Background: Donor-derived cell-free DNA (dd-cfDNA) is a valuable tool for detecting kidney transplant rejection, but its role in monitoring response to rejection therapy is less understood. Subclinical rejection, identified without creatinine changes, presents challenges in assessing treatment response without repeat biopsies. This study evaluates whether dd-cfDNA levels decrease one month after rejection therapy, potentially serving as a non-invasive monitoring method.
Methods: Retrospective chart review of patients with biopsy-confirmed rejection episode(s) followed at a single pediatric kidney transplant(pKTx) program from 1/2021-12/2023. Rejection was defined as any of these: T-cell mediated rejection (TCMR), borderline TCMR(bTCMR), antibody-mediated rejection (ABMR), or mixed rejection (ABMR/TCMR). All patients received appropriate treatment for rejection as per our Center’s protocols. Dd-cfDNA and serum creatinine were collected before the rejection episode and one month after treatment for rejection. Each rejection episode was analyzed separately for patients with more than one rejection episode. Descriptive statistics were applied. 
Results: From 35 pKTx patients followed during the study period, 10 (29%) were diagnosed with rejection, and three patients had two rejection episodes. 10 of 13 rejections (85%) were subclinically diagnosed after being found with dd-cfDNA >1%, and 3 were diagnosed after a for-cause kidney biopsy (2 due to increased creatinine and one due to proteinuria). Kidney biopsies revealed 3 patients with mixed rejection, 5 with TCMR, and 5 with bTCMR. For 10/13 rejection episodes, ddcfDNA decreased one month after treatment from a median dd-cfDNA of 1.6% (interquartile range [IQR] 1-1.9%) to a median dd-cfDNA of 0.7% (IQR 0.5-0.9%). A decrease in creatinine was observed in 2/13 rejection episodes (those with increased creatinine prebiopsy), while no change in serum creatinine was observed from pre-biopsy levels in all other rejection episodes. dd-cfDNA did not decrease in the 3 patients with mixed rejection one month after treatment. At one year after treatment, dd-cfDNA in these 3 patients decreased by 50% (median 3.8% to 1.6%).
Conclusions: Donor-derived cell-free DNA seems useful for monitoring response after treating biopsy-proven kidney rejection in patients with bTCMR/TCMR, as evidenced by levels decreasing below 1% as early as one month after rejection therapy.  However, a slower pattern was observed in patients treated for mixed rejection, and dd-cfDNA levels remained above 1%, correlating with more severe tissue injury and/or ongoing injury. This non-invasive method of monitoring rejection may allow for more tailored and timely adjustments to treatment without the need for repeated kidney biopsies.