The Trifecta Study: Comparing Plasma Levels of Donor-derived Cell-Free DNA with the Molecular Phenotype of Kidney Transplant Biopsies

Abstract

Background: The relationship between the donor-derived cell-free DNA fraction (dd-cfDNA[%]) in plasma in kidney transplant recipients at time of indication biopsy and gene expression in the biopsied allograft has not been defined.

Methods: In the prospective, multicenter Trifecta study, we collected tissue from 300 biopsies from 289 kidney transplant recipients to compare genome-wide gene expression in biopsies with dd-cfDNA(%) in corresponding plasma samples drawn just before biopsy. Rejection was assessed with the microarray-based Molecular Microscope Diagnostic System using automatically assigned rejection archetypes and molecular report sign-outs, and histology assessments that followed Banff guidelines.

Results: The median time of biopsy post-transplantation was 455 days (5 days to 32 years), with a case mix similar to that of previous studies: 180 (60%) no rejection, 89 (30%) antibody-mediated rejection (ABMR), and 31 (10%) T cell-mediated rejection (TCMR) and mixed. In genome-wide mRNA measurements, all 20 top probe sets correlating with dd-cfDNA(%) were previously annotated for association with ABMR and all types of rejection, either natural killer (NK) cell-expressed (e.g., GNLY, CCL4, TRDC, and S1PR5) or IFN-γ-inducible (e.g., PLA1A, IDO1, CXCL11, and WARS). Among gene set and classifier scores, dd-cfDNA(%) correlated very strongly with ABMR and all types of rejection, reasonably strongly with active TCMR, and weakly with inactive TCMR, kidney injury, and atrophy fibrosis. Active ABMR, mixed, and active TCMR had the highest dd-cfDNA(%), whereas dd-cfDNA(%) was lower in late-stage ABMR and less-active TCMR. By multivariate random forests and logistic regression, molecular rejection variables predicted dd-cfDNA(%) better than histologic variables.

Conclusions: The dd-cfDNA(%) at time of indication biopsy strongly correlates with active molecular rejection and has the potential to reduce unnecessary biopsies.

Clinical trial registration number: NCT04239703.

Keywords: biopsy; blood donors; cell-free DNA; genomics; microarrays; phenotype; rejection; transplantation.

Figure 1.

Study design flowchart. Workflow for collection and processing of the 300 biopsies for molecular biopsy assessment and concurrent blood samples processed for percent donor-derived cell-free DNA (%dd-cfDNA) in the Trifecta Study.

Figure 2.

Relationships between %dd-cfDNA, time of biopsy post-transplant, and molecular archetype groups in N=300 samples. Dots represent biopsies and corresponding paired blood sample %dd-cfDNA results, colored by archetype cluster assignments. Relationships between %dd-cfDNA and time post-transplant are represented by linear regression (thick dashed black line), restricted cubic spline (with three knots - blue line) and a supersmoother (red line). Overall, %dd-cfDNA increases slightly over time post-transplant (linear regression P=0.06). %dd-cfDNA ≥ 1 is used as the cutoff throughout this paper for positivity. NR – no rejection.

Figure 3.

Relationships between %dd-cfDNA, molecular archetype groups, and the ABMR_Prob and TCMR_Prob classifier scores in N=300 samples. Dots represent biopsies and corresponding paired blood sample %dd-cfDNA results, colored by archetype cluster assignments. Regression lines (dashed) show the relationship between the (A) ABMR_Prob and (B) TCMR_Prob classifier scores and %dd-cfDNA. Spearman correlations with dd-cfDNA were stronger for ABMR_Prob (0.52, P=6E-22) than TCMR_Prob (0.22, P=9E-5). NR, no rejection.

Figure 4.

Rejection classifier PCA factor maps in N=300 samples, showing %dd-cfDNA as a supplementary variable. (A) PC2 versus PC1. (B) PC2 versus PC3. Input variables (classifier scores) are shown as solid black lines, and %dd-cfDNA in dashed grey as it is not included as input in the PCA. %dd-cfDNA behaves like a molecular rejection measurement, more closely aligned to ABMR than to TCMR.

Figure 5.

Beeswarm/boxplots showing %dd-cfDNA measurements versus molecular rejection archetype groups in N=300 samples. Dots represent biopsies and corresponding paired blood sample %dd-cfDNA results.

Figure 6.

Random forests demonstrating predictive ability of various molecular and histologic features of rejection for %dd-cfDNA ≥1.0 in N=300. (A) Relative variable importance using molecular predictors. (B) Relative variable importance using histologic predictors. In both figures, ABMR and overall rejection related variables are the most important predictors of high %dd-cfDNA.