Surgery (Austin & Northern Health) - Theses

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    Development of a cell-free DNA methodology to assess organ rejection after liver transplantation
    Goh, Su Kah ( 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.