Introduction: Acute rejection (AR) is a major contributor to graft failure following pediatric heart transplant (HTx), but timely detection and treatment of rejection episodes remain a significant clinical challenge.  Initial studies suggest donor-derived cell-free DNA (dd-cfDNA) can sufficiently discriminate AR from no rejection (NR) after pediatric HTx; however, most participants in these studies were older pediatric patients.  To date, no studies have assessed whether dd-cfDNA can successfully identify AR in different pediatric HTx age ranges.  The goal of this analysis was to determine whether dd-cfDNA levels are elevated during AR in different age groups following pediatric HTx.
Methods: This 4-center analysis included single-organ pediatric HT recipients <15 years old at the time of dd-cfDNA testing with ≥1 paired dd-cfDNA and endomyocardial biopsy (EMB) performed on the same day. A second analysis using a dd-cfDNA window of 0-14 days before EMB was also performed. AR was defined as biopsy-proven acute cellular rejection (ACR) grade ≥2R and/or antibody mediated rejection (AMR) grade ≥pAMR1. NR was defined as an ACR grade <2R and pAMR0 on EMB.  dd-cfDNA levels were assigned to one of four groups based on the patient’s age at time of blood draw: 0- to <3-years-old, 3- to <6-years-old, 6- to <11-years-old, 11- to <15-years-old.  A mixed model analysis was conducted to evaluate the interaction between rejection status and age at dd-cfDNA draw on dd-cfDNA test results.
Results: This analysis included 126 HT recipients <15 years old. Amongst patients with complete demographic data, the cohort was 61% white and 50% male, with a median age at HT of 3 (IQR 1-9) years. There were 207 dd-cfDNA paired on the same day as EMB, of which 175 (85%) across 121 patients showed NR and 32 (15%) from 21 patients had AR. AMR-alone accounted for 81% (n=26) of AR, with ACR-alone representing 16% (n=5) of AR cases. One (3%) EMB had both ACR and AMR. The median time post-HT of dd-cfDNA for AR and NR were 3.2 and 1.8 years, respectively. Median same day dd-cfDNA levels were significantly higher during AR than NR in all patients (1.55% vs 0.16%, p<0.001). dd-cfDNA levels were numerically higher during AR when compared to NR samples for all age groups when including dd-cfDNA and EMBs performed on the same day (Figure 1) or dd-cfDNA levels drawn 0-14 days prior to EMB (Figure 2).  There was no significant interaction between dd-cfDNA levels in NR vs AR and age (p=0.30). dd-cfDNA levels associated with NR were higher in patients 0 to<3-years-old compared to other age groups.
Conclusions: dd-cfDNA was consistently elevated in AR compared to NR in all pediatric age groups following HTx.  Further research is needed to understand differences in dd-cfDNA levels between pediatric patients and levels seen in adult studies.