Surgery (Austin & Northern Health) - Theses
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ItemNo Preview AvailableHarnessing cell-free epigenetic and mitochondria-derived DNA signatures to enhance precision in liver transplantation care( 2023-07)Background: Liver transplantation (LT) is a life-saving procedure for people with end-stage liver disease. Ensuring successful, durable health outcomes for LT recipients requires long-term surveillance and monitoring of graft function. The existing, non-invasive techniques used in clinical practice to diagnose complications post-LT are imprecise and lack disease specificity. Ultimately, an invasive graft biopsy is often required to diagnose or exclude complications. The analysis of graft-derived cell-free DNA (gdcfDNA) in plasma has shown promise as an alternative, non-invasive approach for monitoring graft health following solid-organ transplantation. gdcfDNA detection has previously relied on identifying mismatched genotypes in organ donor and recipient DNA. The analysis platforms and laboratory workflows required to perform this genotyping and quantify gdcfDNA are, at present, complex and laborious. This has delayed translation of this technology into clinical practice. Objectives: Hypothesis: gdcfDNA analysis using epigenetic and non-genotyping based techniques is feasible, and might offer a more practical laboratory approach to help facilitate the incorporation of gdcfDNA monitoring into routine LT care. This thesis aimed to develop an assay that harnessed graft-specific epigenetic motifs to detect gdcfDNA in plasma, and then to evaluate its diagnostic accuracy and clinical utility in a study involving LT recipients. A further objective was to explore a role for gdcfDNA analysis to enhance precision in LT care, outside of the milieu of diagnosing or excluding graft immune rejection. Main findings: A novel gdcfDNA quantification assay, based on the detection of liver-specific DNA methylation patterns in plasma cell-free DNA (cfDNA), was developed and validated in a pilot clinical cohort study (Chapter 2). A new laboratory workflow, involving the introduction of a synthetic DNA standard to plasma samples, was implemented to improve pre-analytical quality control prior to gdcfDNA analysis (Chapter 3). In a prospective, cross-sectional clinical study (Chapter 4), plasma gdcfDNA measurement using liver-specific DNA methylation signatures, significantly outperformed conventional liver function tests in the non-invasive prediction of biopsy-proven acute graft rejection following LT. At a cut off of <33% of the total plasma cfDNA fraction, liver methylation-specific gdcfDNA had a 97% specificity for predicting biopsy proven acute rejection requiring treatment (tBPAR); correctly excluding tBPAR in 30/31 LT patients. gdcfDNA monitoring, through the quantification of mitochondria-derived cfDNA, was also demonstrated to act as a potentially useful molecular tool to inform management in other contexts within LT care (Chapters 4 and 5). The analysis of mitochondria-derived cfDNA from plasma and bile during ex-vivo normothermic machine perfusion, prior to LT, was shown to be feasible with a high degree of technical precision. Trends in gdcfDNA levels in this context varied significantly between grafts that were viable for LT and those that were not transplanted (Chapter 5). Conclusions: The detection of liver-specific methylation patterns in cfDNA is shown to represent a non-invasive, precise, rapid-turnover and cost-effective approach for quantifying gdcfDNA in patients following LT. The technique has major practical advantages over previous gdcfDNA quantification methods, particularly as it does not require prior genotyping or sequencing, lending it greater feasibility for translation into LT care. gdcfDNA monitoring has the potential to act as a “second-tier” non-invasive test to stratify the risk of developing tBPAR in LT patients, prior to undertaking an invasive biopsy. This potential clinical niche warrants further study in multi-centre, interventional trials. gdcfDNA monitoring may have a useful role for monitoring graft health in LT, outside of contexts outside of the diagnosis of acute rejection. gdcfDNA monitoring during ex-vivo machine perfusion prior to LT is feasible with a high degree of technical precision, and should be studied further to establish whether monitoring may aid in the evaluation of graft suitability for successful LT.
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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.