Currently, molecular diagnostics in kidney transplantation requires dedicated fresh tissue. This requirement limits use in mainstream clinical settings due to perceived increased patient risk in taking additional biopsies. Recent technical advances enable gene expression analysis from formalin-fixed paraffin-embedded (FFPE) tissue, and initial studies used surplus FFPE tissue from routine diagnostic biopsies to test a panel of immune genes using NanoString. We chose RNAseq for whole transcriptome analysis in FFPE, using ribosomal depletion not polyA extraction, as a discovery platform toward defining molecular classifiers of T cell mediated rejection in FFPE and to investigate long non-coding RNAs as classifiers of rejection. First, we established feasibility of small FFPE biopsies to yield sufficient RNA quantity & quality, using mouse kidney FFPE samples and digital image quantification to estimate RNA yield per unit tissue volume. Next, using surplus clinical biopsies, we assessed relatedness of whole transcriptome profiles from fresh versus FFPE in 11 split pre-reperfusion donor kidney wedge biopsies of two biologic groups (+/- delayed graft function). Principal components analysis (PCA) showed clear separation of fresh from FFPE, as expected, and a persistent parallel relationship between paired samples was seen, suggesting preservation of overall data structure in FFPE. Differential gene expression analysis of FFPE pre-reperfusion biopsies using DESeq2 or DifferentialRankProducts method, revealed a consensus set of 40 transcripts significantly different in DGF versus IGF groups, indicating that FFPE transcriptional profiles retain biologic imprints. Next, we identified a cohort of 23 post-transplant FFPE needle core biopsies taken for cause and representing the histologic spectrum of tubulointerstitial T cell-mediated rejection (without C4d or DSA) or no rejection. Each rejection group consisted of two biologic sub-groups (+/- steroid response). Whole transcriptome data from post-transplant FFPE biopsies showed reliable association with histologic diagnosis by PCA. Although clustering according to steroid response was more variable, differential gene expression analysis did show a low change in steroid-sensitive versus –resistant groups. These preliminary studies reinforce the potential for molecular testing of surplus tissue from routine allograft biopsies, opening the way to development of more universally accessible molecular tests in kidney transplantation.

CITATION INFORMATION: Herzyk P., McMonagle F., Shiels P., Coley S. Novel Approach to Molecular Testing Using Routine Diagnostic Tissue Am J Transplant. 2017;17 (suppl 3).

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