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

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 human leukocyte antigen (HLA)-DSA status and, within DSA-negative cases, non-HLA antibody status.

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 (acute cellular rejection [ACR] ≥ 1B/2R or pAMR ≥ 1).

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).

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.