Cell-Free DNA in Different Clinical Scenarios after Heart Transplantation: Shedding Light or Obscuring the Picture?

Purpose Donor-derived cell-free DNA (dd-cfDNA) as a highly sensitive marker of rejection after heart transplantation (HTx) has gained emerging interest. Recent studies are based on sequencing techniques and use fractional abundance (dd-cfDNA as a fraction of total cfDNA) as their outcome. Here we present patient examples of the BIODRAFT-study (NCT03477383) based on PCR-techniques. Methods Blood samples are taken prospectively in parallel with endomyocardial biopsies (EMB) during the first year after HTx. dd-cfDNA is analyzed using targeted preamplification of 35 single nucleotide polymorphisms followed by digital droplet-PCR. Outcome is fractional abundance as well as total number of DNA copies, both from the donor and the recipient. Results 71 patients (57 adults, 14 children) are so far included and more than 500 blood samples analyzed. In otherwise stable patients, both fractional abundance and total DNA copies seem to follow biopsy patterns with respect to rejection. However, we could also identify different scenarios in which the distribution of cfDNA seems more complicated than hitherto described: Patients with primary graft failure, clinically silent CMV-infection or mild right heart failure show elevated levels of dd-cfDNA, thus complicating the interpretation of results. Some patients present with markedly elevated levels of their own cfDNA without obvious clinical reasons. Conclusion Besides the well-known concept of fractional abundance, our approach gives results that allow following absolute DNA copy numbers of both donor and recipient. This widens the horizon of cfDNA as a marker of graft injury after HTx, and gives an outlook to clinical scenarios with possible false positive (mimicking rejection) and, even worse, false negative results: high levels of recipient-cfDNA could mask ongoing rejection if only fractional abundance can be reported. The biology of cell-free DNA is complex and seems not yet fully understood.