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. 2025 Sep;25(9):1895-1905.
doi: 10.1016/j.ajt.2025.04.021. Epub 2025 May 5.

A two-threshold algorithm using donor-derived cell-free DNA fraction and quantity to detect acute rejection after heart transplantation

Paul J Kim  1 , Michael Olympios  2 , Konstantinos Sideris  3 , Eleni Tseliou  3 , Taylor Y Tran  1 , Spencer Carter  3 , Alison Brann  3 , Navchetan Kaur  2 , Sandra A Carey  2 , Sangeeta Bhorade  2 , Yen-An Chen  2 , David Barnes  2 , Ebad Ahmed  2 , Jing Xie  2 , Adam Prewett  2 , Matthew Rabinowitz  2 , Bernhard G Zimmermann  2 , Michelle S Bloom  2 , Zachary Demko  2 , Eric Adler  1 , Josef Stehlik  4
Affiliations

A two-threshold algorithm using donor-derived cell-free DNA fraction and quantity to detect acute rejection after heart transplantation

Paul J Kim et al. Am J Transplant. 2025 Sep.

Abstract

Donor-derived cell-free DNA (dd-cfDNA) is a promising biomarker of acute rejection (AR) after heart transplantation (HTx). dd-cfDNA, measured as a fraction of total cfDNA, can be affected by changes in total cfDNA whereas dd-cfDNA quantity can mitigate this impact. This study investigated the performance of a 2-threshold algorithm (2TA) that combines dd-cfDNA fraction (dd-cfDNA%) and donor-quantity score (DQS). A total of 808 plasma samples were prospectively collected for dd-cfDNA testing from 187 adult HTx patients with contemporaneous endomyocardial biopsies. cfDNA was analyzed by a single nucleotide polymorphism-based next-generation sequencing workflow; dd-cfDNA% and DQS were measured using the sequencing reads and single nucleotide polymorphism genotypes. Both dd-cfDNA% and DQS were significantly higher in AR than in non-AR samples (P < 10-14). Considering samples exceeding either dd-cfDNA% = 0.26% or DQS = 18 copies/mL as positive, the 2TA demonstrated 86.5% sensitivity and 83.6% specificity for AR detection and an area under the curve of 0.881. Compared to dd-cfdNA% alone, performance improved with a mean net reclassification index of 16.4% (standard deviation: 4.0%; P = .015) and a 37.3% reduction in the number of the false positive cases compared to the previously established cutoff of 0.15%. Combining dd-cfDNA fraction and quantity estimate in a 2TA may improve AR detection accuracy in HTx recipients compared with dd-cfDNA% alone.

Keywords: acute rejection; donor-derived cell-free DNA; heart transplant.

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Conflict of interest statement

Declaration of competing interest The authors of this manuscript have conflicts of interest to disclose as described by American Journal of Transplantation. PK has received grant funding from Natera, Inc and CareDx, and has participated in advisory boards for Natera, Inc. SpC has received consulting fees from Pfizer and BridgeBio. MO, NK, SAC, SB, Y-AC, DB, EbA, JX, AP, BZ, MSB are employees of Natera, Inc, and receive salary and may own stock and/or stock options. ErA has received royalties or licenses from Lexeo Therapeutics, Rocket Pharmaceuticals, is an employee of Lexeo Therapeutics and may own stock and/or stock options in Lexeo Therapeutics, Rocket Pharmaceuticals, Corstasis Therapeutics, Papillion Therapeutics. MR holds leadership roles on the boards Natera, Myome, Marble Therapeutics and reports payments and/or stocks from Natera, Myome, Marble Therapeutics for employment, grants/contracts, and participation in advisory boards. JS reports grant funding from Natera Inc and Merck; consulting fees from Natera, TransMedics, NovoNordisk, Medtronic; and leadership roles in International Society of Heart and Lung Transplant and the Heart Failure Society of America. AB, KS, ET, and TT declare no conflicts of interest.

Figures

Figure 1.
Figure 1.
Overview of dd-cfDNA% and DQS and study cohort. (A) dd-cfDNA% represents the proportion of DQS divided by the total quantity of cfDNA. (B) Flow diagram for the study. ACR, acute cellular rejection; AMR, antibody-mediated rejection; dd-cfDNA, donor-derived cell-free DNA; DQS, donor-quantity score.
Figure 2.
Figure 2.
dd-cfDNA% and DQS in AR and non-AR samples. (A) dd-cfDNA% (%) and (B) DQS (copies/mL) in rejection vs non-rejection samples. Acute rejection samples (biopsy-proven) showed significantly increased dd-cfDNA% and DQS compared to nonrejection samples. The horizontal notched line represents the median value, and the top and bottom of each box show upper and lower limit of the IQR, and the whiskers represent the range. AR, acute rejection; dd-cfDNA, donor-derived cell-free DNA; DQS, donor-quantity score; interquartile range.
Figure 3.
Figure 3.
dd-cfDNA levels in patients stratified by biopsy diagnosis. (A) Both ACR and AMR show significantly increased dd-cfDNA% compared to nonrejection samples (left). ACR 1R and 2R show significantly increased dd-cfDNA% compared to non-rejection samples (right). AMR shows a trend for further increase in dd-cfDNA% compared to ACR. (B) Both ACR and AMR show significantly increased DQS compared to nonrejection samples. DQS shows a similar increase in values compared to dd-cfDNA% across different acute rejection grades (right). ACR, acute cellular rejection; AMR, antibody-mediated rejection; dd-cfDNA, donor-derived cell-free DNA; DQS, donor-quantity score.
Figure 4.
Figure 4.
Performance of a 2-threshold algorithm. (A) Scatterplot of dd-cfDNA test samples; grey vertical line indicates the 0.15% threshold; green vertical line indicates 2TA threshold of 0.26%; green horizontal line indicates 2TA threshold of 18 cp/mL. (B) ROC of dd-cfDNA (%) and 2TA, AUC with bootstrapping. 2TA demonstrated improved testing performance compared to dd-cfDNA% alone. 2TA, 2-threshold algorithm; ACR, acute cellular rejection; AMR, antibody-mediated rejection; AUC, area under the curve; dd-cfDNA, donor-derived cell-free DNA; ROC, receiver operating characteristic.
Figure 5.
Figure 5.
LOWESS smoothed trajectories of (A) dd-cfDNA%, (B) DQS, (C) total cfDNA over time. dd-cfDNA% (A) shows an increasing trend over time after 7 months posttransplant, DQS (B) remains relatively unchanged, and total cfDNA (C) shows a decreasing trend after 7 months posttransplant. dd-cfDNA, donor-derived cell-free DNA; DQS, donor-quantity score; LOWESS, locally weighted scatterplot smoothing.
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