The association between donor-derived cell-free DNA and acute rejection in heart transplant patients

Cardiac transplantation is currently the only effective therapy for patients with end-stage heart failure. Despite its life-saving potential, heart transplantation is associated with several significant challenges, the most prominent of which are post- transplant complications. Among these, the risk of infections is heightened due to the immunosuppressive medications required to prevent organ rejection, while the possibility of graft rejection remains one of the primary concerns. The gold standard for diagnosing transplant rejection remains endomyocardial biopsy (EMB). However, this invasive procedure has several drawbacks, making it a less-than-ideal solution for long-term monitoring of heart transplant recipients. In light of these limitations, the study presented in this thesis explores the potential of a non- invasive approach for detecting transplant rejection: liquid biopsy. This technique involves analyzing blood samples to detect and quantify specific biomarkers of rejection, particularly donor-derived cell-free DNA (dd-cfDNA). dd-cfDNA is DNA that is released into the bloodstream when cells die, and its presence can indicate immune-mediated damage to the transplanted heart. Since dd-cfDNA is of donor origin, its levels in the recipient’s blood can be a direct reflection of transplant rejection, providing a more accessible and less invasive diagnostic method. The methodology employed in this study uses NGS to analyze single nucleotide polymorphisms (SNPs), which are genetic variations that can distinguish between the donor's and the recipient's DNA. By sequencing these SNPs, the study can quantify the fraction of dd-cfDNA present in the patient’s bloodstream. Specialized software is then used to calculate the precise amount of dd-cfDNA, which serves as an indicator of ongoing rejection. The study aims to establish a reliable and early biomarker for transplant rejection, which could potentially replace or complement the traditional biopsy method, offering a less invasive, more cost-effective alternative for monitoring transplant recipients over time.