The Trifecta study: comparing plasma donor-derived cell-free DNA levels with the molecular phenotype of kidney transplant biopsies

Background. The Trifecta study (ClinicalTrials.gov #NCT04239703) is a prospective investigator-initiated trial to evaluate the relationship between plasma %dd-cfDNA at the time of indication biopsy and the molecular phenotype of the biopsy. Results. Median time of biopsy post-transplant was 455 days (5 days - 32 years), with case-mix similar to 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 probesets correlating with %dd-cfDNA were previously annotated for association with ABMR and all rejection, either NK cell-expressed (e.g. GNLY, CCL4, TRDC, and S1PR5) or IFNG-inducible (e.g. PLA1A, IDO1, CXCL11, and WARS). Among gene set and classifier scores, %dd-cfDNA correlated very strongly with ABMR and all rejection, reasonably strongly with active TCMR, and weakly with inactive TCMR, kidney injury and atrophy-fibrosis. %dd-cfDNA was highest in active ABMR, mixed, and active TCMR but 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. %dd-cfDNA at time of indication biopsy strongly correlates with active molecular rejection and has potential to reduce unnecessary biopsies. Methods. We compared genome-wide gene expression (microarrays) in 300 biopsies to plasma %dd-cfDNA drawn at time of biopsy, from 19 centers in Europe and North America. We assessed rejection using the microarray-based Molecular Microscope® Diagnostic System (MMDx) using automatically assigned rejection archetypes (AA) and molecular report signouts, plus histology assessments by the center following current Banff guidelines.