*Purpose: In 2019 a Banff Working Group and Nanostring Technologies developed a panel of 770 mRNA probes for assessment of human organ allografts, based on published data. The advantages of the Nanostring nCounter platform are that 1) routine formalin fixed biopsy tissue (FFPE), including archival tissue, can be tested; 2) no separate core need be taken for molecular studies; and 3) pathology and molecular phenotype can be correlated on adjacent sections. Here we report initial results with the HOT panel.

*Methods: Three 20 μm sections were obtained from 123 kidney FFPE blocks from our institutions (118 routine needle biopsies and 5 normal samples). RNA was extracted and hybridized with the HOT panel reagents and analyzed on an nCounter Max instrument. Data and quality control were analyzed using the Nanostring software, with custom pathways added. The gene set is listed at nanostring.com.

*Results: Adequate mRNA was obtained from 95% of the samples (n=117). Pathway Analysis scores were calculated from normalized counts in groups according to their Banff pathology diagnosis. Representative results are given here. The T cell receptor, cytokine (Fig. 1) and checkpoint signalling pathways were significantly higher in T cell mediated rejection (TCMR) than chronic antibody mediated rejection (CAMR), although the individual values overlapped; all p<.01 (two tailed t-test). The borderline/suspicious category(TCMR-BS) was generally lower for these pathways. Two custom pathways were higher in CAMR than TCMR, DSAST (Hidalgo et al AJT 2010) and CAMR NHP factor (Smith et al AJT 2018) with both p<0.01 (Fig. 2). The biopsies with NER and C4d+ were in the same range as the CAMR cases (Fig. 2). Normal and acute tubular injury had the lowest levels of these pathways as expected. The interferon gamma pathway was not significantly different between CAMR and TCMR. The No Evidence of Rejection (NER) group had marked heterogeneity, suggesting variation in diagnoses not apparent by histology. Limited outcome data is available at this time, however it may be notable that 3/6 of the biopsies with high cytokine scores (>3.0; Fig. 1) developed CAMR later, compared with 2/30 with scores <3.0 (p=0.02 Fisher exact).

*Conclusions: The Nanostring HOT panel provides an efficient method to quantitate gene expression in routine FFPE kidney biopsy samples. When combined with evolving data analysis algorithms, this approach has the potential to provide useful pathogenetic and diagnostic information.

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