Absolute quantification of donor-derived cell-free DNA as a marker of rejection and graft injury in kidney transplantation: Results from a prospective observational study

Abstract

Donor-derived cell-free DNA (dd-cfDNA) is a noninvasive biomarker for comprehensive monitoring of allograft injury and rejection in kidney transplantation (KTx). dd-cfDNA quantification of copies/mL plasma (dd-cfDNA[cp/mL]) was compared to dd-cfDNA fraction (dd-cfDNA[%]) at prespecified visits in 189 patients over 1 year post KTx. In patients (N = 15, n = 22 samples) with biopsy-proven rejection (BPR), median dd-cfDNA(cp/mL) was 3.3-fold and median dd-cfDNA(%) 2.0-fold higher (82 cp/mL; 0.57%, respectively) than medians in Stable Phase patients (N = 83, n = 408) without rejection (25 cp/mL; 0.29%). Results for acute tubular necrosis (ATN) were not significantly different from those with biopsy-proven rejection (BPR). dd-cfDNA identified unnecessary biopsies triggered by a rise in plasma creatinine. Receiver operating characteristic (ROC) analysis showed superior performance (P = .02) of measuring dd-cfDNA(cp/mL) (AUC = 0.83) compared to dd-cfDNA(%) (area under the curve [AUC] = 0.73). Diagnostic odds ratios were 7.31 for dd-cfDNA(cp/mL), and 6.02 for dd-cfDNA(%) at thresholds of 52 cp/mL and 0.43%, respectively. Plasma creatinine showed a low correlation (r = 0.37) with dd-cfDNA(cp/mL). In a patient subset (N = 24) there was a significantly higher rate of patients with elevated dd-cfDNA(cp/mL) with lower tacrolimus levels (<8 μg/L) compared to the group with higher tacrolimus concentrations (P = .0036) suggesting that dd-cfDNA may detect inadequate immunosuppression resulting in subclinical graft damage. Absolute dd-cfDNA(cp/mL) allowed for better discrimination than dd-cfDNA(%) of KTx patients with BPR and is useful to avoid unnecessary biopsies.

Keywords: biomarker; clinical decision-making; clinical research/practice; immunosuppressant; immunosuppression/immune modulation; kidney failure/injury; kidney transplantation/nephrology; rejection.

Figure 1

Selection of patients with samples included in statistical analyses based on compliance with predetermined inclusion criteria. KTx, kidney transplant

Figure 2

Time course in plasma dd‐cfDNA(cp/mL) (A) and dd‐cfDNA(%) (B) during the first year after KTx in patients' samples of Non‐rejecting Phase. Boxes represent median with interquartile range, with whiskers showing the 5th‐95th percentile. Predetermined visits and number of samples (n) are given below each time point. Values for outliers are shown as numbers (either as cp/mL or %). dd‐cfDNA, donor‐derived cell‐free DNA; KTx, kidney transplant

Figure 3

Comparison of post KTx dd‐cfDNA(cp/mL) (A) and dd‐cfDNA(%) (B) data from samples beginning at day 5 post‐KTx. ATN, acute tubular necrosis; BPR, biopsy‐proven rejection; dd‐cfDNA, donor‐derived cell‐free DNA; KTx, kidney transplant; TCMR, T cell‐mediated rejection

Figure 4

ROC curves for dd‐cfDNA(cp/mL) (A) and dd‐cfDNA(%) (B) showing the superior performance (P = .02) of measuring the absolute amount (cp/mL) of dd‐cfDNA rather than the dd‐cfDNA fraction(%). Outliers in the Stable Phase group were excluded from analysis. AUC, area under the curve; BPR, biopsy‐proven rejection; CI, confidence interval; dd‐cfDNA, donor‐derived cell‐free DNA; ROC, receiver operating characteristic

Figure 5

Same‐day plasma creatinine, dd‐cfDNA(%) and dd‐cfDNA(cp/mL) results in samples drawn during the period from 6 days before to 6 days after the biopsy, but excluding the day of the biopsy, in seven patients who had normal biopsy findings. Shaded areas show values below the upper limit of the plasma creatinine reference interval and thresholds for dd‐cfDNA. dd‐cfDNA, donor‐derived cell‐free DNA

Figure 6

Association between low predose tacrolimus concentrations and increased dd‐cfDNA(cp/mL) in patients (N = 24) with a change of tacrolimus concentration >60% in samples (n = 75) collected at three consecutive visits. Lines represent the cut‐off points for Fisher test (Tacrolimus: 8 μg/L; dd‐cfDNA: 50 cp/mL). BPR, biopsy‐proven rejection; dd‐cfDNA, donor‐derived cell‐free DNA