The Trifecta Study: Comparing Plasma Levels of Donor-derived Cell-Free DNA with the Molecular Phenotype of Kidney Transplant Biopsies
Authors: Halloran Philip F. , Reeve Jeff , Madill-Thomsen Katelynn S. , Demko Zachary , Prewett Adam , Billings Paul ,   the Trifecta Investigators
Published: January 2022

Significance Statement
Plasma donor-derived cell-free DNA (cfDNA) measured as a percent of total cfDNA (dd-cfDNA[%]) has been proposed as a screening test for kidney transplant rejection. The prospective Trifecta study examined relationships between dd-cfDNA(%) measured at the time of indication biopsy and the genome-wide molecular findings in 300 biopsies from kidney transplant recipients assessed by microarrays. The dd-cfDNA(%) correlated with active rejection in the biopsy, and molecular scores predicted dd-cfDNA(%) ≥1.0% better than histologic scores. The top transcripts in the biopsy correlating with dd-cfDNA(%) were related to IFN-γ effects and natural killer cells. AKI and atrophy fibrosis were associated with mildly elevated dd-cfDNA(%), although some biopsies with high dd-cfDNA(%) revealed no rejection. These findings indicate that plasma dd-cfDNA levels are strongly related to the active molecular rejection processes in indication biopsies.

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