Development and validation of a peripheral blood mRNA assay for the assessment of antibody-mediated kidney allograft rejection: A multicentre, prospective study

Elisabet Van Loon, Stéphane Gazut, Saleh Yazdani, Evelyne Lerut, Henriette de Loor, Maarten Coemans, Laure-Hélène Noël, Lieven Thorrez, Leentje Van Lommel, Frans Schuit, Ben Sprangers, Dirk Kuypers, Marie Essig, Wilfried Gwinner, Dany Anglicheau, Pierre Marquet, Maarten Naesens, Elisabet Van Loon, Stéphane Gazut, Saleh Yazdani, Evelyne Lerut, Henriette de Loor, Maarten Coemans, Laure-Hélène Noël, Lieven Thorrez, Leentje Van Lommel, Frans Schuit, Ben Sprangers, Dirk Kuypers, Marie Essig, Wilfried Gwinner, Dany Anglicheau, Pierre Marquet, Maarten Naesens

Abstract

Background: Antibody-mediated rejection, a leading cause of renal allograft graft failure, is diagnosed by histological assessment of invasive allograft biopsies. Accurate non-invasive biomarkers are not available.

Methods: In the multicentre, prospective BIOMARGIN study, blood samples were prospectively collected at time of renal allograft biopsies between June 2011 and August 2016 and analyzed in three phases. The discovery and derivation phases of the study (N = 117 and N = 183 respectively) followed a case-control design and included whole genome transcriptomics and targeted mRNA expression analysis to construct and lock a multigene model. The primary end point was the diagnostic accuracy of the locked multigene assay for antibody-mediated rejection in a third validation cohort of serially collected blood samples (N = 387). This trial is registered with ClinicalTrials.gov, number NCT02832661.

Findings: We identified and locked an 8-gene assay (CXCL10, FCGR1A, FCGR1B, GBP1, GBP4, IL15, KLRC1, TIMP1) in blood samples from the discovery and derivation phases for discrimination between cases with (N = 49) and without (N = 134) antibody-mediated rejection. In the validation cohort, this 8-gene assay discriminated between cases with (N = 41) and without antibody-mediated rejection (N = 346) with good diagnostic accuracy (ROC AUC 79·9%; 95% CI 72·6 to 87·2, p < 0·0001). The diagnostic accuracy of the 8-gene assay was retained both at time of stable graft function and of graft dysfunction, within the first year and also later after transplantation. The 8-gene assay is correlated with microvascular inflammation and transplant glomerulopathy, but not with the histological lesions of T-cell mediated rejection.

Interpretation: We identified and validated a novel 8-gene expression assay that can be used for non-invasive diagnosis of antibody-mediated rejection.

Funding: The Seventh Framework Programme (FP7) of the European Commission.

Keywords: Antibody-mediated rejection; Biomarker; Kidney transplantation.

Conflict of interest statement

This manuscript is related to a European patent application (Title: mRNA-based biomarkers for antibody-mediated transplant rejection; Application Number EP19152365.3), which has been filed on January 17, 2019).

Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.

Figures

Fig. 1
Fig. 1
Study design. Peripheral blood samples were obtained at the time of a renal allograft biopsy in four European transplant centres. In the discovery and derivation cohort, samples were selected based on availability and histological criteria of concomitant renal allograft biopsies (excluding cases with diagnosis of glomerulonephritis or polyomavirus nephropathy, and cases with unclear diagnosis), while graft function was not taken into account. In the validation cohort, all samples with concomitant adequate renal allograft biopsy histology, prospectively collected between June 24, 2014 and July 2, 2015, were serially included without selection on histology, demographics or time. The gene expression profile was not complete in seven of these samples, leading to a total of 387 cases in the validation phase. ABMR = antibody-mediated rejection.
Fig. 2
Fig. 2
Diagnostic accuracy of the 8-gene assay for non-invasive diagnosis of antibody-mediated rejection in the validation cohort (N = 387). The left panel shows the 8-gene assay score in cases with versus without antibody-mediated rejection. The middle panel shows the distribution of cases with antibody-mediated rejection across all scores of the 8-gene assay. The right panel shows the ROC curves for samples with versus without antibody-mediated rejection, with presentation of the area under the receiver operating characteristic curve (ROC AUC) and the 95% confidence interval.
Fig. 3
Fig. 3
Diagnostic accuracy of the 8-gene assay for antibody-mediated rejection in specific subgroups in the validation set (N = 387). Post-hoc sensitivity analysis of the 8-gene marker according to time after transplantation is shown in panel A and according to stable graft function vs. graft dysfunction in panel B.
Fig. 4
Fig. 4
Distribution of the 8-gene assay score per histological lesion grade in the validation cohort (N = 387). The 8-gene assay score was significantly associated with lesions of antibody-mediated rejection. Significance was assessed with nonparametric one-way ANOVA and pairwise comparisons with t-test. Significance was apparent for higher severity grades of lesions associated with antibody-mediated rejection (glomerulitis, peritubular capillaritis, microvascular inflammation score, transplant glomerulopathy). No significant association was present with lesions of T-cell mediated rejection (tubulitis, interstitial inflammation) or non-specific chronic damage (interstitial fibrosis, tubular atrophy). g = glomerulitis; ptc = peritubular capillaritis; mvi = microvascular inflammation; cg = transplant glomerulopathy; i interstitial inflammation; t = tubulitis; v = intimal arteritis; C4d = C4d deposition in peritubular capillaries; ci = interstitial fibrosis; ct = tubular atrophy; cv = intimal fibrosis; ah = arteriolar hyalinosis. ns = not significant, *p < 0·05,**p < 0·01,***p < 0·001,****p < 0·0001.

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