Banff Histopathological Consensus Criteria for Preimplantation Kidney Biopsies.

The Banff working group on preimplantation biopsy was established to develop consensus criteria (best practice guidelines) for the interpretation of preimplantation kidney biopsies. Digitally scanned slides were used (i) to evaluate interobserver variability of histopathologic findings, comparing frozen sections with formalin-fixed, paraffin-embedded tissue of wedge and needle core biopsies, and (ii) to correlate consensus histopathologic findings with graft outcome in a cohort of biopsies from international medical centers. Intraclass correlations (ICCs) and univariable and multivariable statistical analyses were performed. Good to fair reproducibility was observed in semiquantitative scores for percentage of glomerulosclerosis, arterial intimal fibrosis and interstitial fibrosis on frozen wedge biopsies. Evaluation of frozen wedge and core biopsies was comparable for number of glomeruli, but needle biopsies showed worse ICCs for glomerulosclerosis, interstitial fibrosis and tubular atrophy. A consensus evaluation form is provided to help standardize the reporting of histopathologic lesions in donor biopsies. It should be recognized that histologic parameters may not correlate with graft outcome in studies based on organs deemed to be acceptable after careful clinical assessment. Significant limitations remain in the assessment of implantation biopsies.

Molecular Assessment of Microcirculation Injury in Formalin-Fixed Human Cardiac Allograft Biopsies With Antibody-Mediated Rejection.

Precise diagnosis of antibody-mediated rejection (AMR) in cardiac allograft endomyocardial biopsies (EMBs) remains challenging. This study assessed molecular diagnostics in human EMBs with AMR. A set of 34 endothelial, natural killer cell and inflammatory genes was quantified in 106 formalin-fixed, paraffin-embedded EMBs classified according to 2013 International Society for Heart and Lung Transplantation (ISHLT) criteria. The gene set expression was compared between ISHLT diagnoses and correlated with donor-specific antibody (DSA), endothelial injury by electron microscopy (EM) and prognosis. Findings were validated in an independent set of 57 EMBs. In the training set (n = 106), AMR cases (n = 70) showed higher gene set expression than acute cellular rejection (ACR; n = 21, p < 0.001) and controls (n = 15, p < 0.0001). Anti-HLA DSA positivity was associated with higher gene set expression (p = 0.01). Endothelial injury by electron microscopy strongly correlated with gene set expression, specifically in AMR cases (r = 0.62, p = 0.002). Receiver operating characteristic curve analysis for diagnosing AMR showed greater accuracy with gene set expression (area under the curve [AUC] = 79.88) than with DSA (AUC = 70.47) and C4d (AUC = 70.71). In AMR patients (n = 17) with sequential biopsies, increasing gene set expression was associated with inferior prognosis (p = 0.034). These findings were confirmed in the validation set. In conclusion, biopsy-based molecular assessment of antibody-mediated microcirculation injury has the potential to improve diagnosis of AMR in human cardiac transplants.

Long-term impact of CMV infection on allografts and on patient survival in renal transplant patients with protocol biopsies.

Cytomegalovirus (CMV) infection is a frequent complication of early posttransplantation. This study examines its impact on chronic allograft changes, long-term graft loss, and patient survival. We studied 594 patients who had protocol biopsies at 6 wk, and 3 and 6 mo posttransplantation. Chronic allograft changes were evaluated according to the updated Banff classification [interstitial fibrosis/tubular atrophy (IF/TA), vascular and glomerular lesions]. Follow-up data were available for up to 10 yr. CMV infection was diagnosed in 153 of 594 patients (26%) in the first year after transplantation, mostly within the first 3 mo. Graft survival was reduced in patients with CMV (P = 0.03) as well as the combined allograft/patient survival (P = 0.008). Prevalence of IF/TA at 6 wk after transplantation was already threefold higher in patients who experienced CMV infection later on compared with patients without CMV (P = 0.005). In multivariate analyses, CMV viremia or disease was not a significant factor for graft loss or death. In conclusion, patients with CMV infection posttransplantation show more chronic allograft changes early on, even before CMV infection, and development of IF/TA is not more prevalent in patients with CMV. Our data do not support a significant role of CMV in patient and graft outcomes.

Banff Initiative for Quality Assurance in Transplantation (BIFQUIT): reproducibility of polyomavirus immunohistochemistry in kidney allografts.

Immunohistochemistry (IHC) is the gold standard for diagnosing (positive vs. negative) polyomavirus BK (BKV) nephropathy and has the potential for disease staging based on staining intensity and quantification of infected cells. This multicenter trial evaluated the reproducibility of BKV IHC among 81 pathologists at 60 institutions. Participants stained tissue microarray slides and scored them for staining intensity and percentage of positive nuclei. Staining protocol details and evaluation scores were collected online. Slides were returned for centralized panel re-evaluation and kappa statistics were calculated. Individual assessment of staining intensity and percentage was more reproducible than combined scoring. Inter-institutional reproducibility was moderate for staining intensity (κ = 0.49) and percentage (κ = 0.42), fair for combined (κ = 0.25) and best for simple positive/negative scoring (κ = 0.78). Inter-observer reproducibility was substantial for intensity (κ = 0.74), percentage (κ = 0.66), positive/negative (κ = 0.78) and moderate for combined scoring (κ = 0.43). Inter-laboratory reproducibility was fair for intensity (κ = 0.37), percentage (κ = 0.40) and combined (κ = 0.24), but substantial for positive/negative scoring (κ = 0.67). BKV RNA copies/cell correlated with staining intensity (r = 0.56) and percentage (r = 0.62). These results indicate that BKV IHC is reproducible between observers but scoring should be simplified to a single-feature schema. Standardization of tissue processing and staining protocols would further improve inter-laboratory reproducibility.

Banff fibrosis study: multicenter visual assessment and computerized analysis of interstitial fibrosis in kidney biopsies.

Increasing interstitial fibrosis (IF) in native and kidney transplant biopsies is associated with functional decline and serves as a clinical trial end point. A Banff 2009 Conference survey revealed a range in IF assessment practices. Observers from multiple centers were asked to assess 30 renal biopsies with a range of IF and quantitate IF using two approaches on trichrome, Periodic acid-Schiff (PAS) and computer-assisted quantification of collagen III immunohistochemistry (C-IHC) slides, as well as assessing percent of cortical tubular atrophy% (TA%) and Banff total cortical inflammation score (ti-score). C-IHC using whole slide scans was performed. C-IHC assessment showed a higher correlation with organ function (r = -0.48) than did visual assessments (r = -0.32--0.42); computerized and visual C-IHC assessment also correlated (r = 0.64-0.66). Visual assessment of trichrome and C-IHC showed better correlations with organ function and C-IHC, than PAS, TA% and ti-score. However, visual assessment of IF, independent of approach, was variable among observers, and differences in correlations with organ function were not statistically significant among C-IHC image analysis and visual assessment methods. C-IHC image analysis correlated among three centers (r > 0.90, p < 0.0001, between all centers). Given the difficulty of visual IF assessment standardization, C-IHC image could potentially accomplish standardized IF assessment in multicenter settings.

Banff 2013 meeting report: inclusion of c4d-negative antibody-mediated rejection and antibody-associated arterial lesions.

The 12th Banff Conference on Allograft Pathology was held in Comandatuba, Brazil, from August 19-23, 2013, and was preceded by a 2-day Latin American Symposium on Transplant Immunobiology and Immunopathology. The meeting was highlighted by the presentation of the findings of several working groups formed at the 2009 and 2011 Banff meetings to: (1) establish consensus criteria for diagnosing antibody-mediated rejection (ABMR) in the presence and absence of detectable C4d deposition; (2) develop consensus definitions and thresholds for glomerulitis (g score) and chronic glomerulopathy (cg score), associated with improved inter-observer agreement and correlation with clinical, molecular and serological data; (3) determine whether isolated lesions of intimal arteritis ("isolated v") represent acute rejection similar to intimal arteritis in the presence of tubulointerstitial inflammation; (4) compare different methodologies for evaluating interstitial fibrosis and for performing/evaluating implantation biopsies of renal allografts with regard to reproducibility and prediction of subsequent graft function; and (5) define clinically and prognostically significant morphologic criteria for subclassifying polyoma virus nephropathy. The key outcome of the 2013 conference is defining criteria for diagnosis of C4d-negative ABMR and respective modification of the Banff classification. In addition, three new Banff Working Groups were initiated.

Molecular diagnosis of antibody-mediated rejection in human kidney transplants.

Antibody-mediated rejection is the major cause of kidney transplant failure, but the histology-based diagnostic system misses most cases due to its requirement for C4d positivity. We hypothesized that gene expression data could be used to test biopsies for the presence of antibody-mediated rejection. To develop a molecular test, we prospectively assigned diagnoses, including C4d-negative antibody-mediated rejection, to 403 indication biopsies from 315 patients, based on histology (microcirculation lesions) and donor-specific HLA antibody. We then used microarray data to develop classifiers that assigned antibody-mediated rejection scores to each biopsy. The transcripts distinguishing antibody-mediated rejection from other conditions were mostly expressed in endothelial cells or NK cells, or were IFNG-inducible. The scores correlated with the presence of microcirculation lesions and donor-specific antibody. Of 45 biopsies with scores>0.5, 39 had been diagnosed as antibody-mediated rejection on the basis of histology and donor-specific antibody. High scores were also associated with unanimity among pathologists that antibody-mediated rejection was present. The molecular score also strongly predicted future graft loss in Cox regression analysis. We conclude that microarray assessment of gene expression can assign a probability of ABMR to transplant biopsies without knowledge of HLA antibody status, histology, or C4d staining, and predicts future failure.

DHEA(S)--a novel marker in Cushing's disease.

INTRODUCTION: Dehydroepiandrosterone sulfate (DHEA(S)) is a multi-functional steroid implicated in a broad range of biological effects, including obesity, diabetes, bone metabolism, neuroprotection, and anti-tumorigenesis. It has not yet undergone wider research in the context of Cushing's disease. The objective of this study was to determine if perioperative blood levels of DHEA(S) correlate with levels of ACTH and cortisol, and therefore may be useful as a new, additional marker for the early definition of cure in patients suffering from Cushing's disease. METHODS: Forty-two consecutive patients undergoing transsphenoidal surgery for Cushing's disease from September 2009 to September 2010 were perioperatively monitored for ACTH, cortisol, and DHEA(S). RESULTS: Pre-operative blood samples revealed ACTH levels of median 65 ng/l (range 11-1,183 ng/l, standard deviation 183.76), cortisol of median 257 µg/l (range 93-803 µg/l, standard deviation 140.88), and DHEA(S) of median 2.22 mg/l (range 0.44-7.79 mg/l, standard deviation 1.82) according to the pathology of Cushing's disease. Postoperative blood samples drawn over a 7-day time span showed a drop in median ACTH to just 14.5 % (median: 9 ng/l, range 2-44, standard deviation 12.75) of its median preoperative figure. Median cortisol levels were reduced to 6.9 % (median: 18 µg/l, range 10-190 µg/l, standard deviation 38.04) of their preoperative values and DHEA(S) levels decreased to 17 % (median: 0.38 mg/l, range 0.05-2.29, standard deviation 0.51). In persistent disease, no patient showed a drop in DHEA(S) below 38 % of its preoperative figures. CONCLUSIONS: DHEA(S) shows the potential to become an additional marker in the diagnosis and follow-up of patients. However, it needs to be examined further, including whether DHEA(S) may also be a useful predictor of recovery of the HPA-axis after successful surgery.

The nature of biopsies with "borderline rejection" and prospects for eliminating this category.

In kidney transplantation, many inflamed biopsies with changes insufficient to be called T-cell-mediated rejection (TCMR) are labeled "borderline", leaving management uncertain. This study examined the nature of borderline biopsies as a step toward eventual elimination of this category. We compared 40 borderline, 35 TCMR and 116 nonrejection biopsies. TCMR biopsies had more inflammation than borderline but similar degrees of tubulitis and scarring. Surprisingly, recovery of function after biopsy was similar in all categories, indicating that response to treatment is unreliable for defining TCMR. We studied the molecular changes in TCMR, borderline and nonrejection using microarrays, measuring four published features: T-cell burden; a rejection classifier; a canonical TCMR classifier; and risk score. These reassigned borderline biopsies as TCMR-like 13/40 (33%) or nonrejection-like 27/40 (67%). A major reason that histology diagnosed molecularly defined TCMR as borderline was atrophy-scarring, which interfered with assessment of inflammation and tubulitis. Decision tree analysis showed that i-total >27% and tubulitis extent >3% match the molecular diagnosis of TCMR in 85% of cases. In summary, most cases designated borderline by histopathology are found to be nonrejection by molecular phenotyping. Both molecular measurements and histopathology offer opportunities for more precise assignment of these cases after clinical validation.

Understanding the causes of kidney transplant failure: the dominant role of antibody-mediated rejection and nonadherence.

We prospectively studied kidney transplants that progressed to failure after a biopsy for clinical indications, aiming to assign a cause to every failure. We followed 315 allograft recipients who underwent indication biopsies at 6 days to 32 years posttransplant. Sixty kidneys progressed to failure in the follow-up period (median 31.4 months). Failure was rare after T-cell-mediated rejection and acute kidney injury and common after antibody-mediated rejection or glomerulonephritis. We developed rules for using biopsy diagnoses, HLA antibody and clinical data to explain each failure. Excluding four with missing information, 56 failures were attributed to four causes: rejection 36 (64%), glomerulonephritis 10 (18%), polyoma virus nephropathy 4 (7%) and intercurrent events 6 (11%). Every rejection loss had evidence of antibody-mediated rejection by the time of failure. Among rejection losses, 17 of 36 (47%) had been independently identified as nonadherent by attending clinicians. Nonadherence was more frequent in patients who progressed to failure (32%) versus those who survived (3%). Pure T-cell-mediated rejection, acute kidney injury, drug toxicity and unexplained progressive fibrosis were not causes of loss. This prospective cohort indicates that many actual failures after indication biopsies manifest phenotypic features of antibody-mediated or mixed rejection and also underscores the major role of nonadherence.

Inflammation lesions in kidney transplant biopsies: association with survival is due to the underlying diseases.

Assessment of kidney transplant biopsies relies on nonspecific inflammatory lesions: Interstitial infiltrates (i), tubulitis (t) and intimal arteritis (v). We studied the relationship between inflammation and prognosis in biopsies for clinical indications from 314 patients (median follow-up 25 months). We used a modified Banff classification, separately assessing inflammation (i-) in nonscarred (i-Banff), scarred (i-IFTA) and whole cortex (i-total), plus tubulitis and intimal arteritis. In early biopsies (<1 year), i- and t-lesions had no association with graft survival. In late (>1 year) biopsies, all i-scores correlated with progression to failure, due to the association of these infiltrates with progressive diseases: antibody-mediated rejection (ABMR) and glomerulonephritis. Tubulitis in nonscarred areas had no impact on survival. Severe tubulitis including scarred areas (tis3) was associated with worse survival, but reflected polyoma virus nephropathy or ABMR, not T-cell-mediated rejection. Intimal arteritis (v-lesions) had no association with allograft loss in early or late biopsies. In multivariate analysis, outcome was better predicted by the presence of progressive disease than by inflammation. Thus inflammation in late kidney transplants has no inherent prognostic impact, but predicts reduced survival because inflammation indicates actively progressing diseases. The most important predictor of outcome is the diagnosis of a progressive disease.

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.

An integrated view of molecular changes, histopathology and outcomes in kidney transplants.

Data-driven approaches to deteriorating kidney transplants, incorporating histologic, molecular and HLA antibody findings, have created a new understanding of transplant pathology and why transplants fail. Transplant dysfunction is best understood in terms of three elements: diseases, the active injury-repair response and the cumulative burden of injury. Progression to failure is mainly attributable to antibody-mediated rejection, nonadherence and glomerular disease. Antibody-mediated rejection usually develops late due to de novo HLA antibodies, particularly anti-class II, and is often C4d negative. Pure treated T cell-mediated rejection does not predispose to graft loss because it responds well, even with endothelialitis, but it may indicate nonadherence. The cumulative burden of injury results in atrophy-fibrosis (nephron loss), arterial fibrous intimal thickening and arteriolar hyalinosis, but these are not progressive without ongoing disease/injury, and do not explain progression. Calcineurin inhibitor toxicity has been overestimated because burden-of-injury lesions invite this default diagnosis when diseases such as antibody-mediated rejection are missed. Disease/injury triggers a stereotyped active injury-repair response, including de-differentiation, cell cycling and apoptosis. The active injury-repair response is the strongest correlate of organ function and future progression to failure, but should always prompt a search for the initiating injury or disease.

The molecular phenotype of heart transplant biopsies: relationship to histopathological and clinical variables.

Histopathology of endomyocardial biopsies (EMB) is the standard rejection surveillance for heart transplants. However, ISHLT consensus criteria for interpreting biopsies are arbitrarily defined. Gene expression offers an independent re-evaluation of existing diagnostic systems. We performed histologic and microarray analysis on 105 EMB from 45 heart allograft recipients. Histologic lesions, diagnosis and transcripts were compared to one another, time posttransplantation, indication for biopsy and left ventricular ejection fraction (LVEF). Histologic lesions presented in two groups: myocyte-interstitial and microcirculation lesions. Expression of transcript sets reflecting T cell and macrophage infiltration, and γ-interferon effects correlated strongly with each other and with transcripts indicating tissue/myocardium injury. This molecular phenotype correlated with Quilty (p < 0.005), microcirculation lesions (p < 0.05) and decreased LVEF (p < 0.007), but not with the histologic diagnosis of rejection. In multivariate analysis, LVEF was associated (p < 0.03) with γ-interferon inducible transcripts, time posttransplantation, ischemic injury and clinically indicated biopsies, but not the diagnosis of rejection. The results indicate that (a) the current ISHLT system for diagnosing rejection does not reflect the molecular phenotype in EMB and lacks clinical relevance; (b) the interpretation of Quilty lesions has to be revisited; (c) the assessment of molecules in heart biopsy can guide improvements of current diagnostics.

NK cell transcripts and NK cells in kidney biopsies from patients with donor-specific antibodies: evidence for NK cell involvement in antibody-mediated rejection.

To explore the mechanisms of antibody-mediated rejection (ABMR) in kidney transplants, we studied the transcripts expressed in clinically indicated biopsies from patients with donor-specific antibody (DSA). Comparison of biopsies from DSA-positive versus DSA-negative patients revealed 132 differentially expressed transcripts: all were associated with class II DSA but none with class I DSA. Many transcripts were expressed in DSA-positive ABMR but were also expressed in T-cell-mediated rejection (TCMR), reflecting shared molecular features. Removal of shared transcripts created 23 DSA selective transcripts (DSASTs). Some DSASTs (6/23) showed selective high expression in NK cells, whereas others (8/23) were expressed in endothelium or in endothelium plus other cell types (7/23). Of 145 biopsies ranked by DSAST expression, the 25 with highest DSAST expression primarily consisted of ABMR (22/25, 88%), either C4d-positive or C4d-negative. By immunostaining, CD56+ and CD68+ cells in peritubular capillaries, but not CD3+ cells, were increased in ABMR compared to TCMR, compatible with a role for NK cells, as well as macrophages, as effectors in endothelial injury during ABMR. Thus, the strategy of using DSASTs in the biopsy to identify mechanism-related transcripts in biopsies from patients with clinical phenotypes indicates the selective involvement of NK cells in ABMR.

Banff '09 meeting report: antibody mediated graft deterioration and implementation of Banff working groups.

The 10th Banff Conference on Allograft Pathology was held in Banff, Canada from August 9 to 14, 2009. A total of 263 transplant clinicians, pathologists, surgeons, immunologists and researchers discussed several aspects of solid organ transplants with a special focus on antibody mediated graft injury. The willingness of the Banff process to adapt continuously in response to new research and improve potential weaknesses, led to the implementation of six working groups on the following areas: isolated v-lesion, fibrosis scoring, glomerular lesions, molecular pathology, polyomavirus nephropathy and quality assurance. Banff working groups will conduct multicenter trials to evaluate the clinical relevance, practical feasibility and reproducibility of potential changes to the Banff classification. There were also sessions on quality improvement in biopsy reading and utilization of virtual microscopy for maintaining competence in transplant biopsy interpretation. In addition, compelling molecular research data led to the discussion of incorporation of omics-technologies and discovery of new tissue markers with the goal of combining histopathology and molecular parameters within the Banff working classification in the near future.

Cluster analysis of lesions in nonselected kidney transplant biopsies: microcirculation changes, tubulointerstitial inflammation and scarring.

Banff classification empirically established scoring of histologic lesions, but the relationships of lesions to each other and to underlying biologic processes remain unclear. We hypothesized that class discovery tools would reveal new relationships between individual lesions, and relate lesions to C4d staining, anti-HLA donor-specific antibody (DSA) and time posttransplant. We studied 234 nonselected renal allograft biopsies for clinical indications from 173 patients. Silhouette plotting and principal component analysis revealed three groups of lesions: microcirculation changes, including inflammation (glomerulitis, capillaritis) and deterioration (double contours, mesangial expansion); scarring/hyalinosis; and tubulointerstitial inflammation. DSA and C4d grouped with microcirculation inflammation, whereas time posttransplant grouped with scarring/hyalinosis lesions. Intimal arteritis clustered with DSA, C4d and microcirculation inflammation, but also showed correlations with tubulitis. Fibrous intimal thickening in arteries clustered with scarring/hyalinosis. Capillary basement membrane multilayering showed intermediary relationships between microcirculation deterioration and time-dependent scarring. Correlation analysis and hierarchical clustering confirmed the lesion relationships. Thus, we propose that the pathologic lesions in biopsies are not independent but are members of groups that represent distinct pathogenic forces: microcirculation changes, reflecting the stress of DSA; scarring, hyalinosis and arterial fibrosis, reflecting the cumulative burden of injury over time; and tubulointerstitial inflammation. Interpretation of lesions should reflect these associations.