Donor-Derived Cell-Free DNA as a Non-Invasive Readout of Activity Across the Rejection Continuum

Recent work demonstrated that kidney allograft rejection unfolds along a biological continuum that can be quantified using histopathology-derived continuous indices. To investigate whether donor-derived cell-free DNA (dd-cfDNA) reflects this rejection continuum and complements these histopathology-derived indices, we analyzed the association between dd-cfDNA measures and the newly developed rejection indices in 249 indication biopsies from two independent cohorts. dd-cfDNA was analyzed as percentage, absolute copies/mL, and as a previously developed combined continuous model (CM) score integrating both measures to mitigate limitations of relative measurements. dd-cfDNA increased with histopathological activity and was highest in biopsies with microvascular inflammation (MVI), including antibody-mediated (AMR) and mixed rejection, paralleling high AMR/MVI and activity indices. T-cell-mediated rejection (TCMR) showed elevated TCMR/tubulointerstitial inflammation (TI) indices but lower and more variable dd-cfDNA, accompanied by increased total cfDNA, providing a plausible explanation for the reduced detectability of low-grade TCMR when dd-cfDNA is expressed as a percentage alone. Interclass correlation analyses revealed the strongest associations between dd-cfDNA and the AMR/MVI and activity indices. The combined CM score achieved the highest overall associations (sum R 2 = 3.4), outperforming absolute and relative dd-cfDNA measures. Thus, dd-cfDNA may serve as a non-invasive readout of graft inflammation and extends the rejection-continuum concept into the non-invasive space.