Surgery (St Vincent's) - Theses

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    Characterisation of sensory corpuscles, vasculature and collagen in the injured scapho-lunate ligament
    Miles, Oliver Joseph ( 2022)
    Background: The scapho-lunate ligament (SLL) is a ligament spanning between the scaphoid and lunate bones of the carpus. It has a well-established role in ligamentous restraint of intercarpal motion and maintenance of carpal alignment under axial load. The SLL also has a role as a sensory organ. Sensory mechanoreceptors are located within the ligament. These receptors are responsible for the afferent input to the reflex arc of extrinsic forearm muscular contraction that provides additional dynamic stability to the wrist joint. The SLL is the most frequently injured ligament within the wrist[1]. Complete disruption of the SLL causes carpal instability and malalignment[2], which eventually leads to osteoarthritis of the wrist. When the ligament is torn, surgical treatment options include reconstruction of the ligament with procedures that usually use tendon grafts or transfers that act as a replacement for critical portions of the damaged ligament complex. These techniques may be able to replicate the static restraint of the SLL, however, they fall short of producing an innervated construct that is capable of co-ordinating neuromuscular reflexes. In search of improved reconstructive surgical options, the ligamentous stump of the damaged SLL has been postulated to be a source of native cells that may drive the process of ligamentisation, the process of transformation of the reconstructed ligament created from alternative tissue such as tendon or synthetic construct, to the histological and functional phenotype of a true ligament. This phenomenon has been investigated in knee ligament reconstruction, with anterior cruciate ligament (ACL) remnant preservation being a popular technique used in ACL reconstruction. Methods: This study aims to investigate the structural and cellular makeup of injured SLLs and compare them to the known structure of normal SLLs to estimate the regenerative potential of the residual SLL stump and its value in developing innervated constructs. Injured SLLs were collected from voluntary human subjects at the time of SLL reconstruction or limited wrist fusion, where the ligament remnants would otherwise be discarded. These specimens were formalin fixed and paraffin embedded for histological analysis and immunostaining was performed to identify the vascular and neural structures of the SLL as well as to determine its collagen constitution. Results: Fifteen ligaments were harvested from subjects at surgery after SLL injury ranging from 39 days to 20 years from time of injury. Eleven ligaments were harvested less than one year after injury and four ligaments were harvested two years or more after injury. A total of 66 mechanoreceptors were identified, 50 in the 11 specimens harvested less than one year following injury. Nine of these 11 specimens within one year contained mechanoreceptors, 54% of which resided in the volar subunit, 20% in the dorsal subunit and 26% in the proximal subunit. Two of the four specimens harvested two years or later from injury contained mechanoreceptors, all of which were located in the dorsal subunit. Blood vessels were found in 13 of the 15 samples. Mean vessel density for all specimens was 1.3%, with the highest average density being 1.8% for the volar subunit. The vessel density in the volar subunit decreased with time after injury, with vessel density of 2.5% seen in specimens less than three months post injury, and vessel density of 0.03% in those specimens that were more than three months post injury. The mean collagen I density was 45.6% of the ligament area for all specimens, with the highest average in the dorsal subunit (53% of the ligament area). Collagen I density did not vary significantly within the first two years of injury. Collagen III density varied significantly with time since injury, with average density of 47% of ligament area in specimens less than three months after injury and and 31% in those specimens that were harvested greater than three months after injury. This decline in Collagen III density after three months was most prominent in the volar subunit. Collagen I:III ratio reflected the pattern of change seen in the collagen III profile. Conclusion: Mechanoreceptors were reliably located in the SLL, particularly in the volar subunit of specimens harvested less than one year after injury. Vessel density in the volar subunits was reliably associated with the mechanoreceptor distribution, as was collagen III distribution in the volar subunit in the initial three months post injury. Scapholunate ligaments demonstrate favourable histological findings that warrant further research into remnant inclusion techniques in earlier injuries, particularly incorporating the volar subunit.
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    Tackling regulation “In situ”: A model for approaching regulation during a 3D bioprinting pre-clinical research translation program
    Mladenovska, Tajanka ( 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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    Multiparametric MRI in Prostate Cancer Diagnosis and Management
    Whish-Wilson, Thomas Ian ( 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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    Colorectal cancer in rural regional Australia
    Ng, Suat Chin ( 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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    Combining TIL and CAR for adoptive cell therapy in metastatic melanoma
    Mills, Jane Kathleen ( 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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    Radiation-associated breast, thyroid and solid malignancies in patients attending the Peter MacCallum Cancer Centre Late Effects service
    Koo, Eva ( 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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    Defining lymphatic microstructure and the genetic basis of lipolymphedema
    Bendon, Charlotte Lucy ( 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.