Molecular Assessment of C4d-Positive Renal Transplant Biopsies Without Evidence of Rejection

Abstract

IntroductionImmunohistochemical staining for C4d in peritubular capillaries has been part of antibody-mediated rejection (AbMR) definition in the Banff Classification for Allograft Pathology since 2003. However, it has limited sensitivity and specificity, therefore the clinical significance of C4d-positive biopsies without evidence of rejection (C4d+ WER) is unknown. We investigated the transcript levels of genes associated with AbMR in C4d+ WER biopsies from both ABO-compatible and incompatible renal transplant patients.MethodsRNA was extracted from formalin-fixed paraffin-embedded renal transplant biopsies (n = 125) and gene expression analysis of 35 AbMR-associated transcripts carried out using the NanoString nCounter system.ResultsAbMR-associated transcripts were significantly increased in samples with AbMR or suspicious AbMR. A subgroup of 17 of 35 transcripts that best distinguished AbMR from C4d-negative biopsies without evidence of rejection was used to study C4d+ WER samples. There was no differential expression between C4d-negative and C4d+ WER from both ABO-incompatible and -compatible transplants. The geometric mean of 17 differentially expressed genes was used to assign the C4d+ WER biopsies a high- or low-AbMR transcript score. Follow-up biopsies showed AbMR within 1 year of initial biopsy in 5 of 7 high-AbMR transcript patients but only 2 of 46 low-AbMR transcript patients. In multivariate logistic regression analysis, elevated transcript levels in a C4d+ WER biopsy were associated with increased odds for biopsy-proven AbMR on follow-up (P = 0.032, odds ratio 16.318), whereas factors including donor-specific antibody (DSA) status and time since transplantation were not.ConclusionGene expression analysis in C4d+ WER samples has the potential to identify patients at higher risk of developing AbMR.

Banff 2019 Meeting Report: Molecular diagnostics in solid organ transplantation–Consensus for the Banff Human Organ Transplant (B-HOT) gene panel and open source multicenter validation

“…Recently, more practical technologies based on FFPE biopsy analysis are now available, in particular the NanoString nCounter system (NanoString Technologies, Seattle, WA). Several NanoString publications using FFPE transplant specimens identify similar transcript associations with the molecular and histologic phenotypes as those reported in microarray studies 3,4,13‐18,27‐29,29‐33 . Among the advantages of NanoString are (1) a separate core processed at the time of biopsy is not required; (2) transcripts are assessed in the same sample analyzed by light microscopy; and (3) large retrospective and longitudinal analyses of archived samples can be readily performed in the setting of multicenter studies, which will enable retrospective randomization with long‐term survival end points available (Table 1).…”

The Banff 2019 Kidney Meeting Report (I): Updates on and clarification of criteria for T cell– and antibody-mediated rejection

“…It is also hoped that implementation of molecular diagnostics could help classifying these cases in a more pathogenesis‐driven way, either employing the Molecular Microscope Diagnostic System (MMDx) 25 or the NanoString platform that can be used with formalin‐fixed, paraffin‐embedded tissue, 26 providing greater insight into factors predictive of the biological behavior of MVI without detectable anti‐HLA DSAs, as well as cases with C4d staining without MVI 27 . Current recommendations of the Banff Molecular Diagnostics Working Group in these and other circumstances are detailed in a separate meeting report.…”

Diagnosis of renal transplant rejection: Banff classification and beyond

“…The observation of 'C4d-positive renal transplant biopsies without evidence of rejection' is also associated with uncertainty. Intriguingly, one study performed gene expression analysis and identified patients at higher risk of developing ABMR among these cases [54].…”