Design of a national Swedish study evaluating donor-derived cell-free DNA as a diagnostic biomarker in heart transplantation using next-generation sequencing
I Haugen Loefman, K L Ljung, O B Braun, E B Bollano, C H Hage, J G Granstrom, S V Vonlanthen, P I Isakson, E L Larzenius, T E Eich, A S L Liedberg, J H Holgersson, M M MelinAbstract
Background
Early detection of rejection after heart transplantation (HTx) is crucial for avoiding graft dysfunction and long-term complications. Traditionally, surveillance for rejection relies on serial endomyocardial biopsies (EMBs) according to center-specific protocols. Recently, non-invasive measurements of biomarkers have been found to detect cellular damage in the graft, with donor-derived cell-free DNA (dd-cfDNA) being the most promising. While different dd-cfDNA tests has gained traction in the United States, its adoption in Europe remains limited. Sweden performs 60–70 Htx annually and there is a need for a locally based, less expensive method to non-invasively monitor the patients.
Purpose
The aim of the study is to establish a robust, nationwide protocol for dd-cfDNA-based rejection monitoring in Swedish HTx recipients and to enhance our understanding of rejection mechanisms and immune activation.
Methods
This prospective, multicenter, investigator initiated study aims to enroll 120 adult HTx recipients, with follow-up conducted at various transplant centers. Enrollment commenced in February 2025 and is expected to conclude within two years. Blood samples for dd-cfDNA and donor-specific antibodies (DSAs) are collected in conjunction with routine EMB, but prior to biopsy collection. Sampling time points are scheduled at weeks 1, 2, 3, and 4; and months 2, 2.5, 3, 4, 5, 6, 8–9, 10–12, 16, 20, and 24 post-transplantation, depending on the local EMB protocol. Quantification of dd-cfDNA is performed using a next-generation sequencing (NGS)-based assay. The dd-cfDNA fraction is expressed as the percentage of dd-cfDNA relative to total circulating cfDNA. DSAs are measured locally at each participating center utilizing the Luminex xMAP platform. EMB specimens are graded for acute cellular rejection (ACR) and antibody-mediated rejection (AMR) according to International Society for Heart and Lung Transplantation (ISHLT) consensus criteria, and correlated with dd-cfDNA levels.
Results
As of January 1st 2026, 72 HTx recipients have been enrolled, with 436 samples collected to date. Comprehensive data, including demographic variables, laboratory results, and immunosuppressive regimens at each sampling point, have been recorded. An interim analysis is planned for May 2026 to provide preliminary findings from the study.
Conclusion
Effective national cooperation is fundamental for conducting national studies and for the development and establishment of new methods in the care of HTx patients. This study will define and validate a standardized, nationwide approach for dd-cfDNA-based rejection surveillance, facilitating early detection of rejection episodes and potentially minimizing the need for invasive procedures. The resulting dataset will also serve as a valuable resource for future research and underscores the importance of close collaboration between centers in smaller countries.Study designFor image description, please refer to the figure legend and surrounding text.