Surgery (St Vincent's) - Theses
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ItemTackling regulation “In situ”: A model for approaching regulation during a 3D bioprinting pre-clinical research translation program( 2021)3D bioprinting uses the techniques of additive manufacturing, but includes living cells, with the goal of creating living 3D tissues for modelling disease or for patient implantation. This ongoing technological revolution presents a major challenge for regulators as the current regulatory frameworks are designed for mass manufactured, standardised devices, and as such are not suited to 3D bioprinted, individual-specific devices, often using the patient’s own cells. The aims of the project were to identify and highlight the challenges of regulating 3D bioprinting technologies, and to develop an approach and the relevant tools that researchers could use for integrating regulatory considerations into the development pipeline for a 3D bioprinting device during a research translation program. By using the Axcelda (formerly known as the Biopen) project example and associated publications, this research highlighted several areas of commonly missed opportunity to consider and integrate relevant regulatory requirements into the initial academic study design. These included both general and specific considerations such as the generation of technical and regulatory documentation to better outline the existing product and components, initial critical ingredient identification and safety profiling, risks and regulatory strategy, as well as to incorporate additional specific biocompatibility testing into early pre-clinical studies, among other considerations. Doing so demonstrated the potential of this process to lead to better designed, faster and more efficient studies (from a regulatory perspective), thus increasing the potential for successful translation into a commercial product.
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ItemMultiparametric MRI in Prostate Cancer Diagnosis and Management( 2021)Prostate canceris the most common cancer affecting men in Australia. Detection has previously relied upon clinical examination and prostate specific antigen (PSA) screening. Addition of MRI of the prostate prior to prostate biopsy has increased our ability to accurately diagnose prostate cancer. This thesis examines the implementation of multiparametric MRI of the prostate in Australia and the impact it has had on prostate cancer diagnosis.
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ItemColorectal cancer in rural regional Australia( 2017)Colorectal cancer (CRC) is the second commonest cancer in Australia. The survival outcome of colorectal cancer patients within Australia is reported to vary with population density and between health services, with some literature showing poorer prognosis in rural regional and remote patients. Chapter one aims to outline some key issues in CRC such as epidemiology, diagnosis, treatment, surveillance, and outcome whilst chapter two aims to present a systematic review of how geographical disparity influences CRC survival. There are many potential factors that contribute to a poorer prognosis in rural regional CRC patients though the literature is limited, and at times, inconsistent. Thus, there is a need for regular audit, reporting and benchmarking of outcomes in CRC patients against agreed standards. I reviewed the long-term outcomes of CRC at Barwon Health, which serves the South West Victoria, a region with a population of some 500,000. My aim was to determine whether changes introduced to the management of CRC translated into improved survival after surgery. The literature to date has suggested that patients living in rural and regional Australia (grouped together) have worse colorectal cancer survival rates than those living in metropolitan Australia. This thesis was based on a prospectively maintained registry kept over a period of thirteen years from 2002 to 2014, that had accumulated 1079 patients who had undergone surgery at the University Hospital Geelong for CRC (744 colon cancers and 335 rectal cancers). The overall number of operations per year increased over time (p=0.037) but with similar proportions of elective and emergency surgery (p=0.75) and tumour stage (P=0.21). This lack of change in the proportion of elective cases was in spite of the Federal Government introducing a National Bowel Cancer Screening Program in 2006. The proportion of patients with severe comorbidities did increase (p=0.015) over the study period. The median survival after surgery by stage was 123 months, 141 months, 76 months and 17 months for stages I to IV CRC respectively. Overall, there were improvements observed in both peri-operative mortality (POMR) (p=0.028) and long- term survival (p=0.0025) of CRC patients in this major regional centre. I then reviewed the outcome of patients with metastatic disease. The Geelong database included 843 patients who had undergone resection and primary anastomosis for their primary tumour (661 colon cancers, 182 rectal cancers). Metastatic disease was present in 16% (135 patients) and was associated with an increased risk of anastomotic leakage (13% vs. 5%, p=0.003) and a higher peri- operative mortality rate (9.6% vs. 2.8%, p=0.0003). Patients with anastomotic leakage had a reduction in the overall survival (121 months vs. 66 months, p=0.02). The fifth chapter aimed to perform a regional study to identify patients with colorectal cancer at higher risk of developing metastatic disease. There were 503 patients (345 colon and 158 rectal) with non-metastatic (stage I-III) CRC who had resections and were followed up for at least five years. Metastatic progression was, as expected, significantly higher for patients with stage III disease (aHR 4.42 for colon cancer 95% CI 1.74 to 11.23, aHR 3.34 for rectal cancer 95% CI 1.36 to 8.22), and those with lymphovascular invasion (aHR 2.94 95% CI 1.70 to 5.06). Metastatic disease was also more likely to eventuate in those with severe comorbidities (aHR 2.18, 95% CI 0.26 to 0.86), and in colon cancer patients with the lowest socioeconomic status (aHR 2.03 95% CI 1.23 to 3.34). Gender, tumour location and geographical location (rural or regional) was not associated with metastatic progression. Before determining surveillance strategies targeting higher-risk patient groups in regional Victoria, these findings would require confirmation from similar studies in other regions of rural/regional Victoria, such as Bendigo, Albury / Wodonga, Latrobe Valley, or Shepparton.
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ItemCombining TIL and CAR for adoptive cell therapy in metastatic melanoma( 2019)Background Metastatic melanoma is a highly lethal disease, and until recently patients had limited therapeutic options. Knowledge and understanding of the role the immune system plays in tumour development and its therapeutic potential has recently gained momentum and immunotherapeutic agents have emerged as the gold standard of therapy in treating this cancer. Adoptive cell therapy (ACT) has been shown to have high rates of tumour regression with durable, complete responses and potential 'cure'. Tumour-infiltrating Lymphocytes (TIL) and Chimeric Antigen Receptor (CAR) therapies are examples of ACT. Each has their own advantages, limitations and toxicities. As the complexity of the immune system and its targets is increasingly appreciated, combining immunotherapies is emerging as a promising avenue for improving patient oncological outcomes. This project explores the efficacy of dual specific T cells by combining TIL and CAR therapies. Aim To establish a model system transducing TIL with anti-Her2 CAR (TIL-CAR) and assessing function against autologous melanoma tumour cells that express Her2 antigen. Method TIL were generated from patient derived metastatic melanoma tumours and tumour cell lines were established in a biobank. TIL were thawed and activated using CD3/28 beads and transduced with second generation anti-Her2 CAR (scFv-erbB2-CD28-zeta) using a retronectin protocol. Patient matched PBMCs were transduced for functional comparison. Melanoma tumour lines in the biobank were found to innately express Her2 antigen to varying degrees. Some melanoma tumour lines were transduced and sorted to create higher expressing Her2 antigen lines for functional comparison. Flow cytometry was used to confirm cell phenotype and antigen/CAR expression. Functional testing was performed using ELISA and chromium release assays. An in vivo ACT model in NSG mice was performed comparing TIL and TIL-CAR. Results TIL were successfully cultured from metastatic melanoma tumour pieces. Despite TIL proliferating at lower rates than PBMCs, both were successfully transduced to express anti-Her2 CAR. When TIL were transduced to express anti-Her2 CAR they were functionally active through both TCR and CAR and produced greater amounts of interferon gamma against Her2 expressing tumour lines. TIL-CAR had greater cytotoxic activity when cultured against autologous melanoma tumour lines, but the benefit transduced TIL over PBMCs varied in response between tested patients. The advantage of TIL-CAR over PBMC-CAR did not demonstrate consistent trends across this limited group of patients. The functional activity may be influenced by the level of Her2 expression in the co-cultured tumour cells as well as by the phenotype of T cell populations. Results of an in vivo pilot study in mice demonstrated reduction in tumour size when TIL-CAR were used in an ACT protocol. The primary limitation of this study was the low proliferation rate of TIL following transduction which required extended periods in culture. Conclusions Combination of TIL-CAR is a novel concept. TIL can be transduced to express anti-Her2 CAR. Metastatic melanoma cells in our biobank constitutively express Her2 antigen. TIL-CAR tend to show greater activity in interferon gamma and cytotoxic functions compared to parental (non-transduced) TIL when cultured against Her2 expressing tumour lines. When compared to the activity of PBMCs transduced with the same CAR the additional benefit conferred by TIL-CAR is inconsistent. Protocols would benefit from further optimisation to generate a 'younger' phenotype capable of more rapid and sustainable proliferative potential and facilitate earlier delivery of therapy if used in a clinical setting.
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ItemRadiation-associated breast, thyroid and solid malignancies in patients attending the Peter MacCallum Cancer Centre Late Effects service( 2017)Background: Survivors of childhood, adolescent and young adulthood (CAYA) malignancies have an increased risk of subsequent primary malignancies, particularly after exposure to therapeutic radiation. The Peter MacCallum Cancer Centre Late Effects (PMCC LE) service provides individualize, multidisciplinary care and surveillance advice for survivors of malignancies, especially CAYA malignancies. Methods: A retrospective review was performed of patients exposed to therapeutic radiation attending the PMCC LE service from 1st January 2000 to 20th February 2013. All invasive malignancies, in-situ malignancies, benign tumours and deaths were evaluated. Separate time-to-event analyses was performed for radiation-associated breast, thyroid and solid malignancies in patients exposed to chest, thyroid and any therapeutic radiation respectively, measured from the date of first attendance to the PMCC LE service and stratified by the interval from completion of radiation to the first attendance. The incidence of breast and thyroid malignancies was compared to the Australian general population. Compliance with breast and thyroid surveillance recommendations was determined by assessing the number of screen events over the period of attendance to the service. Clinicopathological features and management of radiation-associated breast and thyroid and other solid malignancies was examined. Ultrasound and cytological workup of radiation exposed thyroid nodules was assessed. Results: After excluding 187 patients, 534 included patients developed 194 invasive malignancies; 147 were radiation-associated and 47 non-radiation associated. The most common malignancies were non-melanoma skin (37.1%), thyroid (17.0%) and breast (12.9%) malignancies. Patients whose first attendance was ≥15+ years after radiation exposure experienced the highest incidence of radiation-associated breast, thyroid and solid malignancies, with 23%, 8% and 27% affected after 10 years of subsequent follow-up respectively. The incidence of breast and thyroid malignancy was elevated 11.2 and 57.6 times respectively compared to the Australian general population (both p<0.001). Compliance with breast surveillance using mammography or any screening modality was observed in 18.4% and 28.6% of women at risk respectively. Twenty-eight radiation-associated breast malignancies occurred in 24 women (16.7% bilaterality). Breast malignancies diagnosed after the first attendance to the PMCC LE service were more likely screen-detected (p=0.002). Most were hormone receptor positive (87.5%), invasive ductal carcinomas (82.1%) managed with mastectomy (89.3%). Compliance with thyroid surveillance was observed in 76.9% of patients at risk. Ultrasound features of microcalcification and increased internal vascularity had a low sensitivity (62.5%) for predicting a malignant nodule, which improved when used in conjunction with a Bethesda IV-VI result (91.7%), although cytological assessment was not performed in 45.6% of operative cases. Thirty-three patients had a radiation-associated thyroid malignancy; 45.4% (n=15) were incidental. The majority were papillary thyroid cancers (88.9%); of which 12.5% were node positive and 34.4% were multifocal. Node positive thyroid cancers were more likely to present symptomatically (p=0.03). There were 36 deaths in the cohort (6.7%), most commonly attributable to radiation-associated malignancies (41.9%), especially brain, breast and sarcomatous malignancies. Conclusions: Patients attending the PMCC LE service have a high burden of subsequent malignancies that typically occur after a long latency. Ongoing long-term surveillance is essential and judicious management with adherence to guidelines is advocated in this unique population of patients.
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ItemDefining lymphatic microstructure and the genetic basis of lipolymphedema( 2017)The lymphatic system regulates tissue fluid homeostasis, intestinal fat absorption, and immune cell trafficing. Lymphedema is soft tissue swelling secondary to lymphatic dysfunction, which results in the accumulation of tissue fluid in the interstitial space. This might occur as a primary disorder of the developing lymphatic system, or alternatively lymphedema might be an acquired disorder secondary to lymphatic injury. For example, secondary lymphedema is a common problem following cancer and cancer treatments such as lymph node surgery and radiotherapy, resulting in significant morbidity. Radiotherapy is an established risk factor for lymphedema, and in addition to causing direct injury to the lymphatic vessel, it is possible that alternative mechanisms might also contribute to radiation‐induced lymphatic dysfunction, such as localized ischemia of the lymphatic wall. It is also likely that predisposing genetic risk factors are at play, as not all individuals exposed to the same risk factors will develop secondary lymphedema. Lipoedema is a different form of soft tissue swelling due to the abnormal accumulation of adipose tissue. Lipoedema and lymphatic dysfunction appear to be linked, as individuals frequently develop a degree of lymphedema, particularly as the condition progresses in severity, where it may be decribed as lipo‐lymphedema. The cause of lipoedema and the genetic basis of the condition are currently unknown. This thesis aims to discover and define alternative mechanisms for lymphtic dysfunction in the context of secondary lymphedema, particularly focussing on the supply of oxygenated blood to the lymphatic vessel wall. We also aim to describe inheritance patterns and the genetic factors involved in lipoedema and lipo‐lymphedema. Such knowledge might uncover therapeutic targets and facilitate the development of treatments for lymphedema and lipoedema, including gene therapy.
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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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ItemInvestigations of the patello-femoral joint in the osteoarthritic knee( 2013)Background Patello-femoral arthritis and anterior knee pain is common in patients with tibio- femoral arthritis. It is presumed that anterior knee pain is caused by patello-femoral arthritis and or malalignment. The predictors of anterior knee pain post total knee arthroplasty are unknown. There are no studies describing the nature and location of anterior knee pain. Methods A retrospective analysis of a prospectively followed cohort of 334 patients undergoing knee arthroplasty was performed. Radiological analysis of measures of patello- femoral malalignment and joint space narrowing was performed. These measures were analysed for association with anterior knee pain and function. A prospective cohort study of 105 patients was performed with minimum 12 months follow-up to analyse post-operative patello-femoral alignment in knees with and without patellar resurfacing. Statistical analysis was performed for possible clinical and radiological predictors of post-operative anterior knee pain. The location of anterior knee pain was reported in this cohort via drawings performed by patients and analysis of pre and post arthroplasty anterior knee pain location was performed. Results Patello-femoral malalignment as measured by lateral patellar tilt and lateral displacement were strongly associated with severe patello-femoral arthritis. Anterior knee pain was not associated with arthritis or malalignment of the patello-femoral joint. Knees that underwent patellar resurfacing during total knee arthroplasty had better post-operative patellar alignment compared to patellae left non-resurfaced. Better patello-femoral alignment did not correlate with improvement in function or decrease in anterior knee pain. Female gender was an independent factor associated with anterior knee pain both before and after arthroplasty. Anterior knee pain is felt more often peripherally around the patella than centrally. Anterior knee pain post-arthroplasty is most commonly similar in nature and location to pain felt pre arthroplasty. Conclusion Patello-femoral arthritis is more severe in knees with patello-femoral malalignment. The cause of anterior knee pain is multi-factorial and predicting anterior knee pain after total knee arthroplasty is difficult. Female gender was the only strong statistical predictor of anterior knee pain pre and post arthroplasty. Better patellar alignment post arthroplasty does not guarantee better function or less anterior knee pain at 12 months follow-up.
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ItemCardiac surgery using cardiopulmonary bypass in adult patients, effects of pre-operative insulin resistance and the metabolic syndrome, dimethylamine dimethylaminohydrolase activity, and vasoreactivity of regional adipose tissue( 2013)Introduction: Atherosclerosis and coronary artery disease (CAD) are accelerated in Diabetes mellitus (DM) and the Metabolic Syndrome (MS). Insulin Resistance (IR) is thought to be central in its pathogenesis. Recently, Asymmetric Dimethylarginine (ADMA) has linked IR and CAD. ADMA inhibits the vasodilating and vasoprotective agent Nitric Oxide (NO), while the enzyme Dimethylarginine Dimethylaminohydrolase (DDAH)reduces circulating levels of ADMA. As an increase in visceral adiposity is associated with MS and IR, we sought to determine if there were clinically relevant differences in ADMA and DDAH levels in adipose tissue from patients undergoing cardiac surgery, with and without DM and MS. Adipose tissue may alter vascular function via production of vasoreactive paracrine factors. This may explain the acceleration of atherosclerosis in patients with DM and IR. We sought to determine if vasoreactive factors were altered in the adipose tissue of patients with IR or the MS. Methods: Between January 2002 and December 2011, clinical data was prospectively collected on all patients undergoing open cardiac surgery at St Vincent’s Hospital. During 2012, 40 patients undergoing non-emergency cardiac surgery were recruited and adipose tissue was harvested intra-operatively, as well as peri-operative blood and IR parameters. Adipose samples were analysed for DDAH activity, and serum was processed for measurement of ADMA levels. Adipose tissue was incubated with culture medium, and the resulting conditioned medium was used for vasoreactivity experiments. Rat aortic, as well as autologous bypass conduit vessel vasoreactivity was measured using organ bath methodology in the presence of conditioned medium. Results: Of 4542 patients who underwent cardiac surgery in the last 10 years, 1640 (36.1%) had 3 or more surrogate markers of the MS, and 1360 (29.9%) had DM. There was a greater incidence of atrial fibrillation (43.3% vs. 38.6%, p=0.026), renal failure (6.5% vs. 3.8%, p=0.004) and low systemic vascular resistance (16.5% vs. 11.7%, p=0.04) in elective patients undergoing CABG who had the MS. Pre-operative fasting glucose (r=0.63, p<0.001) and HbA1c (r=0.57, p=0.001) correlated with intra-operative hyperglycaemia. HbA1c also predicted post-operative hyperglycaemia (r=0.40, p=0.02). IR measures involving serum insulin (HOMA-IR, QUICKI, LogHOMA) did not predict peri-operative dysglycaemia. Erythrocyte DDAH activity did not appear to change over the course of Cardio-Pulmonary Bypass (CPB). There was an increase in subcutaneous adipose DDAH activity over the course of CPB in patients with the MS (0.02419-0.04318 μg Cit/μg protein/min, p=0.047, n=6), but no change in mediastinal or epicardial adipose DDAH activity was detected. There was also an increase in subcutaneous adipose DDAH activity in diabetic patients (0.02024-0.03127μg Cit/μg protein/min, p=0.013, n=10).There were positive correlations between pre- and post-operative DDAH activity in all depots in patients undergoing CABG with CPB, but not in visceral depots in patients who did not have CPB (OPCAB). Organ bath experiments revealed a significant factor in culture medium that delayed vaso-relaxation to acetylcholine. Vaso-constrictive responses to phenylephrine were not significantly different when conditioned medium was added to the organ bath. Conclusions: There is an increased incidence of post-operative renal failure, atrial fibrillation and low vascular resistance in patients with the MS undergoing CABG. These factors are common with the literature, and may reflect an increased inflammatory response to CPB and surgery. Simple measures of pre-operative dysglycaemia (fasting glucose and HbA1c) correlate with peri-operative dysglycaemia. Levels of DDAH activity in human subjects sustaining an inflammatory stimulus are reported, and appear to increase in subcutaneous adipose tissue. Culture medium utilised for adipose culture contains a significant vaso-active substance that needs to be considered in future experiments.