Cell-Free DNA and Active Rejection in Kidney Allografts

Abstract

Histologic analysis of the allograft biopsy specimen is the standard method used to differentiate rejection from other injury in kidney transplants. Donor-derived cell-free DNA (dd-cfDNA) is a noninvasive test of allograft injury that may enable more frequent, quantitative, and safer assessment of allograft rejection and injury status. To investigate this possibility, we prospectively collected blood specimens at scheduled intervals and at the time of clinically indicated biopsies. In 102 kidney recipients, we measured plasma levels of dd-cfDNA and correlated the levels with allograft rejection status ascertained by histology in 107 biopsy specimens. The dd-cfDNA level discriminated between biopsy specimens showing any rejection (T cell-mediated rejection or antibody-mediated rejection [ABMR]) and controls (no rejection histologically), P<0.001 (receiver operating characteristic area under the curve [AUC], 0.74; 95% confidence interval [95% CI], 0.61 to 0.86). Positive and negative predictive values for active rejection at a cutoff of 1.0% dd-cfDNA were 61% and 84%, respectively. The AUC for discriminating ABMR from samples without ABMR was 0.87 (95% CI, 0.75 to 0.97). Positive and negative predictive values for ABMR at a cutoff of 1.0% dd-cfDNA were 44% and 96%, respectively. Median dd-cfDNA was 2.9% (ABMR), 1.2% (T cell-mediated types ≥IB), 0.2% (T cell-mediated type IA), and 0.3% in controls (P=0.05 for T cell-mediated rejection types ≥IB versus controls). Thus, dd-cfDNA may be used to assess allograft rejection and injury; dd-cfDNA levels <1% reflect the absence of active rejection (T cell-mediated type ≥IB or ABMR) and levels >1% indicate a probability of active rejection.

Keywords: DART; biomarker; cell-free DNA; kidney; rejection; transplant.

Figure 1.

Banff elementary lesions and clinical features correlate with dd-cfDNA level. The 107 samples (27 patients with 27 samples with active rejection; 75 patients with 80 samples with no active rejection) are rank-ordered and color-coded by dd-cfDNA level. White indicates the element was not associated with that biopsy/visit. For each sample (x axis), associated elements (y axis) are shown as a colored box, by the level of dd-cfDNA associated with the sample; highest dd-cfDNA in red, lowest in blue, with a vertical dashed line at the 1% cutoff. The significance (P value) of association of dd-cfDNA >1% with each element is shown. BM, (glomerular) basement membrane; CNI, calcineurin inhibitor; DGF, delayed graft function; ENDATs, (gene expression profiles of) endothelial activation (and injury) transcripts; Inflam, inflammation; ptc, peritubular capillary.

Figure 2.

Patients, blood samples, and biopsies used in this study.

Figure 3.

dd-cfDNA discriminates active rejection. (A) Fraction of dd-cfDNA in active rejection (n=27) versus no active rejection (n=80). Box and whisker plots; horizontal line represents the median; bottom and top of each box represents 25th and 75th percentiles. Dots are individual results. Median dd-cfDNA in active rejection 1.6% versus 0.3% for no rejection (P<0.001). (B) Serum creatinine (milligrams per deciliter) in active rejection (n=27) versus no active rejection (n=80). Box and whisker plots; horizontal line represents the median; bottom and top of each box represents 25th and 75th percentiles. Dots are individual results. Serum creatinine was not significantly different in median values between two groups (P=0.23). (C) ROC curve for dd-cfDNA to discriminate active rejection. AUC=0.74 (95% CI, 0.61 to 0.86). (D) ROC curve for serum creatinine to discriminate active rejection. AUC=0.54 (95% CI, 0.43 to 0.66). (E) The sensitivity (%) and specificity (%) for dd-cfDNA to discriminate active rejection versus no active rejection status. (F) The PPV and NPV for dd-cfDNA for discriminating active rejection from no active rejection.

Figure 4.

dd-cfDNA levels are higher in ABMR than TCMR. (A) dd-cfDNA in 27 biopsy-based rejections: 10 chronic, active ABMR; six acute/active ABMR; 16 TCMR, types IA (6, ▲), IB (7, ▪), and IIA (3, ♦). Biopsy specimens diagnosed with AMBR and TCMR (mixed) are shown in the ABMR plots, with points colored to indicate the TCMR diagnosis also made on the same biopsy specimen. ABMR without TCMR is shown as a circle (●). Median dd-cfDNA 2.9% (ABMR). Median for TCMR-only, 1.2% (types ≥IB), 0.2% (TCMR type IA). (B) All data for samples classified as TCMR, including TCMR mixed with ABMR.

Figure 5.

dd-cfDNA discriminates ABMR. (A) Fraction of dd-cfDNA in ABMR (n=16) versus no ABMR (n=91). Box and whisker plots; horizontal line represents the median; bottom and top of each box represents 25th and 75th percentiles. Dots are individual results. Median dd-cfDNA in ABMR 2.9% versus 0.29% for no ABMR (P<0.001). (B) Serum creatinine (milligrams per deciliter) in ABMR (n=16) versus no ABMR (n=91). Serum creatinine was not significantly different in median values between two groups (P=0.41). (C) ROC curve for dd-cfDNA to discriminate active ABMR. AUC=0.87 (95% CI, 0.75 to 0.97). (D) ROC curve for serum creatinine to discriminate ABMR. AUC=0.57 (95% CI, 0.42 to 0.71). (E) The sensitivity (%) and specificity (%) for dd-cfDNA to discriminate active ABMR versus no active ABMR. (F) The PPV and NPV for dd-cfDNA to discriminate active ABMR from no active ABMR.

Figure 6.

dd-cfDNA levels in plasma from patients with active rejection are not correlated with other histopathological findings. ◊, chronic, active ABMR; △, acute/active ABMR; ◻, TCMR only.

Figure 7.

dd-cfDNA levels in plasma from patients without active rejection are not correlated with other histopathological findings. Each circle represents a biopsy specimen. BK, BK virus; Tx Glom, transplant glomerulopathy.