Circulating, Cell-Free, MicroRNA Sequencing to Diagnose Cardiac Allograft Rejection and Distinguish Rejection Subtype

Summary of Objectives Genomic biomarkers are used to non-invasively screen cardiac transplant recipients for allograft rejection. Gene expression profiling has no ability to diagnose antibody-mediated rejection (AMR) and has a poor positive predictive value for T-cell mediated rejection (TCMR). An emerging class of genomic biomarkers are circulating, cell-free microRNAs (cf-miR). MicroRNAs modulate gene expression and have been implicated in rejection, they are found in the circulation, and early studies, using targeted genomic approaches suggest that cf-miRs may be a helpful biomarker to detect rejection. We hypothesize that cf-miRs can non-invasively diagnose rejection and distinguish AMR from TCMR. Methods The Genomic Research Alliance for Transplantation (GRAfT) is a prospective, multicenter study enrolling transplant recipients since 2014. All patients with AMR or TCMR were included and a group of controls without rejection were selected based on age, sex and race. Each biopsy was reviewed by two blinded, independent, expert cardiac pathologists. Plasma aliquots will undergo small molecular RNA isolation and the circulating cf-miR transcriptome will be sequenced using a next-generation platform. We expect to generate ∼6 million, single-end, 50bp reads per sample. Sequence data will be annotated using the FASTX_Toolkit and DESeq2 will be used to identify differentially expressed cf-miRs. Machine learning algorithms will be used to refine the cf-miR panel and maximize the C-statistic. Endpoints The study includes 35 patients with allograft rejection and 70 controls (Table). For the TCMR cohort there were 27 episodes of grade ≥ 2R rejection. For AMR there are 11 episodes of ≥ pAMR2 and 16 episodes of pAMR1 h or i. In total ∼300 plasma samples will be sequenced. Our primary objective is to develop a cf-miR panel that diagnoses allograft rejections, distinguishes rejection subtypes and allows for a blood-based, biochemical assessment of rejection severity.