Beyond Donor-Specific Antibodies: Molecular Characterization of Antibody-Mediated Rejection Using Tissue-Based Transcriptomics in Heart Transplantation.

BACKGROUND: Antibody-mediated rejection (AMR) contributes to graft dysfunction after heart transplantation, yet histopathology and circulating donor-specific antibodies (DSA) may incompletely capture intragraft biology. We characterized molecular ABMR by HLA-DSA status and, within DSA-negative cases, non-HLA antibody status. METHODS: We analyzed 579 for-cause endomyocardial biopsies with paired Molecular Microscope Diagnostic System (MMDx) and histopathology. Molecular scores, transcripts, donor-derived cell-free DNA (dd-cfDNA), and gene-expression profiling (GEP) were compared using patient-clustered models with false-discovery-rate correction. Multivariable Cox models evaluated a 2-year composite of death, retransplantation, graft dysfunction (LVEF<50%), or subsequent histologic rejection (ACR≥1B/2R or pAMR≥1). RESULTS: MMDx-defined ABMR was present in 83 biopsies (14.3%); 30 (36.1%) were DSA-negative. DSA-negative and DSA-positive ABMR had similar ABMR probabilities (0.66 vs 0.66), global rejection scores, dd-cfDNA (0.83% vs 0.63%), GEP (35 vs 33), and transcriptomic profiles. Within DSA-negative ABMR, non-HLA antibodies were detected in 20/30 (66.7%) and associated with higher ABMR probability (0.69 vs 0.63; P=0.041), dd-cfDNA (0.91% vs 0.62%; P=0.048), GEP (35 vs 33; P=0.019), and greater endothelial, ABMR/NK-cell, interferon-γ, and injury-related transcript activation. Histologic-molecular agreement was limited (κ=0.14); 70/83 molecular ABMR biopsies lacked histologic pAMR. In joint models, molecular ABMR, but not pAMR, was associated with higher dd-cfDNA (b=0.38%, P=0.039). Conversely, histologic pAMR independently predicted the composite outcome (adjusted HR 2.68, 95% CI 1.34-5.35; P=0.005), whereas molecular ABMR did not (adjusted HR 0.93, 95% CI 0.56-1.56; P=0.795). CONCLUSIONS: Molecular ABMR exhibited similar profiles regardless of HLA-DSA status. Non-HLA antibodies identified greater activation and dd-cfDNA levels within DSA-negative ABMR. Molecular assessment may complement conventional evaluation by refining ABMR phenotyping.