Pathogenesis-based Transcript Research Articles

#2683 NEXT GENERATION SEQUENCING: FROM BANFF SCORE MOLECULAR SIGNATURES AND CLASSIFIERS TO PREDICTED HISTOLOGICAL ARCHETYPES OF KIDNEY BIOPSIES

Abstract Background and Aims To improve risk stratification in kidney transplantation, molecular diagnostic tools are increasingly investigated for their feasibility and gain in diagnostic power. Nonetheless, previously published studies focused on sequencing technologies and gene panels with limited vision of the transcriptome (pathogenesis-based transcripts [PBTs], Banff Human Organ Transplant [BHOT] panel). The EU-TRAIN consortium was built to discover new predictive and informative biomarkers for kidney transplant histology and rejection diagnoses, leveraging a next generation sequencing technology. Method EU-TRAIN (NCT03652402) is a prospective multicentric study including unselected kidney transplant cohorts from 11 centres from 4 countries (France, Spain, Germany, Switzerland). We performed a bulk RNA sequencing on the polyadenylated probes of 770 kidney biopsies (n = 540 kidney recipients) collected between 2018 and 2021. Differential gene expression analyses were computed to obtain a molecular signature for all Banff score lesions. We then derived three different feature selections by either i) training an ElasticNet model on all differentially-expressed genes (DEGs), or by taking the top 30 ii) overall DEGs or iii) the top 30 DEGs focusing on transcripts included in the BHOT gene panel. From them, we trained four machine learning (ML) classifiers through a 10-times repeated 3-fold cross-validation. Models’ performances were assessed on a hold-out test set accounting for 30% of the total samples. Finally, we derived prototypic histological profiles using an archetypal analysis on the samples’ predicted Banff score probabilities from the best classifier. Results The ElasticNet feature selection lowered the number of DEGs to be included from a range of [859; 10,839] to [52; 867]. These selections were composed of a mixture of PBTs [55.2%; 84.2%], BHOT genes [2.2%;13.5%] and new transcripts [12.4%; 37.2%]. Based on these findings, four ML classifiers (Naïve Bayes, Extreme Gradient Boosting, Linear Support Vector Machine and K-Nearest Neighbours) were trained on the three different feature selections and their performances in predicting Banff score lesions were compared using the precision-recall area under the curve (PRAUC). In all settings, the ElasticNet feature selection outperformed the other two methods with a minimal/maximal increase in PRAUC of 0.068 (t) / 0.726 (cg). Excluding the ah score, the best discriminations were obtained with the Linear Support Vector Machine with a PRAUC in the interval [0.708; 0.980] (t/ptc). Excluding cv and ah, all models calibrated properly (Hosmer and Lemeshow goodness of fit p-value > 0.05). The archetypal analysis resulted in 8 profiles: acute and chronic antibody-mediated rejection (presence of circulating donor-specific antibodies, C4d deposition, g, ptc and cg lesions), acute T-cell mediated rejection (i, t and ti lesions), chronic TCMR (i, t, ti, ci, ct and i-IFTA lesions), mixed rejection (g, ptc, i, t and ti lesions), vascular injuries (cv and ah), fibrosis (ci and ct lesions, older donors with history of hypertension), minimal fibrotic change (ci and ct) and minor changes (no lesions). Conclusion From new transcripts, we managed to develop models that predict accurately the Banff score lesions and 8 profiles were identified among these predictions. External validation and archetypes’ association with graft loss will be addressed in the future.

Molecular patterns of isolated tubulitis differ from tubulitis with interstitial inflammation in early indication biopsies of kidney allografts

The Banff 2019 kidney allograft pathology update excluded isolated tubulitis without interstitial inflammation (ISO-T) from the category of borderline (suspicious) for acute T cell-mediated rejection due to its proposed benign clinical outcome. In this study, we explored the molecular assessment of ISO-T. ISO-T or interstitial inflammation with tubulitis (I + T) was diagnosed in indication biopsies within the first 14 postoperative days. The molecular phenotype of ISO-T was compared to I + T either by using RNA sequencing (n = 16) or by Molecular Microscope Diagnostic System (MMDx, n = 51). RNA sequencing showed lower expression of genes related to interferon-y (p = 1.5 *10–16), cytokine signaling (p = 2.1 *10–20) and inflammatory response (p = 1.0*10–13) in the ISO-T group than in I + T group. Transcripts with increased expression in the I + T group overlapped significantly with previously described pathogenesis-based transcript sets associated with cytotoxic and effector T cell transcripts, and with T cell-mediated rejection (TCMR). MMDx classified 25/32 (78%) ISO-T biopsies and 12/19 (63%) I + T biopsies as no-rejection. ISO-T had significantly lower MMDx scores for interstitial inflammation (p = 0.014), tubulitis (p = 0.035) and TCMR (p = 0.016) compared to I + T. Fewer molecular signals of inflammation in isolated tubulitis suggest that this is also a benign phenotype on a molecular level.

Molecular phenotyping of rejection-related changes in mucosal biopsies from lung transplants.

Diagnosing lung transplant rejection currently depends on histologic assessment of transbronchial biopsies (TBB) with limited reproducibility and considerable risk of complications. Mucosal biopsies are safer but not histologically interpretable. Microarray-based diagnostic systems for TBBs and other transplants suggest such systems could assess mucosal biopsies as well. We studied 243 mucosal biopsies from the third bronchial bifurcation (3BMBs) collected from seven centers and classified them using unsupervised machine learning algorithms. Using the expression of a set of rejection-associated transcripts annotated in kidneys and validated in hearts and lung transplant TBBs, the algorithms identified and scored major rejection and injury-related phenotypes in 3BMBs without need for labeled training data. No rejection or injury, rejection, late inflammation, and recent injury phenotypes were thus scored in new 3BMBs. The rejection phenotype correlated with IFNG-inducible transcripts, the hallmarks of rejection. Progressive atrophy-related changes reflected by the late inflammation phenotype in 3BMBs suggest widespread time-dependent airway deterioration, which was especially pronounced after two years posttransplant. Thus molecular assessment of 3BMBs can detect rejection in a previously unusable biopsy format with potential utility in patients with severe lung dysfunction where TBB is not possible and provide unique insights into airway deterioration. ClinicalTrials.gov NCT02812290.

Molecular signatures and clinical outcomes of transplant glomerulopathy stratified by microvascular inflammation and donor-specific antibody.

We investigated clinical outcomes and molecular signatures of transplant glomerulopathy (TG) stratified by microvascular inflammation (MVI) and donor-specific antibody (DSA) status. We performed a retrospective review of 749 kidney transplant patients who received a for-cause kidney biopsy from 2009 to 2014. We classified TG as MVI positive (MVI+) or MVI negative (MVI-), and with or without DSA. We obtained gene expression profiles for 44 biopsies by Affymetrix HuGene 1.0 ST expression arrays. A total of 100 patients had TG; 49 were MVI+, and 51 were MVI-. After a median post-biopsy follow-up of 2.08years (range 0.43-4.59), Kaplan-Meier survival analysis demonstrated worse allograft survival in MVI+ TG patients compared with MVI- TG patients (P=0.01), and time to graft failure was significantly shorter in MVI+ patients (1.08±1.01years vs 2.3±1.8years; P=0.002). DSA status did not affect graft survival within MVI+ or MVI- groups. Analysis of pathogenesis-based transcripts (PBT) showed that MVI+ TG biopsies had increased expression of gamma interferon and rejection (GRIT) and DSA-associated transcripts (DSAST), as observed in antibody-mediated rejection. MVI- TG biopsies had increased expression of cytotoxic and regulatory T cell- and B cell-associated transcripts but not GRIT or DSAST. DSA status had no effect on expression of any PBTs studied in MVI- TG biopsies. Graft survival in TG is significantly worse in the presence of MVI. Gene expression profiles of MVI+ TG resemble antibody-mediated rejection while gene expression profiles of MVI- TG resemble cell-mediated rejection regardless of DSA status.

Effect of Prolonged Cold Ischemia on Intragraft Gene Expression Profiles

Background Deceased-donor (DD) kidneys are at higher risk for ischemia/reperfusion injury leading to increased inflammatory mediators and innate immune response. We aimed to investigate the effects of prolonged cold ischemia on intragraft molecular gene expression profiles of DD kidneys comparing to living-donors (LD) pre-implantation biopsies using microarrays. Methods There were 48 pre-implantation kidney biopsy samples (29 LD and 19 DD). The cold ischemia time (CIT) was < 16 hours in 10 and > 16 hours in 9 DD kidneys. The gene expression profiles were studied by Affymetrix HuGene 1.0 ST expression arrays. Results There were 198 differentially expressed genes when pre-implantation DD biopsies were compared to LD biopsies (Both False discovery rate p<0.05 and fold change >2). Pathogenesis based transcripts (PBT) showed increased transcripts associated with injury and response (IRIT), Constitutive Macrophage (CMAT), and Gamma Interferon (GRIT) in DD samples compared to pre-implantation LD biopsies. There was no difference in Cytotoxic T-cell (CAT), Natural-Killer cell (NKAT), B-cell (BAT), and Endothelial cell (ENDAT) associated gene transcripts between 2 groups. However, there was no statistically significant difference in expression of any PBTs studied when biopsies with CIT > 16 hours compared to biopsies with CIT < 16 hours. Gene Ontology analysis showed that DD preimplantation biopsies had increased expression of pathways related to acute inflammatory response, lymphocyte mediated immunity, innate and humoral immune response, complement activation and regulation of immune response and IL-6 activation. Conclusions Pre-implantation DD kidney biopsies showed increased expression of gene transcripts associated with inflammatory mediators, cytokines, macrophages, innate immune response and response to injury compared to LD kidneys. However, increased cold ischemia time does not change the intragraft gene expression profiles.

Gene Expression Profiling for the Identification and Classification of Antibody-Mediated Heart Rejection.

Antibody-mediated rejection (AMR) contributes to heart allograft loss. However, an important knowledge gap remains in terms of the pathophysiology of AMR and how detection of immune activity, injury degree, and stage could be improved by intragraft gene expression profiling. We prospectively monitored 617 heart transplant recipients referred from 4 French transplant centers (January 1, 2006-January 1, 2011) for AMR. We compared patients with AMR (n=55) with a matched control group of 55 patients without AMR. We characterized all patients using histopathology (ISHLT [International Society for Heart and Lung Transplantation] 2013 grades), immunostaining, and circulating anti-HLA donor-specific antibodies at the time of biopsy, together with systematic gene expression assessments of the allograft tissue, using microarrays. Effector cells were evaluated with in vitro human cell cultures. We studied a validation cohort of 98 heart recipients transplanted in Edmonton, AB, Canada, including 27 cases of AMR and 71 controls. A total of 240 heart transplant endomyocardial biopsies were assessed. AMR showed a distinct pattern of injury characterized by endothelial activation with microcirculatory inflammation by monocytes/macrophages and natural killer (NK) cells. We also observed selective changes in endothelial/angiogenesis and NK cell transcripts, including CD16A signaling and interferon-γ-inducible genes. The AMR-selective gene sets accurately discriminated patients with AMR from those without and included NK transcripts (area under the curve=0.87), endothelial activation transcripts (area under the curve=0.80), macrophage transcripts (area under the curve=0.86), and interferon-γ transcripts (area under the curve=0.84; P<0.0001 for all comparisons). These 4 gene sets showed increased expression with increasing pathological AMR (pAMR) International Society for Heart and Lung Transplantation grade (P<0.001) and association with donor-specific antibody levels. The unsupervised principal components analysis demonstrated a high proportion of molecularly inactive pAMR1(I+), and there was significant molecular overlap between pAMR1(H+) and full-blown pAMR2/3 cases. Endothelial activation transcripts, interferon-γ, and NK transcripts showed association with chronic allograft vasculopathy. The molecular architecture and selective AMR transcripts, together with gene set discrimination capacity for AMR identified in the discovery set, were reproduced in the validation cohort. Tissue-based measurements of specific pathogenesis-based transcripts reflecting NK burden, endothelial activation, macrophage burden, and interferon-γ effects accurately classify AMR and correlate with degree of injury and disease activity. This study illustrates the clinical potential of a tissue-based analysis of gene transcripts to refine diagnosis of heart transplant rejection.

Microarray Analysis of Crohnʼs Disease and Correlation With Traditional Clinical and Histologic Features

Introduction: As a T cell-mediated disease of the gastrointestinal epithelium, Crohn's disease (CD) is likely to share pathogenic elements with other T cell-mediated inflammatory diseases. Recently we showed that ulcerative colitis (UC) manifested large-scale molecular disturbances that correlated with endoscopic and histologic features(IBD 20: 2353, 2014). We hypothesized that ileal CD would manifest similar disturbance. Methods: We studied 27 patients in 31 biopsies with ileal CD, characterizing the clinical, endoscopic and histological features and defined the mRNA phenotype using microarray analysis of ileal biopsies. We measured the expression of pathogenesis-based transcript sets (PBTs) previously published for ulcerative colitis representing effector T cells, macrophages, IFNG effects, and parenchymal injury-repair response and dedifferentiation (table 1). The molecular features were then correlated with conventional assessments including clinical features (modified Harvey Bradshaw index(HBI), simple endoscopic score for CD(SES-CD), c-reactive protein, albumin) and histologic features (lamina propria neutrophilic and lymphoplasmacytic infiltrate, crypt abscess, ulcers present and crypt architectural distortion). Results: CD ileal biopsies arranged by injury-repair score (IRRAT) manifested coordinate transcript changes with IFNG-induced transcripts (GRIT), macrophage transcripts (QCMAT), and injury-repair transcripts increasing while parenchymal transcripts (PT) decreased (figure 1). Lymphoplasmacytic infiltrate was significantly correlated with IRRAT (P=0.005) and negatively correlated with parenchymal transcript expression (P=0.01). Neutrophilic lamina propria infiltrate (p=0.03) and number of ulcers (p=0.03) also correlated with IRRAT. No significant correlation was seen between the molecular features and the HBI (P=0.5), SES-CD(P=0.8) or CRP (0.2).Figure 1Table 1: Pathogenesis-based transcript sets (PBTs)Conclusion: The molecular phenotype of CD manifests a large-scale coordinate disturbance similar to that in UC and other T cell-mediated diseases, reflecting changes in inflammatory cells and parenchymal elements and correlating with histologic assessment, especially the lymphoplasmacytic and neutrophilic lamina propria infiltrate, but not with the clinical and endoscopic features. This raises further questions about our clinical and endoscopic assessments of CD. Novel molecular systems for quantitating and staging the disease elements in the tissues in CD may add a significant new dimension to patient management beyond our current standards.

Molecular patterns in human ulcerative colitis and correlation with response to infliximab.

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, microarray analysis revealed large-scale molecular changes in T cell-mediated rejection of kidney and heart transplants. We hypothesized that similar disturbances might be operating in UC and could provide insights into responsiveness to therapy. We studied 56 colon biopsies from patients with colitis characterizing the clinical and histological features and using microarrays to define the messenger RNA phenotype. We expressed the microarray results using previously defined pathogenesis-based transcript sets. We also studied 48 published microarray files from human colon biopsies downloaded from the Gene Expression Omnibus database, classified by response to infliximab therapy, to examine whether the molecular measurements derived from our studies correlated with nonresponsiveness to treatment. UC biopsies manifested coordinate transcript changes resembling rejecting transplants, with effector T cell, IFNG-induced, macrophage, and injury transcripts increasing while parenchymal transcripts decreased. The disturbance in gene expression, summarized as principal component 1 (PC1), correlated with conventional clinical and histologic assessments. When assessed in microarray results from published studies, the disturbance (PC1) predicted response to infliximab: patients with intense disturbance did not achieve clinical response, although quantitative improvement was seen even in many clinical nonresponders. Similar changes were seen in Crohn's colitis. 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.

A pathogenesis-based transcript signature in donor-specific antibody-positive kidney transplant patients with normal biopsies.

Affymetrix Human Gene 1.0-ST arrays were used to assess the gene expression profiles of kidney transplant patients who presented with donor-specific antibodies (DSAs) but showed normal biopsy histopathology and did not develop antibody-mediated rejection (AMR). Biopsy and whole-blood profiles for these DSA-positive, AMR-negative (DSA +/AMR−) patients were compared to both DSA-positive, AMR-positive (DSA +/AMR +) patients as well as DSA-negative (DSA −) controls. While individual gene expression changes across sample groups were relatively subtle, gene-set enrichment analysis using previously identified pathogenesis-based transcripts (PBTs) identified a clear molecular signature involving increased rejection-associated transcripts in AMR − patients. Results from this study have been published in Kidney International (Hayde et al., 2014 [1]) and the associated data have been deposited in the GEO archive and are accessible via the following link: http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE50084

Effects of Prolonged Cold Ischemia On Intragraft Gene Expression Profiles.

Background: Deceased-donor (DD) kidneys are at higher risk for ischemia/reperfusion injury leading to increased inflammatory mediators and innate immune response. We aimed to investigate the effects of prolonged cold ischemia on intragraft molecular gene expression profiles of DD kidneys comparing to living-donors (LD) before and after transplantation using microarrays. Methods: There were 20 pre-implantation kidney biopsy samples (10 LD and 10 DD). The cold ischemia time (CIT) was < 24 hours in 7 and > 24 hours in 3 DD kidneys. There were 15 post-transplant biopsy samples from DD (8 had CIT < 24 h and 7 had > 24 h) with the diagnosis of acute tubular necrosis (ATN) which were compared to 9 normal transplant biopsies. The gene expression profiles were studied by Affymetrix HuGene 1.0 ST expression arrays. Results: There were 336 differentially expressed genes when pre-implantation DD biopsies were compared to LD biopsies (Both False discovery rate p<0.05 and fold change >2). Pathogenesis based transcripts (PBT) showed increased transcripts associated with injury and response (IRIT), Cytotoxic T-cell (CAT), Natural-Killer cell (NKAT), Constitutive Macrophage (CMAT), and Gamma Interferon (GRIT) in DD samples compared to pre-implantation LD biopsies. There was no difference in T-regulatory cell (TREG), B-cell (BAT), and Endothelial cell (ENDAT) associated gene transcripts between 2 groups. However, there was no statistically significant difference in expression of any PBTs studied when biopsies with CIT > 24 hours compared to biopsies with CIT < 24 hours. There was also no difference in expression of any PBTs studied in post-transplant DD biopsies with the diagnosis of ATN compared to normal transplant biopsies (Table, P-value for significance <0.05). Conclusions: Pre-implantation DD biopsies showed increased expression of gene transcripts associated with inflammatory mediators (IRIT and GRIT) and innate immune response involving NKAT and CAT compared to LD kidneys. However, increased CIT does not change the intragraft gene expression profiles before and after transplantation.Table: No Caption available.

The Molecular Phenotype of Antibody-Mediated Rejection in Heart Transplants.

Background ABMR is a major cause of organ transplant failure. Recently, the molecular landscape of ABMR has been defined in kidney transplantation. We hypothesized that there are common molecular signatures and overlapping rejection mechanisms across these two organs. Methods We enrolled heart transplant recipients from four French centers with a diagnosis of biopsy proven ABMR and adequateendomyocardial (EMB) material for microarray processing (n=21). ABMR diagnosis was based on the presence of circulating DSA using Luminex SA together with histopathology evidence of intra-vascular mononuclear cells and/or C4d/CD68 IHC positivity (ISHLT 2013). We used the Molecular Microscope system of diagnostic classifier equations (ABMR Score) and pathogenesis-based transcript sets (PBTs) derived from literature and validated in kidney transplantation: endothelial DSA-selective transcripts (eDSAST), macrophage transcripts (QCMAT), gamma-interferon response (GRIT), injury-repair response transcripts (IRRAT) and the ABMR Score. We compared the gene expression in EMB in the ABMR group to a matched control group of heart recipients with normal EMB (n=37). Results All patients with ABMR EMB had DSA at the time of biopsy: class I in 4 (19%), class II in 10 (47.6%) and class I and II in 7 (33.3%). The mean class I or II immunodominant DSA MFI was 4940 ± 718. The histopathological features of ABMR revealed 15 cases of pAMR1 (71%), 5 cases of pAMR2 (24%) and 1 case of pAMR3 (5%). Eight patients had positive C4d/CD68 staining. The gene expression analysis revealed that as compared EMB from heart transplant patients without rejection, EMB with ABMR showed distinct molecular signal characterized by higher expression of eDSAST (p<0.0001), QCMAT (p<0.0001), GRIT (p<0.0001), IRRAT (p=0.0001) and increase of the ABMR Score that reflects interferon-effects, microcirculation stress and NK burden (p<0.0001). Conclusion The molecular signature of ABMR in heart shows common features with relevant gene scores and PBTs identified in kidney transplant biopsies. This suggest common mechanisms involved in ABMR - endothelial response to injury, NK transcripts, IFNG effects - and may provide basis for improving diagnosis and identifying adapted therapeutic strategies in heart transplantation. DISCLOSURE:Halloran, P.: Other, Astellas, Lecturing, Novartis, Lecturing, One Lambda, Lecturing.

Evidence for Decreased Subclinical Antibody-Mediated Injury in Kidney Biopsies After Simultaneous Liver-Kidney Transplantation.

In positive crossmatch kidney transplantation (+XM-KT) with high levels of donor-specific alloantibody (DSA), subclinical chronic injury is demonstrable at 1-yr in almost all grafts when sensitive methods are used (e.g. gene expression and protocol biopsies). Our goal was to determine if similar low levels of chronic injury are present in the kidney at 1-yr after +XM simultaneous liver-kidney transplantation (+XM-SLK) where the liver appears to protect the kidney from clinical injury. Methods: Surveillance 1-yr protocol kidney biopsies of 9 +XM-SLK and 15 age-, sex- and race-matched +XM-KT recipients with similar graft function (eGFR 42±12.6 and 53±21.9, respectively) were scored using Banff ‘97 and intragraft gene expression was assessed by Illumina microarrays. At transplant, all had a positive flow cytometric XM and DSA demonstrated by LABscreen solid phase assay (mean highest DSA MFI=10022 in +XM-SLK; 6339 in +XM-KT). Inflammatory cell-related (Ingenuity Knowledge base) and Pathogenesis-based transcript sets were used to calculate gene set scores of all detectable transcripts. Results: By light microscopy, peritubular capillaritis (PTC score>1) was less common after +XM-SLK (25 vs 83%, P<0.05). Gene expression also showed a lower incidence of subclinical microvascular inflammation (e.g. Macrophage adhesion and NK pathways) in +XM-SLK compared to +XM-KT. In addition, other individual transcripts associated with microvascular inflammation, such as vascular endothelial adhesion (VE-cadherin, CD34), activation (TEK-tyrosine kinase), leukocyte migration (PECAM) and monocyte chemoattractant protein were significantly lower in +XM-SLKT. Endothelial cell activation, a process associated with DSA in +XM-KT, was also less common after +XM-SLK (P<0.05). Two +XM-SLK had persistent circulating DSA at the time of the biopsy (* in Fig. 1); both had profiles similar to +XM-KT and PTC >1. Transcripts for T cell activation, proliferation and adhesion were not different between two groups. Conclusions: The data suggest that the liver truly protects kidneys from chronic antibody-associated injury. This effect appears to be related to the lack of circulating DSA after SLK.Figure: No Caption available.

Genomics of BK Viremia in Kidney Transplant Recipients

This study aimed to investigate global gene expression profiles of BK viremia and nephropathy (BKVN) samples using microarrays to investigate the immunologic response to BK virus. Patients were monitored for BK viremia in the blood monthly for 6 months, then at 9 and 12 months after kidney transplantation. BKVN and normal transplant kidney biopsy samples, and whole blood samples of patients with and without BK viremia were analyzed by Affymetrix Human Gene 1.0 ST Arrays. During a mean follow-up of 917±325 days, 61 of the 289 patients (21%) developed BK viremia at a median 149 (27, 1,113) days after transplantation with a median peak PCR titers of 35,900 (1,000, 2,677,000). The only significant risk factor for development of BK viremia was induction with anti-thymocyte globulin (P=0.03). Only four patients developed BKVN (1.3%). Pathogenesis-based transcript analysis revealed a significant increased expression of interferon-gamma and rejection induced (GRIT), quantitative cytotoxic T-cell (QCAT), quantitative constitutive and alternate macrophage, B-cell and natural killer cell-associated transcripts (NKAT), indicating an active inflammatory immune response in BKVN biopsies (n=3) compared to normal transplant kidney biopsies with (n=3) and without BK viremia (n=11). The whole blood gene expression profiles of 19 BK viremia patients revealed significant increased expression of GRIT, QCAT, and NKAT compared to 14 patients without viremia. The results showed increased activity of cytotoxic T cells and natural killer cells in BKVN and viremia samples resembling acute rejection and suggested the involvement of both adaptive and innate immunity.

The Clinical and Molecular Significance of C4d Staining Patterns in Renal Allografts

We investigated the clinical and molecular significance of minimal peritubular capillary (PTC) and isolated glomerular C4d+ staining using microarrays. Two hundred fifty-five clinically indicated transplant biopsies were included in the analyses. C4d staining was performed on paraffin sections using a polyclonal rabbit anti-C4d antibody. Gene expression profiles in a subset of patients were studied using Affymetrix HuGene 1.0ST arrays. Immunohistochemistry for C4d of 255 biopsies showed 51% C4d negative, 4% minimal PTC C4d+, 15% focal or diffuse PTC C4d+, and 31% isolated glomerular C4d+ biopsies. Patients with minimal and focal/diffuse PTC C4d+ staining had higher frequency of donor-specific anti-HLA antibodies (DSA) (67% and 82%) and antibody mediated rejection (AMR) (66% and 89%) when compared with C4d-negative biopsies (25% and 19%, respectively) (P<0.001). The glomerulitis, interstitial inflammation, and peritubular capillaritis scores were also significantly higher in minimal (0.88, 1.25, and 1.5) and focal/diffuse PTC C4d+ biopsies (0.65, 1.41, and 1.5), compared with C4d-negative biopsies (0.25, 079, and 0.34), respectively. There were no differences in the DSA frequency, AMR rate, or Banff scores between isolated glomerular C4d+ and C4d-negative patients. Although both minimal and focal/diffuse C4d+ biopsies showed increased expression of genes related to the immune response, interferon-gamma and rejection-induced, cytotoxic T cell and constitutive macrophage-associated pathogenesis-based transcripts, there was no activation of immune-response-related genes in isolated glomerular C4d+ biopsies. Minimal PTC C4d+ staining but not isolated glomerular C4d+ staining is associated with AMR, circulating DSAs and immune-response-related gene activation.

The Molecular Phenotype of 6-Week Protocol Biopsies from Human Renal Allografts: Reflections of Prior Injury but Not Future Course

We assessed the molecular phenotype of 107 6-week protocol biopsies from human renal allografts, using Affymetrix microarrays. Transcript changes were summarized as nonoverlapping pathogenesis-based transcript sets (PBTs) reflecting inflammation (T cells, macrophages, IFNG effects) and the injury-repair response of the parenchyma, stroma and microcirculation-increased ('injury-up') and decreased ('injury-down') transcripts. The molecular changes were highly correlated with each other, even when all rejection and borderline cases were excluded. Inflammation and injury-down PBTs correlated with histologic inflammation and tubulitis, and the inflammation transcripts were greater in kidneys diagnosed as T cell-mediated or borderline rejection. Injury-up PBTs did not correlate with histopathology but did correlate with kidney function: thus functional disturbances are represented in transcript changes but not in histopathology. PBT changes correlated with prior delayed graft function. However, there was little difference between live donor kidneys and deceased donor kidneys that had not shown delayed graft function. Molecular changes did not predict future biopsies for clinical indications, rejection episodes, functional deterioration or allograft loss. Thus while detecting T cell-mediated inflammation, the molecular phenotype of early protocol biopsies mostly reflects the injury-repair response to implantation stresses, and has little relationship to future events and outcomes.

The Molecular Phenotype of Kidney Transplants

Microarray studies of kidney transplant biopsies provide an opportunity to define the molecular phenotype. To facilitate this process, we used experimental systems to annotate transcripts as members of pathogenesis-based transcript sets (PBTs) representing biological processes in injured or diseased tissue. Applying this annotation to microarray results revealed that changes in single molecules and PBTs reflected a large-scale coordinate disturbance, stereotyped across various diseases and injuries, without absolute specificity of individual molecules or PBTs for rejection. Nevertheless, expression of molecules and PBTs was quantitatively specific: IFNG effects for rejection; T cell and macrophage transcripts for T cell-mediated rejection; endothelial and NK transcripts for antibody-mediated rejection. Various diseases and injuries induced the same injury-repair response, undetectable by histopathology, involving epithelium, stroma and endothelium, with increased expression of developmental, cell cycle and apoptosis genes and decreased expression of differentiated epithelial features. Transcripts reflecting this injury-repair response were the best correlates of functional disturbance and risk of future graft loss. Late biopsies with atrophy-fibrosis, reflecting their cumulative burden of injury, displayed more transcripts for B cells, plasma cells and mast cells. Thus the molecular phenotype is best described in terms of three elements: specific diseases, including rejection; the injury-repair response and the cumulative burden of injury.

Using gene arrays in diagnosis of rejection

In the last decade, microarray technology has revolutionized biological research by allowing the screening of tens of thousands of genes simultaneously. This article reviews recent studies in organ transplantation using microarrays and highlights the issues that should be addressed in order to use microarrays in diagnosis of rejection. Microarrays have been useful in identifying potential biomarkers for chronic rejection in peripheral blood mononuclear cells, novel pathways for induction of tolerance, and genes involved in protecting the graft from the host immune system. Microarray analysis of peripheral blood mononuclear cells from chronic antibody-mediated rejection has identified potential noninvasive biomarkers. In a recent study, correlation of pathogenesis-based transcripts with histopathologic lesions is a promising step towards inclusion of microarrays in clinics for organ transplants. Despite promising results in diagnosis of histopathologic lesions using microarrays, the low dynamic range of microarrays and large measured expression changes within the probes for the same gene continue to cast doubts on their readiness for diagnosis of rejection. More studies must be performed to resolve these issues. Dominating expression of globin genes in whole blood poses another challenge for identification of noninvasive biomarkers. In addition, studies are also needed to demonstrate effects of different immunosuppression therapies and their outcomes.

Analysis of independent microarray datasets of renal biopsies identifies a robust transcript signature of acute allograft rejection

Transcriptomics could contribute significantly to the early and specific diagnosis of rejection episodes by defining 'molecular Banff' signatures. Recently, the description of pathogenesis-based transcript sets offered a new opportunity for objective and quantitative diagnosis. Generating high-quality transcript panels is thus critical to define high-performance diagnostic classifier. In this study, a comparative analysis was performed across four different microarray datasets of heterogeneous sample collections from two published clinical datasets and two own datasets including biopsies for clinical indication, and samples from nonhuman primates. We characterized a common transcriptional profile of 70 genes, defined as acute rejection transcript set (ARTS). ARTS expression is significantly up-regulated in all AR samples as compared with stable allografts or healthy kidneys, and strongly correlates with the severity of Banff AR types. Similarly, ARTS were tested as a classifier in a large collection of 143 independent biopsies recently published by the University of Alberta. Results demonstrate that the 'in silico' approach applied in this study is able to identify a robust and reliable molecular signature for AR, supporting a specific and sensitive molecular diagnostic approach for renal transplant monitoring.

FOXP3 Expression in Human Kidney Transplant Biopsies Is Associated with Rejection and Time Post Transplant but Not with Favorable Outcomes

Expression of the transcription factor forkhead box P3 (FOXP3) in transplant biopsies is of interest due to its role in a population of regulatory T cells. We analyzed FOXP3 mRNA expression using RT-PCR in 83 renal transplant biopsies for cause in relationship to histopathology, clinical findings and expression of pathogenesis-based transcript sets assessed by microarrays. FOXP3 mRNA was higher in rejection (T-cell and antibody-mediated) than nonrejection. Surprisingly, some native kidney controls also expressed FOXP3 mRNA. Immunostaining for FOXP3 was consistent with RT-PCR, showing interstitial FOXP3+ lymphocytes, even in some native kidney controls. FOXP3 expression correlated with interstitial inflammation, tubulitis, interstitial fibrosis, tubular atrophy, C4d positivity, longer time posttransplant, younger donors, class II panel reactive antibody >20% and transcript sets reflecting inflammation and injury, but unlike these features was time dependent. In multivariate analysis, higher FOXP3 mRNA was independently associated with rejection, T-cell-associated transcripts, younger donor age and longer time posttransplant. FOXP3 expression did not correlate with favorable graft outcomes, even when the analysis was restricted to biopsies with rejection. Thus FOXP3 mRNA expression is a time-dependent feature of inflammatory infiltrates in renal tissue. We hypothesize that time-dependent entry of FOXP3-positive cells represents a mechanism for stabilizing inflammatory sites.

Comparing Microarray Versus RT-PCR Assessment of Renal Allograft Biopsies: Similar Performance Despite Different Dynamic Ranges

In renal allografts, assessing gene expression can add relevant diagnostic information to histopathology. Results can be expressed as single genes or gene sets, representing pathogenesis-based transcript sets (PBTs): cytotoxic T-cell-associated, interferon gamma- induced or decreased kidney parenchymal transcripts. Two technology platforms are available: RT-PCR and microarrays. We compared RT-PCR, U133plus2.0 microarrays and histopathology in 86 biopsies. We compared 13 potentially diagnostic genes as RT-PCR probes to microarray-derived PBTs, 'mini'-PBTs (small sets of 3-5 transcripts) and a histology classifier. Most RT-PCR probes (10/13) correlated well with the corresponding microarray probe sets (r > 0.8). Exceptions included FASLG and CD8B1 microarray probe sets, which were not performing on microarrays but were detectable by RT-PCR most likely due to differences in sensitivity. In general, RT-PCR showed greater dynamic range, detecting small changes in normal kidneys, but RT-PCR and microarrays gave similar results in abnormal kidneys. Individual transcripts or mini-PBTs assessed by either platform correlated well with one another, with microarray PBTs and the histology classifier. Thus, microarrays and RT-PCR assessments agree strongly with one another and histopathology in assessing transplant inflammation, particularly, when results are expressed as PBTs or mini-PBTs. The dynamic range of both platforms was sufficient to detect the relevant changes in rejection.