P048 Gene expression profiling of transplant formalin-fixed paraffin-embedded biopsies: comparison of a custom TaqMan® low density array and quantitative nuclease-protection assay

Abstract

Aim We developed a custom TLDA to monitor changes of gene expression in FFPE renal and small bowel allografts. Here we carried out a parallel comparison of expression profiles of marker genes obtained with TaqMan® technology and with the automated qNPA system. Methods Kidney, small bowel and heart transplant FFPE biopsies with different levels of rejection were selected. 5–6 curls of 10 μm were cut from each biopsy for analysis in TLDA (47 markers including immune, inflammatory and apoptosis genes), and in HTG Edge System using the Immuno-Oncology Expression assay (HTGIOE, 26 markers), or HTG EdgeSeq Oncology Biomarker assay (HTGESOB, 2558 genes, format for NGS). Data was presented as differential expression. Results There are 10 overlapping markers between our TLDA and the HTGIOE assay. Analysis of 30 kidney biopsies with both technologies gave similar results for all 10 markers. Several markers were not detected in some samples analyzed with the HTG Edge System, mostly in normal donor and non-rejection samples. 11 unique markers of HTGIOE assay showed potential value for monitoring gene expression in kidney. 55 samples of heart biopsies were analyzed in TLDA and HTGIOE. Similar results were obtained with both technologies for all overlapping markers. 15 useful markers for monitoring gene expression in heart were found in HTGIOE assay. HTGIOE markers often did not express in non-rejection samples. We also analyzed 17 small bowel biopsies in HTGESOB, which contains 39 of the 47 markers in our TLDA (8 for SB, 17 for both kidney and small bowel, and 14 for kidney). 17.3% of the 2558 genes in the panel showed 2-fold increase expression relative to non-rejection. 15 markers in our TLDA (6 small bowel specific, 7 for both kidney and small bowel, and 2 kidney specific) were in the 5% of markers with the highest fold change. Conclusions We showed that expression analysis carried out with TLDA and qNPA are comparable. The qNPA technology developed by HTG Molecular Diagnostics, Inc. is a good alternative to assess expression in biopsies. We found that the HTGIOE could be an excellent automated tool to evaluate panels with low number of markers in a clinical lab. The HTGESOB allows evaluation of a large number of genes, but it includes a NGS step that makes it costlier and time consuming. It is a good exploratory tool for searching new markers.