Surgery (Austin & Northern Health) - Theses
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ItemDevelopment of a cell-free DNA methodology to assess organ rejection after liver transplantation( 2019)Background: Liver transplantation has revolutionised the prognosis of patients with fulminant liver failure, chronic liver disease, and liver cancer. Although liver transplantation is safe, organ rejection is a common complication after such a procedure. The gold-standard for diagnosing organ rejection after liver transplantation is a tissue biopsy. Liver biopsies are invasive. There is thus an unmet clinical need for accurate blood tests to diagnose the episodes of organ rejection after liver transplantation. Donor-specific cell-free DNA (dscfDNA) is an emerging biomarker of organ rejection. Measuring dscfDNA using current methodologies such as next generation sequencing can be both complex and expensive. Novel tests that overcome these limitations would favour adoption of such methodologies for the quantification of dscfDNA and implementation for the surveillance of organ rejection after transplantation. Objectives: The first objective of this thesis was to develop a cell-free DNA based assay for the accurate quantification of dscfDNA that could overcome some of the limitations of existing methodologies. The second objective of this thesis was to deploy this assay to monitor episodes of organ rejection in a prospective cohort of recipients. Main findings: A probe-free droplet digital PCR-based methodology was developed. The methodology overcame some of the common limitations that were observed in next generation sequencing-based and other PCR-based methodologies. The newly developed approach was accurate, economical, and rapid which facilitated the rapid turnaround of results as well as enabled early clinical decision-making (Chapters 3 and 4). The application of this approach to measure dscfDNA was shown to be feasible for the monitoring of dscfDNA in a prospective cohort of forty recipients after liver transplantation (Chapter 5). The levels of dscfDNA were reflective of organ health. Furthermore, a calculated threshold of 898 copies of dscfDNA per mL of recipient plasma identified majority of the recipients with biopsy-proven acute rejection requiring treatment. The diagnostic performance of dscfDNA, in this cohort, was superior compared to routine liver function tests in identifying organ rejection. Conclusion: This thesis presented the application of a novel cfDNA methodology to measure dscfDNA in a prospective cohort of recipients after liver transplantation. The results demonstrated the promising utility of dscfDNA as a marker of organ rejection after liver transplantation. These pertinent findings warrant further validation with a view towards clinical implementation.
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ItemThe role of hypoxia inducible factor 1 alpha (HIF1α) in prostate cancer( 2016)Prostate cancer (PC) is one of the most prevalent cancers in men. Although many PCs are indolent, a significant proportion will metastasize and develop resistance to therapy. Contemporary screening tests lack the finesse to accurately differentiate aggressive PCs from indolent tumours, potentially leading to over-diagnosis and over-treatment. While cellular hypoxia often plays an integral role in carcinogenesis and tumour progression, this connection has been difficult to demonstrate in PC. However, a downstream marker of hypoxia, Hypoxia inducible factor 1α (HIF1α), which is a transcription factor that protects cells against noxious stimuli, is frequently over expressed in PC. Therefore, the role of HIF1α in PC was investigated in this thesis. The Castrate resistant PC (CRPC)-like human PC cell lines PC3 and DU145 were found to over-express HIF1α protein compared to an androgen-sensitive cell line LNCaP under normoxic conditions. Using HIF1α 5’UTR-luciferase constructs in PC3 cells, further experiments revealed that increased translation of HIF1α mRNA regulated by a 70bp GC-rich, secondary structure in the 5’UTR of the HIF1α promoter may be responsible for normoxic HIF1α overexpression. Cell proliferation assays revealed that PC3 cells over-expressing HIF1α were more resistant to destruction by cytotoxic agents (H2O2 and 5-fluorouracil) than androgen-dependent LNCaP cells. Reduction of HIF1α expression in PC3 cells using RNA interference decreased both the resistance towards cytotoxic agents and cell migration. Conversely, in the androgen-dependent LNCaP cells overexpression of HIF1α increased the resistance to cytotoxic agents. One hundred prostate tumours were then immune-stained for HIF1α and outcomes measured. On multivariate analysis HIF1α was an independent risk factor for progression to metastatic PC (Hazard ratio (HR) 9.8, p = 0.017) and development of CRPC (HR 10.0, p = 0.021) in patients on androgen-deprivation therapy (ADT). Notably the tumours that did not express HIF1α did not metastasise or develop CRPC. Next, the effects of non-specific HIF1α inhibitors (digoxin, metformin and angiotensin-2 receptor blockers) were investigated in ninety-eight patients who had continuous ADT as first line therapy and developed CRPC. The median CRPC-free survival was longer in men using HIF1α inhibitors compared to those not on inhibitors (6.7 yrs vs. 2.7yrs, p=0.01) and there was a 71% reduction in the risk of developing CRPC (p=0.02) and an 81% reduction in the risk of developing metastases (p=0.02) after adjustment for Gleason score, age and PSA. Finally, the effects of metformin were investigated in 2055 men treated for PC with external beam radiotherapy. Surprisingly, metformin did not result in any improvement in time to biochemical failure, time to metastases or overall survival in men undergoing radiotherapy, but there was an 1.5 fold increase in PC-specific deaths (p<0.05) in men on metformin who received ADT when adjusted for cancer risk and co-morbidities. In conclusion, the results presented in this thesis indicate that HIF1α is a promising marker in PC, which may be used for early identification of cancers that potentially will progress to metastases and develop resistance to ADT. HIF1α is likely to contribute to metastasis and chemo-resistance of CRPC, targeted reduction of HIF1α may improve outcomes of aggressive PC.