Molecular Classifiers for Acute Kidney Transplant Rejection in Peripheral Blood by Whole Genome Gene Expression Profiling

Abstract

There are no minimally invasive diagnostic metrics for acute kidney transplant rejection (AR), especially in the setting of the common confounding diagnosis, acute dysfunction with no rejection (ADNR). Thus, though kidney transplant biopsies remain the gold standard, they are invasive, have substantial risks, sampling error issues and significant costs and are not suitable for serial monitoring. Global gene expression profiles of 148 peripheral blood samples from transplant patients with excellent function and normal histology (TX; n = 46), AR (n = 63) and ADNR (n = 39), from two independent cohorts were analyzed with DNA microarrays. We applied a new normalization tool, frozen robust multi-array analysis, particularly suitable for clinical diagnostics, multiple prediction tools to discover, refine and validate robust molecular classifiers and we tested a novel one-by-one analysis strategy to model the real clinical application of this test. Multiple three-way classifier tools identified 200 highest value probesets with sensitivity, specificity, positive predictive value, negative predictive value and area under the curve for the validation cohort ranging from 82% to 100%, 76% to 95%, 76% to 95%, 79% to 100%, 84% to 100% and 0.817 to 0.968, respectively. We conclude that peripheral blood gene expression profiling can be used as a minimally invasive tool to accurately reveal TX, AR and ADNR in the setting of acute kidney transplant dysfunction.

A Molecular Signature of Two Long Non-Coding RNAs in Peripheral Blood Predicts Acute Renal Allograft Rejection

“…In the PRTx cohort, all patients received the renal transplantation at Stanford University and routinely followed up for two years [6]. In the ARTx cohort, the subjects were randomly recruited from five different clinical centers including Scripps Clinic, Cleveland Clinic, St. Vincent Medical Center, University of Colorado, and Mayo Clinic Arizona [7]. The renal biopsies were scored by at least two pathologists blindly and independently according to Banff classification [21].…”

“…The renal biopsies were scored by at least two pathologists blindly and independently according to Banff classification [21]. Finally, a total of 150 biopsy-defined AR and stable (STA) recipients with corresponding PB samples were included (PRTx: n=75, ARTx: n=75), and the detailed selection criteria have been described previously [6, 7]. All subjects were well informed and provided written consent, and the appropriate approvals were obtained from the corresponding institutional review boards [6, 7].…”

“…Finally, a total of 150 biopsy-defined AR and stable (STA) recipients with corresponding PB samples were included (PRTx: n=75, ARTx: n=75), and the detailed selection criteria have been described previously [6, 7]. All subjects were well informed and provided written consent, and the appropriate approvals were obtained from the corresponding institutional review boards [6, 7]. Furthermore, the protocol of current study was approved by the institutional review board of Nanjing First Hospital, Nanjing Medical University.…”

“…Each PB sample was paired with renal allograft biopsy (in PRTx cohort: within 48 hours; in ARTx cohort: at the time of biopsy), and collected in 2.5 ml PAXgene Blood RNA Tubes (PreAnalytiX, Qiagen, Valencia, CA; or PreAnalytiX GmbH, Hombrechtikon, Switzerland), and total RNA was extracted using the PAXgene Blood RNA Kit according to the manufacturer’s instructions [6, 7]. The well-prepared cDNA was hybridized to Affymetrix Human Genome U133 (HG-U133) Plus 2.0 GeneChips (Affymetrix Inc., Santa Clara, CA), and the raw expression data were deposited in Gene Expression Omnibus database (accession numbers: GSE14346 for PRTx cohort and GSE15296 for ARTx cohort).…”

“…In the current study, we comprehensively analyzed the lncRNA expression profiles in the PB samples of patients from two cohorts: adult renal transplant (ARTx) and pediatric renal transplant (PRTx) cohorts [6, 7], with the aim to: 1) explore the differentially expressed lncRNAs in PB of renal transplant recipients with and without AR; 2) identify the circulating lncRNAs that could distinguish recipients with AR from those without; 3) generate a lncRNA signature in PB which could serve as a novel diagnostic biomarker of AR in both adult and pediatric renal transplant recipients.…”