Potential impact of microarray diagnosis of T cell-mediated rejection in kidney transplants: the INTERCOM study
ABSTRACT: The authors report that in INTERCOM, a prospective international study of 300 kidney transplant biopsies, a microarray-based molecular score for T cell-mediated rejection changed the assessment of 26% of all biopsies, illustrating the potential of precision diagnostics to impact practice. Microarray analysis of 300 kidney transplant biopsies (non-TCMR, TCMR) and 6 nephrectomies.
Project description:Microarray analysis of human kidneys with acute kidney injury (AKI) has been limited because such kidneys are seldom biopsied. However, all kidney transplants experience AKI, and early kidney transplants without rejection are an excellent model for human AKI: they are screened to exclude chronic kidney disease, frequently biopsied, and have extensive follow-up. We used histopathology and microarrays to compare indication biopsies from 28 transplants with AKI to 11 pristine protocol biopsies of stable transplants. Kidneys with AKI showed increased expression of 394 injury-repair response associated transcripts, including many known epithelial injury molecules (e.g. ITGB6, LCN2), tissue remodeling molecules (e.g. VCAN), and inflammation molecules (S100A8, ITGB3). Many other genes also predict the phenotype, depending on statistical filtering rules, including AKI biomarkers as HAVCR1 and IL18. Most mouse orthologs of the top injury-repair transcripts were increased in published mouse AKI models. Pathway analysis of the injury-repair transcripts revealed similarities to cancer, development, and cell movement. The injury-repair transcript score AKI kidneys correlated with reduced function, future recovery, brain death, and need for dialysis, but not future graft loss. In contrast, histologic features of "acute tubular injury" did not correlate with function or with the molecular changes. Thus the injury-repair associated transcripts represent a massive coordinate injury-repair response of kidney parenchyma to AKI, similar to mouse AKI models, and provide an objective measure for assessing the severity of AKI in kidney biopsies and validation for the use of many AKI biomarkers. AKI biopsies sample names and CEL files are from GSE21374. All consenting renal transplant patients undergoing biopsies for cause as standard of care between 09/2004 and 10/2007 at the university of Alberta or between 11/2006 and 02/2007 at the University of Illinois were included in the analysis. In addition to the cores required for standard histopathology, we collected one core for gene expression studies. the relationship between gene expression in the biopsy and subsequent graft loss was analyzed.
Project description:Histologic diagnosis of T cell-mediated rejection in kidney transplant biopsies has limited reproducibility because it is based on non-specific lesions using arbitrary rules that are subject to differing interpretations. We used microarray results from 403 indication biopsies previously given histologic diagnoses to develop a molecular classifier that assigned a molecular T cell-mediated rejection score to each biopsy. Independent assessment of the biopsies by multiple pathologists confirmed considerable disagreement on the presence of TCMR features: 79-88% accuracy and 35-69% sensitivity. The agreement of the molecular T cell-mediated rejection score with the histology diagnosis was similar to agreement among individual pathologists: accuracy 89%, sensitivity 51%. However, the score also predicted the consensus among pathologists, being highest when all agreed. Many discrepancies between the scores and the histologic diagnoses were in situations where histology is unreliable e.g. scarred biopsies. The score correlated with histologic lesions and gene sets associated with T cell-mediated rejection. The transcripts most often selected by the classifier were expressed in effector T cells, dendritic cells, or macrophages or inducible by interferon-gamma. Thus the T cell-mediated rejection score offers an objective assessment of kidney transplant biopsies, predicting the consensus opinion among multiple pathologists, and offering insights into underlying disease mechanisms. Antibody-mediated rejection is a major cause of kidney transplant failure, but the current diagnostic system misses most cases due to dependency on subjective non-standardized tests. We hypothesized that molecular features could provide a test to address this problem. We classified 403 biopsies by a reference standard based on microcirculation lesions and donor-specific HLA antibody, and used microarray analysis to develop a classifier that assigned each biopsy a score reflecting the probability of antibody-mediated rejection. The scores correlated with donor-specific antibody and histologic lesions: 42/45 biopsies with antibody-mediated rejection scores >0.5 had both donor-specific antibody and microcirculation lesions. Intermediate scores (0.2-0.5) were more ambiguous, but became more specific combined with donor-specific antibody. Compared to diagnoses based on histology-plus-donor-specific antibody, the scores had sensitivity 0.67; specificity 0.90. Donor-specific antibody improved the specificity to 0.97. The score correlated not only with diagnoses of individual pathologists but with the consensus among multiple pathologists. The classifier used transcripts expressed in endothelial cells (e.g. CDH13, DARC, ROBO4) and NK cells (e.g. CX3CR1, FGFBP2), as well as IFNG-inducible transcripts e.g. CXCL11. Thus the molecular phenotype of antibody-mediated rejection provides not only an objective test that predicts microcirculation lesions and donor-specific HLA antibody, but also offers mechanistic insights. All consenting renal transplant patients undergoing biopsies for cause as standard of care. 403 samples and 8 controls (nephrectomies).
Project description:Kidney transplants that develop dysfunction or proteinuria after one year post transplant are at considerable risk for progression to renal failure. Identifying the molecules associated with graft failure could potentially lead to interventions that would slow the progression of organ failure. We analyzed the relationship between gene expression in late biopsies for cause in human kidney transplants and subsequent graft loss, and assessed the predictive value of gene expression. We evaluated the performance of these genes in an independent , and in a population of early biopsies that have a very low risk of subsequent graft failure. All consenting renal transplant patients undergoing biopsies for cause as standard of care between 09/2004 and 10/2007 at the university of Alberta or between 11/2006 and 02/2007 at the University of Illinois were included in the analysis. In addition, biopsies obtained from Minnesota between 09/2006 and 09/2007 were used as an independent . In addition to the cores required for standard histopathology, we collected one core for gene expression studies. the relationship between gene expression in the biopsy and subsequent graft loss was analyzed.
Project description:In deceased donor kidney transplantation, acute kidney injury (AKI) prioir to surgery is a major determinant of delayed graft function (DGF), but AKI is histologically silent and difficult to assess. We hypothesized that a molecular measurement of AKI would add power to conventional risk assessments to predict the early poor allograft function at first week post transplantation. We performed microarrays on implantation biopsies taken during reperfusion in 70 deceased donor kidneys from 53 donors. Early poor function was classified by two definitions on day 7 post-transplantation: serum creatinine greater than 265 umol/L (3 mg/dL) or the requirement for dialysis. Donor age and related risk scores (Irish, Schold, KDRI) associated with worse early function, as expected, but histologic features (glomerulosclerosis; pathology risk scores (Remuzzi, MAPI)) correlated with donor age but not with poor function. However, molecular AKI signal, previously defined in kidneys with early injury, was the best single predictor of poor allograft function. The combination of donor age and the AKI signal improved the prediction of poor function. In addition, asssessments of tissue quality particularly donor age, Banff ct, Irish and KDRI scores, showed negative correlative trend with late graft function, whereas the AKI signal did not. Thus donor age and the molecular AKI signal are the main predictors of early impaired function, but have little impact on survival.
Project description:Treatment with angiotensin converting enzyme inhibitors (ACEI) or angiotensin receptor blockers (ARB) has been shown to have anti-inflammatory effects such as decreased growth factors and cytokines in animal models, this effect however, has not been investigated in kidney transplant recipients. We aimed to study the effect of ACEI or ARB treatment on intragraft gene expression profiles of transplant kidney biopsies using microarrays. Both groups had similar demographic characteristics in terms of age, race, sex, type of transplant, previous history of transplantation or acute rejection, panel reactive antibody levels, and immunosuppressive treatment. There were no differences in acute and chronic Banff allograft injury scores between the 2 Groups. Intragraft gene expression profiles of ACEI or ARB treated Group 2 biopsies showed decreased gene transcripts of interferon-gamma and rejection-associated transcripts (GRIT) and constitutive macrophage-associated transcripts (CMAT) compared to Group 1 biopsies. There were no statistically significant differences in expression of cytotoxic T cell (CAT), regulatory T cell (TREG), B-cell (BAT), natural killer cell (NKAT), or endothelial cell-associated transcripts (ENDAT) between the 2 Groups. Our data suggest that exposure to ACEI or ARB was associated with down-regulation of GRIT and CMAT. This anti-inflammatory effect of ACEI or ARB treatment could be an additional benefit in kidney transplant recipients. We identified 29 near normal biopsies with chronic sum allograft injury score (ct+ci+cv) ≤ 3 for gene expression profiling comparing 2 groups; Group 1 (n=16), patients with no exposure of ACEI or ARB treatment and Group 2 patients (n=13) with exposure to ACEI or ARB at least 6 months prior to kidney biopsy. Biopsies with a diagnosis of acute or chronic rejection, recurrent or de novo glomerular disease, or polyoma nephropathy were excluded.
Project description:Background & Aims. As a T cell-mediated disease of the colonic epithelium, ulcerative colitis (UC) is likely to share pathogenic elements with other T cell-mediated inflammatory diseases. Recently we showed T cell-mediated rejection of kidney and heart transplants share large scale molecular changes. We hypothesized that UC would manifest a similar disturbance, and that these features would correlate with response to infliximab. Results. UC biopsies manifested coordinate transcript changes resembling rejecting transplants, with T cell, IFNG-induced, macrophage, and injury transcripts increasing while parenchymal transcripts decreased. The disturbance expressed as principal component 1 correlated with conventional assessments e.g. Mayo scores, serum albumin, and lymphoplasmacytic infiltrate. When assessed in published microarray studies, the disturbance predicted response to infliximab: patients with intense disturbance did not achieve clinical response, although quantitative improvement was usually seen even in non-responders. Similar changes were seen in Crohn’s colitis (CDc). Conclusions. The molecular phenotype of UC manifests a large scale coordinate disturbance reflecting changes in inflammatory cells and parenchymal elements that correlates with conventional features and predicts response to infliximab. We studied 56 colon biopsies from patients with colitis, including 43 with UC, characterizing the clinical and histological features, and defined the mRNA phenotype with Affymetrix expression microarrays. We measured the expression of previously defined pathogenesis-based transcript sets representing effector T cells, macrophages, IFNG effects, and parenchymal injury response and dedifferentiation. We also studied 48 microarray files from human colon biopsies from the Gene Expression Omnibus database, classified by response to infliximab therapy to look for molecular changes that predict unresponsiveness.
Project description:Males are 50% more likely to develop end stage kidney failure compared to women. In this study we wanted to find out the molecular mechanism responsible for this increased risk. We collected kidney samples from patients with and without kidney disease and performed a comprehensive gene expression analysis in healthy and diseased male and female kidneys. Interestingly, the set of gender biased genes in healthy kidneys were different from those in diseased kidneys indicating not only baseline gene expression differences but also that the male and female kidney respond differently to disease condition. Our studies indicate that men and women with kidney problems might need to be treated differently. Keywords: Gender difference We collected 42 kidney samples from healthy living transplant donors, nephrectomies and from diagnostic kidney biopsies. Preliminary studies did not show significant gene expression differences in control kidneys based on the collection method (i.e. living kidney biopsy vs. unaffected portion of tumor nephrectomy). We grouped the tissue samples based on the histological readings of the kidney biopsies. Samples with evidence of glomerular and tubulointerstitial fibrosis were assigned into the diseased group. Characteristics of the research participants indicate diverse ethnic and disease groups and mild (StageIII) CKD. The tissue was microdissected into glomerular and tubulointerstitial fractions and expression arrays were performed separately.
Project description:Using meta-analysis of eight independent transplant datasets (236 graft biopsy samples) from four organs, we identified a common rejection module (CRM) consisting of 11 genes that were significantly overexpressed in acute rejection (AR) across all transplanted organs. The CRM genes could diagnose AR with high specificity and sensitivity in three additional independent cohorts (794 samples). In another two independent cohorts (151 renal transplant biopsies), the CRM genes correlated with the extent of graft injury and predicted future injury to a graft using protocol biopsies. Inferred drug mechanisms from the literature suggested that two FDA-approved drugs (atorvastatin and dasatinib), approved for non-transplant indications, could regulate specific CRM genes and reduce the number of graft infiltrating cells during acute rejection. We treated mice with HLA-mismatched murine cardiac transplant with atorvastatin and dasatinib and showed reduction of the CRM genes, significant reduction of graft infiltrating cells, and extended graft survival. We further validated the beneficial effect of atorvastatina on graft survival by retrospective analysis of electronic medical records of a single-center cohort of 2,515 renal transplant patients. In conclusion, we identified a CRM in transplantation that provides new opportunities for diagnosis, drug repositioning and rational drug design. The eight transplant data sets used for discovery include GSE4315, GSE2596, GSE4470, GSE9377, GSE1563, GSE9493, GSE13440, GSE6095. The two additional independent cohorts of 151 transplant biopsies are GSE25902 and GSE1563. Not all samples from all studies were used. Only the samples marked as either AR or STA in each data set were used for analysis. The 101 samples in this study were used as case-control experiment between acute rejection (AR) and stable (STA) in renal transplant. The samples in this series are one of the three independent validation cohorts. The other two cohorts are described in GSE21374 and GSE36059
Project description:Glomerular abnormalities have been demonstrated in kidney biopsies of patient after orthotopic liver transplantation (OLT). We hypothesize that these changes exist prior to OLT and may play a role in the development of renal failure after OLT. We use gene expression microarrays to investigate the mechanism of kidney disease in patients listed for OLT. Gene expression profiles of biopsies of cirrhotic patients were compared with pre-implantation living donor biopsies. Glomerular abnormalities were seen in 92% of the biopsies, the most common being increase in mesangial matrix. Electron microscope showed effacement of podocytes (93%) and duplication (35%) and widening of glomerular basement membrane (45%). Gene Ontology analysis revealed significant up-regulation of genes implicated in immune response, including T-cell, leucocyte and platelet activation and differentiation. Pathogenesis-based transcripts analysis revealed significantly increased expression of cytotoxic T-cell, macrophage, B-cell, natural killer cell, and endothelial cell associated transcripts, indicating an ongoing inflammatory immune response. Eight patients with cirrhotic livers listed for transplantation were referred to the kidney transplant clinic at Montefiore Medical Center, Bronx, New York, for evaluation of renal dysfunction between April 2010 and March 2013. Patients consented prior to clinically indicated kidney biopsy for enrollment into the study. Information and lab results were collated for each patient including: age, sex, race, etiology of liver disease, history of diabetes mellitus (DM) and hypertension (HTN), metabolic profile, liver function tests, glycosylated hemoglobin (HgbA1c), C3 and C4 complement concentrations, antinuclear antibody titers and spot urine protein to creatinine ratio. Patients completed a 24-hr urine collection for protein and creatinine, which was used to assess proteinuria and creatinine clearance. eGFR was calculated using the Modified Diet of Renal Disease (MDRD) formula. Hematuria was defined as presence of any red cells in urine analysis. Proteinuria was defined as protein excretion of greater than 100mg in 24 hour. HTN and DM were defined by use of anti-hypertensive and insulin or oral hypoglycemic agents respectively in past or at time of kidney biopsy. Biopsies from cirrhotic patients were then compared to nine pre-implantation biopsies from living kidney donors. The de-identified patient clinical characteristics are provided in each sample records; For Hematuria, HTN, MM (mesangial matrix expansion), MC (mesangial cell increase), II (interstitial inflammation), podocyte, BM (basement membrane thickening), the value 0 and 1 represents 'ABSENT' and 'PRESENT', respectively. For C3 and C4, the value 0 and 1 represents NORMAL and LOW C3 (or C4) LEVELS, repectively. For TA (tubular atrophy), IF (interstital fibrosis), AS (arteiosclerosis), the value 0, 1, and 2 represents ABSENT, PRESENT MILD, and PRESENT MODERATE, respectively. For IgG, IgA, IgM, C, and EmPE, the values represent 1 (ABSENT), 1 (MILD STAINING), 2 (MODERATE), 3 (SEVERE), NA (NOT AVAILABLE), P (PENDING). For BMWId, BMd, and EDD, the values represent 0 (ABSENT), 1 (PRESENT), NA (NOT AVAILABLE), P (PENDING).
Project description:One and four month formalin-fixed paraffin embedded biopsies from 48 kidney transplant recipients (24 AKI donors, 24 non-AKI) underwent global gene expression profiling using DNA microarrays (96 arrays). At one month, there were 898 differentially expressed genes in the AKI group (p-value <0.005; FDR <10%), but by 4-months there were no longer any differences. One and four month formalin-fixed paraffin embedded (FFPE) biopsies from 48 kidney transplant recipients (24 AKI donors, 24 controls; 96 total samples) were profiled using Affymetrix U133PM Plus PM plate arrays on a GeneTitan MC instrument. All samples were selected from the SCD cohort only. AKI donor and controls samples were matched by date of transplantation. Total RNA was extracted from four 20-micron sections using Ambion RecoverAll Total Nucleic Acid Kit and processed for chip hybridization using the new Affymetrix SensationPlus™ FFPE Amplification and 3’ IVT Labeling kit according to the manufacturer’s protocol. Quantile data normalization by RMA and analysis was performed using Partek Genomics Suite for PDR-corrected differential gene expression by ANOVA. Pathway mapping was performed using Ingenuity Pathway Analysis (IPA).