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. 2023 Feb;165(2):460-468.e2.
doi: 10.1016/j.jtcvs.2022.04.027. Epub 2022 Apr 30.

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

Marc E Richmond  1 , Shriprasad R Deshpande  2 , Steven D Zangwill  3 , David P Bichell  4 , Steven J Kindel  5 , William T Mahle  6 , Jacob N Schroder  7 , Mark A Wigger  8 , Kenneth R Knecht  9 , Elfriede Pahl  10 , Nunzio A Gaglianello  11 , Mary A Goetsch  12 , Pippa Simpson  13 , Mahua Dasgupta  13 , Liyun Zhang  13 , Paula E North  14 , Aoy Tomita-Mitchell  15 , Michael E Mitchell  15
Affiliations

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

Marc E Richmond et al. J Thorac Cardiovasc Surg. 2023 Feb.

Abstract

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.

Keywords: cell-free DNA; endomyocardial biopsy; heart transplantation; pediatric heart transplantation; rejection.

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Figures

Figure 1.
Figure 1.
CONSORT Flow Diagram illustrating exclusion of samples according to predefined clinical and samples-based criteria, resulting in final cohort of 745 biopsy associated DF cfDNA samples from 130 participants. DF cfDNA=Donor fraction cell-free DNA
Figure 2.
Figure 2.
Box-plot showing the relationship between log DF cfDNA and acute allograft rejection (as defined by ACR ≥2R and/or pAMR ≥2) in all participants (a), adults (b) aad children (c). Median DF cfDNA was significantly higher in samples associated with acute allograft rejection as compared to healthy allografts. The upper and lower borders of the box represent the upper and lower quartiles, respectively. The horizontal line represents the median value. The upper and lower whiskers represent the maximum and minimum values of the nonoutliers, respectively. DF cfDNA=Donor Fraction Cell-Free DNA, ACR=Acute Cellular Rejection, pAMR=Antibody Mediated Rejection
Figure 3.
Figure 3.
Receiver operating characteristic curves for detection of acute allograft rejection (as defined by ACR ≥2R and/or pAMR ≥2) using a DF cfDNA threshold of 0.14% in all participants (a), adults (b), and children (c). DF cfDNA=Donor Fraction Cell-Free DNA, ACR=Acute Cellular Rejection, pAMR=Antibody Mediated Rejection, AUC=area under the curve, PPV=positive predictive value, NPV=negative predictive value
Figure 4.
Figure 4.
Box-plot showing relationship between log DF cfDNA and acute cellular rejection (ACR ≥2R) in all participants (a), adults (b), and children (c). Median DF cfDNA was significantly higher in samples associated with acute cellular rejection as compared to healthy allografts. The upper and lower borders of the box represent the upper and lower quartiles, respectively. The horizontal line represents the median value. The upper and lower whiskers represent the maximum and minimum values of the nonoutliers, respectively DF cfDNA=Donor Fraction Cell-Free DNA, ACR=Acute Cellular Rejection
Figure 5.
Figure 5.
Box-plot showing relationship between log DF cfDNA and Antibody Mediated Rejection (pAMR ≥2) in all participants (a), adults (b), and children (c). Median DF cfDNA was significantly higher in samples associated with biopsy proven antibody mediated rejection as compared to healthy allografts. The upper and lower borders of the box represent the upper and lower quartiles, respectively. The horizontal line represents the median value. The upper and lower whiskers represent the maximum and minimum values of the nonoutliers, respectively DF cfDNA=Donor Fraction Cell-Free DNA, pAMR=Antibody Mediated Rejection
Figure 6.
Figure 6.
Summary of the validation of donor fraction cell-free DNA to detect acute allograft rejection. The upper and lower borders of the box represent the upper and lower quartiles, respectively. The horizontal line represents the median value. The upper and lower whiskers represent the maximum and minimum values of the nonoutliers.
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References

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