A novel INDEL-based next-generation sequencing assay for monitoring donor-derived cell-free DNA in renal transplant recipients-from bedside to results: a UK pilot study.

Background: Monitoring donor-derived cell-free DNA (dd-cfDNA) is a promising noninvasive method for assessing allograft health in renal transplant recipients. This UK pilot study evaluated a novel insertion and deletion (INDEL)-based next-generation sequencing (NGS) assay for detecting dd-cfDNA and explored its association with potentially injurious concomitant pathologies, including donor-specific antibodies. Current methods are limited to first and only transplant recipients, as other assays cannot distinguish graft injury in the context of transplantation from multiple donors. Methods: Fourteen high-risk renal transplant recipients (level IV human leukocyte antigen mismatch, calculated reaction frequency >20%, retransplant) were recruited between October 2023 and July 2024 at Liverpool University Hospitals. Plasma samples were collected 6 months posttransplant, and cfDNA was extracted using QIAsymphony DSP Circulating DNA Kit (Qiagen). dd-cfDNA was quantified using the Devyser Accept cfDNA assay (Devyser), and NGS was performed using MiSeq (Illumina). Results: We present preliminary observations from the first 14 patients included in this proof-of-concept arm of the study. A dd-cfDNA level ≤0.5% correlated with stable graft function (n=11). Patients with dd-cfDNA ≥1.0% had supratherapeutic tacrolimus levels (n=2). Intermediate dd-cfDNA levels (0.5%-1.0%) were found in the setting of Conclusions: The INDEL-based NGS assay is a promising novel tool for detecting and monitoring dd-cfDNA in renal transplant recipients with an easy-to-implement workflow. These preliminary results support its clinical utility in a high-immunological-risk setting. These findings are consistent with emergent literature; however, longitudinal data and further validation in a larger cohort of patients are required.