*Purpose: Endomyocardial biopsy (EMB) remains the gold standard in acute rejection (AR) surveillance in heart transplantation (HT) despite interpretation subjectivity and low concordance rates between pathologists, both of which could affect clinical treatment decisions. Gene expression (GE) profiling has a high negative predictive value for ruling out AR, but this method is based on peripheral leukocyte testing. A literature-derived 34-gene set GE profile test on formalin-fixed paraffin-embedded (FFPE) renal biopsy tissue was recently reported and found to correlate with histology, but this technology has not been utilized in FFPE EMB specimen. This study reports our initial feasibility findings of this NanoString® testing on FFPE EMB samples.

*Methods: Tissue sections were dissected from 26 FFPE blocks of EMBs and included no rejection [(NR), grade 0R; n=9], mild AR (grade 1R; n=12), and moderate to severe AR [(grade 2R; n=4) and (grade 3R; n=1)]. The expression of 758 genes in the Banff Human Organ Transplant gene panel was quantified on the NanoString nCounter® system. Differential GE, defined as a false discovery rate (FDR)-adjusted p-value <0.05, was assessed between 1) NR and mild AR samples and 2) NR and moderate to severe AR specimen. Gene set enrichment analysis (GSEA) was performed to identify pathways with significant changes (FDR p<0.05) during AR.

*Results: This retrospective study included n=17 patients (88% men, 94% Caucasian, 49.1±14.9 yrs). Thirty-seven genes (mean±SD fold change: 2.6±1.0) had significantly different GE between NR and mild AR samples, with most (36 genes) exhibiting higher expression during mild AR. One hundred and twenty-eight genes had higher gene expression (FDR p<0.05) during moderate to severe AR compared to NR (fold change: 3.1±1.4), including 34 genes with differential GE in the NR versus mild AR analysis. GSEA identified upregulation of the allograft rejection and IFNg signaling pathways during both mild and moderate to severe AR compared to NR.

*Conclusions: Our results suggest GE in EMBs could help differentiate various grades of AR from NR after HT. We also identified multiple pathways upregulated during AR that have previously been shown to play a role in allograft rejection after kidney transplantation, suggesting allograft rejection across various types of solid organ transplants might share similar molecular mechanisms. Future studies will aim to validate these preliminary findings in a larger selection of HT EMBs.

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