD39 is also critical to immune regulation we hypothesised that transgenic expression of hCD39 would modify lymphocyte development and/or function and consequently impact on ischemia reperfusion injury. Flow cytometric analysis was used to assess the number and phenotype of lymphocytes within the thymus and in the periphery of hCD39 transgenic mice. In vitro and in vivo assays were used to test the function of CD4+ T cells and invariant NKT cells from hCD39 transgenic mice. Bone marrow adoptive transfers experiments defined the role of hCD39 expression on bone marrow progenitor cells in comparison to tissue expression. The importance of adenosine signalling through the A2a receptor was studied by crossing hCD39 transgenic mice with A2a receptor knock-out (KO) mice. The effect of hCD39 expression on ischemia reperfusion injury was evaluated in a model of murine liver transplantation A high level of hCD39 expression in the transgenic thymus resulted in lymphocyte maturation blockade and peripheral lymphopenia of CD4+ T cells and invariant NKT cells. Both lymphocyte populations were functionally deficient. The observed phenotype resulted from the expression of hCD39 on bone marrow progenitor cells but was independent of A2a receptor signalling. Over-expression of hCD39 in transgenic livers was protective against ischemia reperfusion injury induced by cold storage and liver transplantation.