Validation of donor fraction cell-free DNA with biopsy-proven cardiac allograft rejection in children and adults.

OBJECTIVES: Donor-specific cell-free DNA shows promise as a noninvasive marker for allograft rejection, but as yet has not been validated in both adult and pediatric recipients. The study objective was to validate donor fraction cell-free DNA as a noninvasive test to assess for risk of acute cellular rejection and antibody-mediated rejection after heart transplantation in pediatric and adult recipients. METHODS: Pediatric and adult heart transplant recipients were enrolled from 7 participating sites and followed for 12 months or more with plasma samples collected immediately before all endomyocardial biopsies. Donor fraction cell-free DNA was extracted, and quantitative genotyping was performed. Blinded donor fraction cell-free DNA and clinical data were analyzed and compared with a previously determined threshold of 0.14%. Sensitivity, specificity, negative predictive value, positive predictive value, and receiver operating characteristic curves were calculated. RESULTS: A total of 987 samples from 144 subjects were collected. After applying predefined clinical and technical exclusions, 745 samples from 130 subjects produced 54 rejection samples associated with the composite outcome of acute cellular rejection grade 2R or greater and pathologic antibody-mediated rejection 2 or greater and 323 healthy samples. For all participants, donor fraction cell-free DNA at a threshold of 0.14% had a sensitivity of 67%, a specificity of 79%, a positive predictive value of 34%, and a negative predictive value of 94% with an area under the curve of 0.78 for detecting rejection. When analyzed independently, these results held true for both pediatric and adult cohorts at the same threshold of 0.14% (negative predictive value 92% and 95%, respectively). CONCLUSIONS: Donor fraction cell-free DNA at a threshold of 0.14% can be used to assess for risk of rejection after heart transplantation in both pediatric and adult patients with excellent negative predictive value.

Relationship between donor fraction cell-free DNA and clinical rejection in heart transplantation.

BACKGROUND: Clinical rejection (CR) defined as decision to treat clinically suspected rejection with change in immunotherapy based on clinical presentation with or without diagnostic biopsy findings is an important part of care in heart transplantation. We sought to assess the utility of donor fraction cell-free DNA (DF cfDNA) in CR and the utility of serial DF cfDNA in CR patients in predicting outcomes of clinical interest. METHODS: Patients with heart transplantation were enrolled in two sequential, multi-center, prospective observational studies. Blood samples were collected for surveillance or clinical events. Clinicians were blinded to the results of DF cfDNA. RESULTS: A total of 835 samples from 269 subjects (57% pediatric) were included for this analysis, including 28 samples associated with CR were analyzed. Median DF cfDNA was 0.43 (IQR 0.15, 1.36)% for CR and 0.10 (IQR 0.07, 0.16)% for healthy controls (p < .0001). At cutoff value of 0.13%, the area under curve (AUC) was 0.82, sensitivity of 0.86, specificity of 0.67, and negative predictive value of 0.99. There was serial decline in DF cfDNA post-therapy, however, those with cardiovascular events (cardiac arrest, need for mechanical support or death) showed significantly higher levels of DF cfDNA on Day 0 (2.11 vs 0.31%) and Day 14 (0.51 vs 0.22%) compared to those who did not have such an event (p < .0001). CONCLUSION: DF cfDNA has excellent agreement with clinical rejection and, importantly, serial measurement of DF cfDNA predict clinically significant outcomes post treatment for rejection in these patients.

Increase in nuclear cell-free DNA is associated with major adverse events in adult and pediatric heart transplant recipients.

BACKGROUND: Cell-free DNA is an emerging biomarker. While donor fraction may detect graft events in heart transplant recipients, the prognostic value of total nuclear cell-free DNA (ncfDNA) itself is largely unexplored. OBJECTIVE: Explore the relationship between ncfDNA and clinical events in heart transplant recipients. METHODS: We conducted a multi-center prospective study to investigate the value of cell-free DNA in non-invasive monitoring following heart transplantation. Over 4000 blood samples were collected from 388 heart transplant patients. Total ncfDNA and donor fraction were quantified. Generalized linear models with maximum likelihood estimation for repeated measures with subjects as clusters were used to explore the relationship of ncfDNA and major adverse events. Receiver operating characteristic curves were used to help choose cutpoints. RESULTS: A ncfDNA threshold (50 ng/ml) was identified that was associated with increased risk of major adverse events. NcfDNA was elevated in patients who suffered cardiac arrest, required mechanical circulatory support or died post heart transplantation as well as in patients undergoing treatment for infection. CONCLUSIONS: Elevated ncfDNA correlates with risk for major adverse events in adult and pediatric heart transplant recipients and may indicate a need for enhanced surveillance after transplant.

Donor fraction cell-free DNA and rejection in adult and pediatric heart transplantation.

BACKGROUND: Endomyocardial biopsy (EMB) is the current standard for rejection surveillance in heart transplant recipients. The quantification of donor-specific cell-free DNA (cfDNA) may be an appropriate biomarker for non-invasive rejection surveillance. A multicenter prospective blinded study (DNA-Based Transplant Rejection Test, DTRT) investigated the value of donor fraction (DF), defined as the ratio of cfDNA specific to the transplanted organ to the total amount of cfDNA present in a blood sample. METHODS: A total of 241 heart transplant patients were recruited from 7 centers. Age at transplant ranged from 8 days to 73 years, with 146 subjects <18 years and 95 ≥18 years. All the patients were followed for at least 1 year, with blood samples drawn at routine and for-cause biopsies. A total of 624 biopsy-paired samples were included for analysis through a commercially available cfDNA assay (myTAIHEART, TAI Diagnostics Inc.). A blinded analysis of repeated measures compared the outcomes using receiver operating characteristic (ROC) curves. All primary clinical end-points were monitored at 100%. All analysis and conclusions were reviewed by both an independent external oversight committee and the National Institutes of Health-mandated DTRT steering committee. RESULTS: DF in acute cellular rejection (ACR) 1R/2R (n = 15) was higher than ACR 0R (n = 42) (p = 0.02); DF in antibody-mediated rejection pAMR1 (n = 8) and pAMR2 (n = 12) (p = 0.05) were higher than pAMR0 (n = 466) (p = 0.04 and p = 0.05 respectively). An optimal DF threshold was determined by the use of an ROC analysis, which ruled out the presence of either ACR or antibody-mediated rejection. CONCLUSIONS: The cell-free DNA DF holds promise as a non-invasive diagnostic test to rule out acute rejection in both adult and pediatric heart transplant populations.