*Purpose: Optimization of effective anti-rejection therapies to prevent allograft rejection and to minimize drug toxicity may be aided by utilizing in situ biomarkers for detection of acute rejection. To this end, we previously developed a Taqman® Low Density Array (TLDA) containing a limited number of immune, inflammatory and apoptosis marker genes (47), to assess T cell mediated rejection (TCMR) in kidney and small bowel transplants, by measuring alterations in gene expression in FFPE biopsies.

*Methods: In this work we present a NGS-based Ion AmpliSeq RNA custom panel assay of 129 marker genes that includes the previous 47. The scope of the panel was increased by adding markers described in the literature to be associated with universal organ rejection, selective for antibody mediated rejection (ABMR), endothelial donor specific markers, and selective for TCMR. We evaluated the expression panel using FFPE biopsies of renal allografts submitted for histopathological evaluation and that showed predominant acute cellular rejection (TCMR, 15 samples) or humoral rejection (ABMR, 6 samples), and donor kidney biopsies (DO, 30 samples). The sequencing data was analyzed using the ampliSeqRNA plugin in S5 Ion Torrent Suite™ Software, and the differential expression analysis was carried out using the DESeq2 package in RStudio software. Significant differential expression was considered as adjusted p-values (padj) < 0.05.

*Results: The analysis DO vs. TCMR showed differential expression in 112 markers (68 upregulated and 44 downregulated), while analysis of DO vs. ABMR showed similar results with differential expression in 92 markers (53 upregulated and 39 downregulated). On the other hand, TCMR vs. ABMR revealed differential expression in only 6 markers. These genes behaved as described in the literature, e.g. the genes SH2D1B, KLRF1 and FGFBP2, defined selective for ABMR, were upregulated in ABMR samples. Also, the gene ADAMDEC1, considered a selective TCMR, was down regulated in ABMR samples.

*Conclusions: Our results demonstrate that this NGS-based expression panel was robust for prediction of TCMR and TCMR with superimposed ABMR. The expression panel could also discriminate between TCMR and ABMR, but the relation was weaker, however, the number of FFPE biopsies with predominant ABMR analyzed in this ongoing study was small. We expect a better separation of the TCMR and ABMR categories with a larger sample size. Further evaluation of this expression panel will allow the development of a micro-panel of the most selective markers for prediction of TCMR and ABMR from formalin-fixed kidney transplant biopsies, as well as other markers for potential complications.

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