Noninvasive Diagnosis Of Acute Rejection Research Articles

474 An Investigation of Novel Urinary Cell mRNA Profiles for Noninvasive Diagnosis of Acute Rejection in Kidney Transplant Recipients

OBJECTIVES/GOALS: RNA-seq of urine and kidney allograft biopsies (bx) found that urinary cell immune landscape reflects intragraft molecular events and we discovered a shared set of 127 mRNAs in urine matched to T cell mediated and antibody mediated rejection bx. We prioritized ITM2A, SLAMF6 and IKZF3 mRNAs and herein investigate if these accurately predict rejection. METHODS/STUDY POPULATION: We collected urine samples from adult kidney allograft (KA) recipients at the time of KA bx. KA bx were classified by pathologists by Banff criteria. Total RNA was isolated from KA bx-matched urine samples. Absolute copy numbers of ITM2A, SLAMF6, and IKZF3 mRNAs and 18S rRNA were measured using our customized RT-qPCR assays. Logistic regression used to derive an equation for a combined signature score of 18S-normalized urinary cell mRNA levels of ITM2A, IKZF3, and SLAMF6 that best predicts Acute Rejection (AR= both T cell mediated rejection and antibody mediated rejection). Area under the ROC curve (AUC) was calculated to discriminate between AR and No Rejection (NR) biopsies for 18S-normalized urinary cell levels of ITM2A, IKZF3 and SLAMF6 and the composite signature score. AUCs were compared by DeLong Method. RESULTS/ANTICIPATED RESULTS: Urinary cell 18S-normalized levels of ITM2A, IKZF3, and SLAMF6 mRNAs in urine discriminated KA recipients with AR biopsies (n=95) from those with NR biopsies (n=160) (All P values <0.05, Mann-Whitney test) and the AUC was 0.69 (95%CI, 0.62 to 0.76) for ITM2A, 0.61 (95%CI, 0.53 to 0.68) for IKZF3, and 0.60 (95%CI, 0.53 to 0.68) for SLAMF6. The derived combination signature score of urinary cell 18S-normalized levels of ITM2A, IKZF3, and SLAMF6 mRNA discriminated KA recipients with AR from those with NR (P<0.0001, Mann Whitney test) and the combined signature score AUC was 0.72 (95%CI, 0.65 to 0.79). The combination signature score discriminated AR vs NR better than IKZF3 and SLAMF6 alone, but was not significantly different than ITM2A alone (DeLong method). (Additional results/figures to be included in poster) DISCUSSION/SIGNIFICANCE: Our RNA-seq offered a unique opportunity to diagnose AR by measuring the mRNAs in urine. We now found that urinary cell mRNA levels of ITM2A, IKZF3, SLAMF6 and the combined signature are diagnostic of AR, a major and serious post-transplant complication. This allows for much-needed KA molecular surveillance and personalization of immunosuppression.

Noninvasive Diagnosis of Acute Rejection in Renal Transplant Patients Using Mass Spectrometric Analysis of Urine Samples: A Multicenter Diagnostic Phase III Trial.

Background.Timely recognition and treatment of acute kidney graft rejection is important to prevent premature graft failure. A predefined urinary marker set for acute T cell–mediated rejection (TCMR) containing 14 peptides was tested for this purpose in a multicenter in-place validation study.Methods.Three hundred twenty-nine prospectively collected and 306 archived urine samples from 11 transplant centers in Germany, France, and Belgium were examined. Samples were taken immediately before a biopsy, performed for graft dysfunction within the first transplant year. Primary outcomes were sensitivity and specificity of the marker set for the diagnosis of biopsy-proven acute TCMR, with prespecified thresholds of 83% for sensitivity and 70% for specificity.Results.Eighty-two patients (13%) had acute TCMR grade I–III. In relation to the biopsy diagnosis of TCMR, the sensitivity of the urine test was 0.66 (95% confidence interval, 0.56-0.76) and the specificity 0.47 (95% confidence interval, 0.43-0.51), with an area under the curve (AUC) of 0.60. The different TCMR grades I–III were not reflected by the marker set, and borderline TCMR was not specifically detected. Secondary independent masked assessment of biopsies consented by 2 pathologists revealed an interobserver kappa value of 0.49 for diagnosing TCMR, compared with the local center’s diagnosis. Using this consensus diagnosis, the AUC of the urine test was 0.63 (sensitivity 0.73, specificity 0.45). Post hoc optimization of the marker set improved the diagnostic performance in the study cohort (AUC 0.67) and in an independent patient cohort (AUC 0.69).Conclusions.This study illustrates the difficulty of proteomics-based diagnosis of TCMR and highlights the need for rigorous independent in-place validation and optimization of diagnostic biomarkers.

Expanding the Non-Invasive Diagnosis of Acute Rejection in Kidney Transplants Through Detection of Donor-Derived DNA in Urine: Proof-of-Concept Study.

BackgroundApproximately 10%-20% of kidney transplant (KT) recipients suffer from acute rejection (AR); thus, sensitive and accurate monitoring of allograft status is recommended. We evaluated the clinical utility of donor-derived DNA (dd-DNA) detection in the urine of KT recipients as a non-invasive means for diagnosing AR.MethodsUrine samples serially collected from 39 KT recipients were tested for 39 single-nucleotide variant loci selected according to technical criteria (i.e., high minor allele frequency and low analytical error) using next-generation sequencing. The fraction of dd-DNA was calculated and normalized by the urine creatinine (UCr) level (%dd-DNA/UCr). The diagnostic performance of %dd-DNA/UCr for AR was assessed by ROC curve analysis.ResultsThere was an increasing trend of %dd-DNA/UCr in the AR group before subsequent graft injury, which occurred before (median of 52 days) histological rejection. The serum creatinine (SCr) level differed significantly between the AR and non-AR groups at two and four months of follow-up, whereas %dd-DNA/UCr differed between the groups at six months of follow-up. The combination of %dd-DNA/UCr, SCr, and spot urine protein (UPtn)/UCr showed high discriminating power, with an area under the ROC curve of 0.93 (95% confidence interval 0.81-1.00) and a high negative predictive value of 100.0%.ConclusionsAlthough the dd-DNA-based test cannot eliminate the need for biopsy, the high negative predictive value of this marker could increase the prebiopsy probability of detecting treatable injury to make biopsy an even more effective diagnostic tool.

Serum biomarkers of acute rejection: Towards precision medicine in heart transplant

Despite the important changes in the management of heart transplantation in the recent decades, the use of endomyocardial biopsy is still necessary for the follow-up of these patients. However, this technique has several limitations, the most important being the substantial interobserver variability. In the last years multiple attempts have been made to find non-invasive assays for cardiac allograft surveillance, such as imaging modalities and serum biomarkers. This state-of-the-art review focuses on describing the different serum biomarkers that have been proposed for non-invasive diagnosis of acute rejection and that are paving the way towards precision medicine in the field of heart transplantation.

The Regulatory/Cytotoxic T Cell Profiles in Blood of Pediatric Kidney-Transplant Recipients with Acute Rejection

Background: The equilibrium between regulatory cells and cytotoxic cells may define graft consequence. We investigated the relationship between the expression of main regulatory and cytotoxic markers (i.e., FOXP3 and granzyme B (GZM-B), respectively) and acute rejection (AR) in the peripheral blood of pediatric renal transplant recipients. Methods: In this retrospective study, FOXP3 mRNA expression and serum GZM-B levels in peripheral blood samples from 47 first-time pediatric kidney-transplant recipients were measured, with 17 children classified as possessing AR; whereas the remaining 30 children had functionally stabilized allografts. Results: Levels of the FOXP3 mRNA vs. the expression levels GADPH mRNA (FOXP3 mRNA/GADPH mRNA) were significantly elevated in children with AR than those with stabilized renal allograft (0.48 ± 0.26 vs.0.23 ± 0.18, respectively, P=0.002) Also, serum GZM-B levels in the AR group were elevated than those in the functionally stabilized children (120.07 ± 91.42p g/ml and, 60.16 ± 46.29 pg/ml respectively, P=0.01). ROC curve evidenced that measuring FOXP3 mRNA may have a scope as a decision-taking agent in clinical proceedings to diagnose AR. Measuring peripheral blood FOXP3 mRNA elucidated scope to help in the noninvasive diagnosis of AR. Conclusions: Our results emphasize FOXP3 mRNA as a biomarker for AR in pediatrics. Assessment of regulatory/cytotoxic profiles in the peripheral blood of pediatric renal transplant recipients is a potentially useful tool for patient selection and early detection of rejection. Depending on many variables, such as the method of sample normalization, the technique used, the extent of graft inflammation, the immunosuppression regimen, depletion/ repletion of T-lymphocyte component, the importance of FOXP3 may differ.

GC/MS-based urine metabolomics analysis of renal allograft recipients with acute rejection

BackgroundAcute renal allograft rejection is a common complication after renal transplantation that often leads to chronic rejection and ultimate graft loss. While renal allograft biopsy remains the gold standard for diagnosis of acute rejection, the possibility of biopsy-associated complications cannot be overlooked. The development of noninvasive methods for accurate detection of acute renal allograft rejection is thus of significant clinical importance.MethodsGas chromatography–mass spectrometry (GC/MS) was employed for analysis of urine metabolites in 15 renal allograft recipients with acute rejection and 15 stable renal transplant recipients. Partial least squares (PLS) regression and leave-one-out analyses were performed to ascertain whether the metabolites identified could be exploited to distinguish acute rejection from stable groups as well as their sensitivity and specificity.ResultsOverall, 14 metabolites were significantly altered in the acute rejection group (11 and 3 metabolites displayed higher and lower levels, respectively) relative to the stable transplant group. Data from PLS and leave-one-out analyses revealed that the differential metabolites identified not only distinguished acute rejection from stable transplant recipients but also showed high sensitivity and specificity for diagnosis of renal allograft recipients with acute rejection.ConclusionUrine metabolites identified with GC/MS can effectively distinguish acute rejection from stable transplant recipients, supporting the potential utility of metabolome analysis in non-invasive diagnosis of acute rejection.

Diagnostic Performance of Fas Ligand mRNA Expression for Acute Rejection after Kidney Transplantation: A Systematic Review and Meta-Analysis.

BackgroundThe value of Fas ligand (FASL) as a diagnostic immune marker for acute renal rejection is controversial; this meta-analysis aimed to clarify the role of FASL in acute renal rejection.MethodsThe relevant literature was included by systematic searching the MEDLINE, EMBASE, and Cochrane Library databases. Accuracy data for acute rejection (AR) and potential confounding variables (the year of publication, area, sample source, quantitative techniques, housekeeping genes, fluorescence staining, sample collection time post-renal transplantation, and clinical classification of AR) were extracted after carefully reviewing the studies. Data were analyzed by Meta-DiSc 1.4, RevMan 5.0, and the Midas module in Stata 11.0 software.ResultsTwelve relevant studies involving 496 subjects were included. The overall pooled sensitivity, specificity, positive likelihood ratio (LR), negative LR, and diagnostic odds ratio, together with the 95% CI were 0.64 (0.57–0.70), 0.90 (0.85–0.93), 5.66 (3.51–9.11), 0.30 (0.16–0.54), and 30.63 (14.67–63.92), respectively. The area under the summary receiver operating characteristic curve (AUC) was 0.9389. Fagan’s nomogram showed that the probability of AR episodes in the kidney transplant recipient increased from 15% to 69% when FASL was positive, and was reduced to 4% when FASL was negative. No threshold effect, sensitivity analyses, meta-regression, and subgroup analyses based on the potential variables had a significant statistical change for heterogeneity.ConclusionsCurrent evidence suggests the diagnostic potential for FASL mRNA detection as a reliable immune marker for AR in renal allograft recipients. Further large, multicenter, prospective studies are needed to validate the power of this test marker in the non-invasive diagnosis of AR after renal transplantation.

A Meta-analysis of the Significance of Granzyme B and Perforin in Noninvasive Diagnosis of Acute Rejection After Kidney Transplantation.

Previous studies have reported that granzyme B (GZMB) and perforin (PRF) could serve as noninvasive biomarkers in the diagnosis of acute rejection (AR) after kidney transplant. Yet, their noninvasive diagnostic value in clinical practice is still unknown. To assess the noninvasive diagnostic performance of GZMB and PRF for AR, we performed a systematic search. After reviewing published studies in which both GZMB and PRF were detected, data on the diagnostic accuracy of separate and combined evaluation of GZMB and PRF were pooled. Across 16 studies (680 subjects), summary sensitivity, specificity, positive likelihood ratios, and negative likelihood ratios with 95% confidence intervals were calculated. For overall GZMB analysis, the indices were 0.76 (0.71-0.81), 0.86 (0.82-0.89), 4.58 (3.36-6.25), and 0.32 (0.22-0.47), respectively. For overall PRF analysis, the indices were 0.83 (0.78-0.88), 0.86 (0.82-0.89), 4.82 (3.66-6.35), and 0.26 (0.18-0.37), respectively. Subgroup analyses showed similar results compared to overall study analyses. In analyses of combined evaluation of GZMB and PRF, the above indices were 0.65 (0.53-0.76), 0.96 (0.91-0.98), 12.66 (5.83-27.50), and 0.40 (0.23-0.69), respectively, when both markers were positive. The probability of developing AR in kidney transplant recipients increased from 15% to 73% when both GZMB and PRF tests were positive and was reduced to 2% if that were negative. Currently, neither GZMB nor PRF, if evaluated alone, could be a convincing noninvasive diagnostic marker for AR in clinical practice. Combined use of PRF and GZMB post-kidney transplant may be a better choice in AR evaluation to direct allograft biopsy execution and earlier therapeutic intervention.

Apoptosis and Acute Cellular Rejection in Human Heart Transplants

Apoptosis has been implicated in the pathophysiology of various forms of heart disease. Acute cellular rejection leads to morbidity after heart transplantation and invasive techniques are needed for its diagnosis. We investigated the presence of cardiomyocyte apoptosis in transplanted hearts, its progression, its relationship with rejection, and the possibility that serological markers of apoptosis can be used to detect rejection noninvasively. Overall, 130 endomyocardial biopsies obtained sequentially from 14 consecutive patients during the first 6 months following heart transplantation underwent histochemical analysis. The degree of acute rejection was determined, myocyte apoptosis was assessed using the TUNEL method, and caspase-3 activity was measured. In the first 10 patients, soluble Fas, tumor necrosis factor-alpha (TNFα) and interleukin 6 levels were determined in serum collected at biopsy. Apoptotic cells were detected in 81.5% of biopsies. No significant correlation was found between the apoptotic index and either the degree of rejection or the time from transplantation; there was only a trend to higher values during prolonged episodes of rejection, which did not reach statistical significance. An inverse correlation was observed between the degree of rejection and the TNFα level (rs=-0.33; P=.003). There was no correlation with any other variable. Cardiomyocyte loss due to apoptosis was observed in transplanted hearts, but no correlation was observed with either acute rejection or the time from transplantation. Our findings suggest there could be an inverse correlation between rejection and the serum TNFα level. No serum parameter evaluated was regarded as suitable for the noninvasive diagnosis of acute rejection.

Non-invasive diagnosis of acute rejection in kidney transplants with delayed graft function

Delayed graft function (DGF) often occurs in kidney transplants from deceased donors. We wanted to provide studies giving more accurate non-invasive tests for acute rejection (AR). Using real-time PCR, we examined the expression of cytolytic molecules such as perforin, granzyme B, and fas-ligand along with serpin proteinase inhibitor-9. We also measured the expression of FOXP3, a characteristic gene of T-regulatory cells known to be involved in AR. These studies were conducted on peripheral blood monocytes, urinary cells, and 48 surveillance kidney biopsies taken from a total of 35 patients with DGF. Of these patients, 20 had a histopathological diagnosis of AR, whereas other 28 had characteristics of acute tubular necrosis (ATN). Expression of cytolytic and apoptotic-associated genes in the biopsy tissue, peripheral blood leukocytes, and urinary cells was significantly higher in patients with AR than that in patients with ATN. Diagnostic parameters associated with FOXP3 gene expression were most accurate in peripheral blood leukocytes and urine cells with sensitivity, specificity, positive and negative predictive values, and accuracy between 94 and 100%. Our study shows that quantification of selected genes in peripheral blood leukocytes and urinary cells from renal transplant patients with DGF may provide a useful and accurate non-invasive diagnosis of AR.

1854: Non-Invasive Diagnosis of Acute Rejection by Measuring Chemokinegene Expression in Urine ofKidney Transplant Recipients

You have accessJournal of UrologyDiscussed Poster, Wednesday, May 12, 2004, 8:00am - 12:00 pm1 Apr 20041854: Non-Invasive Diagnosis of Acute Rejection by Measuring Chemokinegene Expression in Urine ofKidney Transplant Recipients Masayoshi Miura, Tetsuo Hirano, Naohiko Shimada, Ken Morita, Yoshihiko Watarai, and Katsuya Nonomura Masayoshi MiuraMasayoshi Miura More articles by this author , Tetsuo HiranoTetsuo Hirano More articles by this author , Naohiko ShimadaNaohiko Shimada More articles by this author , Ken MoritaKen Morita More articles by this author , Yoshihiko WataraiYoshihiko Watarai More articles by this author , and Katsuya NonomuraKatsuya Nonomura More articles by this author View All Author Informationhttps://doi.org/10.1016/S0022-5347(18)39046-3AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail "1854: Non-Invasive Diagnosis of Acute Rejection by Measuring Chemokinegene Expression in Urine ofKidney Transplant Recipients." The Journal of Urology, 171(4S), p. 490 © 2016 by American Urological AssociationFiguresReferencesRelatedDetails Volume 171Issue 4SApril 2004Page: 490 Advertisement Copyright & Permissions© 2016 by American Urological AssociationMetricsAuthor Information Masayoshi Miura More articles by this author Tetsuo Hirano More articles by this author Naohiko Shimada More articles by this author Ken Morita More articles by this author Yoshihiko Watarai More articles by this author Katsuya Nonomura More articles by this author Expand All Advertisement Loading ...

Noninvasive diagnosis of acute rejection of solid organ transplants.

Accurate diagnosis of acute rejection remains a formidable challenge in organ transplantation. The current gold standard diagnostic test for acute rejection is histological examination of the transplanted organ. However, biopsy procedures are invasive and complications occur. Furthermore, sampling errors may bias the histological diagnosis. Not uncommonly, empiric anti-rejection therapy has to be provided prior to the availability of a confirmatory histological report. Thus, there is an urgent need for specific and sensitive noninvasive biomarkers of acute rejection. Herein, we review noninvasive strategies for the diagnosis of acute rejection of solid organ transplants.

Acute rejection after heart transplantation: Noninvasive echocardiographic evaluation

Objectives. The purpose of this study was to assess the reliability of echocardiography in the noninvasive diagnosis of acute rejection in heart transplant recipients.Background. Although echocardiographic results seem to correlate well with allograft rejection, published data are limited and contradictory.Methods. In 130 transplant recipients, 1,400 serial echocardiograms were recorded within 24 h of endomyocardial biopsy. Increased wall thickness, myocardial echogenicity, pericardial effusion, shorter pressure half-time, isovolumetric relaxation time and a decrease in left ventricular ejection fraction were considered markers of rejection.Results. The distribution of echocardiographic markers revealed highly significant differences between bioptically graded moderate, mild and no rejection and between untreated and treated rejection episodes (both chi-square test, p < 0.0001). Specificity was 98.6% for two markers, but sensitivity was good (80%) for only moderate rejection because of the large number of false negatives in untreated patients with mild rejection. In untreated patients, there was a highly significant difference in the number of echocardiographic criteria between a benign and nonbenign outcome (chi-square test, p < 0.0001). In treated patients, the significant difference in the variation in echocardiographic criteria between favorable and unfavorable responses after 1 week was more pronounced after 2 weeks (t test, p < 0.01 vs. < 0.081). Diastolic indexes and pericardial effusion at 2 weeks seemed to be predictive of therapeutic response.Conclusions. Poor sensitivity to mild rejection indicates that serial echocandiography cannot supplant endomyocardial biopsy in the early diagnosis of acute rejection, but it seems to be a reliable noninvasive means of identifying acute rejection requiring intensified immunosuppressive therapy and of evaluating outcome.