Molecular Heterogeneity and Prognostic Implications of Low-Grade Acute Cellular Rejection (1R/1A) in Heart...

International Society for Heart & Lung Transplantation (ISHLT) grade 1R/1A cellular rejection (ACR) is generally considered clinically low risk and does not routinely prompt treatment. Whether this histological finding reflects biologically meaningful alloimmune activation and confers prognostic risk remains uncertain. We sought to define the molecular and clinical significance of low-grade ACR by integrating intragraft transcriptomic profiling with circulating biomarkers and post-biopsy outcomes.

We analyzed consecutive for-cause endomyocardial biopsies (EMBs) with paired histology and Molecular Microscope Diagnostic System (MMDx) assessment obtained between November 2022 and March 2025. Biopsies with ISHLT grade ≥2R/1B, pathological antibody-mediated rejection (pAMR ≥1), or Quilty lesions were excluded. Molecular phenotypes (MMDx sign-outs), molecular rejection scores, transcript expression profiles, and circulating biomarkers (dd-cfDNA) and gene-expression profiling (GEP) were compared between histological 1R/1A ACR and 0R biopsies using patient-clustered multivariable models adjusted for sex, time from transplant, and prior treated rejection. The primary endpoint was the composite of graft dysfunction, retransplantation, or death. Secondary endpoints included subsequent rejection and dd-cfDNA elevation. Short-term outcomes were evaluated at 90 and 180 days by using generalized estimating equation models, and longitudinal outcomes were assessed by using cluster-robust Cox proportional hazards models stratified by treatment status. Exploratory analyses evaluated molecular activity, transcript expression, and biomarker associations, including risk stratification using an MMDx-derived global activity score (1-NRI >0.6). Sensitivity analyses were performed using first-biopsy-per-patient and time-varying histology models.

Among 454 biopsies from 280 patients, 91 (20%) were classified as 1R/1A and 363 (80%) as 0R. Compared with 0R, 1R/1A biopsies exhibited higher dd-cfDNA levels (0.29% vs 0.20%; P = 0.019) and higher GEP scores (33.0 vs 32.0; P = 0.045). Molecular rejection was more common in 1R/1A than in 0R (38.5% vs 12.9%; adjusted OR 3.45, 95% CI 1.99-5.97; P < 0.001), driven predominantly by antibody-mediated molecular phenotypes. Transcriptomic profiling demonstrated upregulation of alloimmune activation transcripts, including QCAT, GRIT, CTLA4, IFNG, and CXCL13. Despite these biological differences, histological 1R/1A was not associated with the primary composite endpoint of graft dysfunction, retransplantation, or death (adjusted HR 0.96, 95% CI 0.56-1.67; P = 0.89). However, histological 1R/1A was associated with an increased risk of subsequent rejection (adjusted HR 2.74, 95% CI 1.30-5.78; P = 0.008) and subsequent dd-cfDNA elevation (adjusted HR 1.43, 95% CI 1.05-1.94; P = 0.025). Importantly, molecular activity further stratified risk within histological 1R/1A. Biopsies with elevated molecular activity (1-NRI >0.6) had a more than threefold higher risk of subsequent rejection compared with histologically negative, molecularly inactive biopsies (adjusted HR 3.32, 95% CI 1.17-9.41; P = 0.02), whereas molecularly inactive 1R/1A biopsies demonstrated rejection-free survival comparable to 0R.

Histological 1R/1A represents a biologically heterogeneous state, rather than a uniformly low-risk finding. Compared with 0R biopsies, 1R/1A was associated with higher dd-cfDNA levels, higher GEP scores, and greater transcriptomic alloimmune activation. Although histological 1R/1A was not associated with graft dysfunction, retransplantation, or death, it was associated with an increased risk of subsequent rejection and dd-cfDNA elevation. Importantly, MMDx identified a biologically active subset of 1R/1A biopsies in patients who were at increased risk of future rejection, and it provided incremental prognostic information beyond conventional histology. These findings support the integration of molecular diagnostics into post-transplant risk stratification and surveillance.