A Probabilistic Approach to Histologic Diagnosis of Antibody‐Mediated Rejection in Kidney Transplant Biopsies

Histologic diagnosis of antibody‐mediated rejection (ABMR) in kidney transplant biopsies uses lesion score cutoffs such as 0 versus >0 rather than actual scores and requires donor‐specific antibody (DSA); however, cutoffs lose information, and DSA is not always reliable. Using microarray‐derived molecular ABMR scores as a histology‐independent estimate of ABMR in 703 biopsies, we reassessed criteria for ABMR to determine relative importance of various lesions, the utility of equations using actual scores rather than cutoffs, and the potential for diagnosing ABMR when DSA is unknown or negative. We confirmed that the important features for ABMR diagnosis were peritubular capillaritis (ptc), glomerulitis (g), glomerular double contours, DSA and C4d staining, but we questioned some features: arterial fibrosis, vasculitis, acute tubular injury, and sum of ptc+g scores. Regression equations using lesion scores predicted molecular ABMR more accurately than score cutoffs (area under the curve 0.85–0.86 vs. 0.75). DSA positivity improved accuracy, but regression equations predicted ABMR with moderate accuracy when DSA was unknown. Some biopsies without detectable DSA had high probability of ABMR by regression, although most had HLA antibody. We concluded that regression equations using lesion scores plus DSA maximized diagnostic accuracy and can estimate probable ABMR when DSA is unknown or undetectable.     

Reassessing the Significance of Intimal Arteritis in Kidney Transplant Biopsy Specimens

Intimal arteritis (the presence of v-lesions) in kidney transplant biopsy specimens is believed to have major prognostic and diagnostic significance. We assessed the relationship of v-lesions to prognosis in 703 indication biopsy specimens and used microarray-based molecular tests to re-examine the relationship of v-lesions to rejection. v-Lesions were noted in 49 specimens (7%) and were usually mild (v1). The presence of v-lesions had no effect on graft survival compared with the absence of v-lesions. Pathologists using current conventions almost always interpreted v-lesions as reflecting T cell–mediated rejection (TCMR), either pure or mixed with antibody-mediated rejection (ABMR). The molecular scores questioned the conventional diagnoses in 29 of 49 specimens (59%), including ten that were conventional TCMR with no molecular rejection and nine that were conventional TCMR mixed with pure ABMR molecularly. The presence of tubulointerstitial inflammation (i-t) meeting TCMR criteria allowed subclassification of v-lesion specimens into 21 i-t-v-lesion specimens and 28 isolated v-lesion specimens. Molecular TCMR scores were positive in 95% of i-t-v-lesion specimens but only 21% of isolated v-lesion specimens. Molecular ABMR scores were often positive in isolated v-lesion biopsies (46%). Time of biopsy after transplantation was critical for understanding isolated v-lesions: most early isolated v-lesion specimens had no molecular rejection and were DSA negative, whereas most isolated >1 year after transplantation had positive DSA and ABMR scores. Therefore, v-lesions in indication biopsy specimens do not affect prognosis and can reflect TCMR, ABMR, or no rejection. Time after transplantation, DSA, and accompanying inflammation provide probabilistic basis for interpreting v-lesions.     

Discrepancy analysis comparing molecular and histology diagnoses in kidney transplant biopsies

Discrepancy analysis comparing two diagnostic platforms offers potential insights into both without assuming either is always correct. Having optimized the Molecular Microscope Diagnostic System (MMDx) in renal transplant biopsies, we studied discrepancies within MMDx (reports and sign‐out comments) and between MMDx and histology. Interpathologist discrepancies have been documented previously and were not assessed. Discrepancy cases were classified as “clear” (eg, antibody‐mediated rejection [ABMR] vs T cell–mediated rejection [TCMR]), “boundary” (eg, ABMR vs possible ABMR), or “mixed” (eg, Mixed vs ABMR). MMDx report scores showed 99% correlations; sign‐out interpretations showed 7% variation between observers, all located around boundaries. Histology disagreed with MMDx in 37% of biopsies, including 315 clear discrepancies, all with implications for therapy. Discrepancies were distributed widely in all histology diagnoses but increased in some scenarios; for example, histology TCMR contained 14% MMDx ABMR and 20% MMDx no rejection. MMDx usually gave unambiguous diagnoses in cases with ambiguous histology, for example, borderline and transplant glomerulopathy. Histology lesions or features associated with more frequent discrepancies (eg, tubulitis, arteritis, and polyomavirus nephropathy) were not associated with increased MMDx uncertainty, indicating that MMDx can clarify biopsies with histologic ambiguity. The patterns of histology‐MMDx discrepancies highlight specific histology diagnoses in which MMDx assessment should be considered for guiding therapy.     

Evaluation of C1q Status and Titer of De Novo Donor‐Specific Antibodies as Predictors of Allograft Survival

De novo donor‐specific antibodies (dnDSAs) that develop after renal transplantation are independent predictors of allograft loss. However, it is unknown if dnDSA C1q status or titer at the time of first detection can independently predict allograft loss. In a consecutive cohort of 508 renal transplant recipients, 70 developed dnDSAs. Histologic and clinical outcomes were correlated with the C1q assay or dnDSA titer. C1q positivity correlated with dnDSA titer (p < 0.01) and mean fluorescence intensity (p < 0.01) and was more common in class II versus class I dnDSAs (p < 0.01). C1q status correlated with tubulitis (p = 0.02) and C4d status (p = 0.03) in biopsies at the time of dnDSA development, but not T cell–mediated rejection (TCMR) or antibody‐mediated rejection (ABMR). De novo DSA titer correlated with Banff g, i, t, ptc, C4d scores, TCMR (p < 0.01) and ABMR (p < 0.01). Post‐dnDSA graft loss was observed more frequently in recipients with C1q‐positve dnDSA (p < 0.01) or dnDSA titer ≥ 1:1024 (p ≤ 0.01). However, after adjustment for clinical phenotype and nonadherence in multivariate models, neither C1q status nor dnDSA titer were independently associated with allograft loss, questioning the utility of these assays at the time of dnDSA development.     

The Banff 2019 Kidney Meeting Report (I): Updates on and clarification of criteria for T cell– and antibody‐mediated rejection

The XV. Banff conference for allograft pathology was held in conjunction with the annual meeting of the American Society for Histocompatibility and Immunogenetics in Pittsburgh, PA (USA) and focused on refining recent updates to the classification, advances from the Banff working groups, and standardization of molecular diagnostics. This report on kidney transplant pathology details clarifications and refinements to the criteria for chronic active (CA) T cell–mediated rejection (TCMR), borderline, and antibody‐mediated rejection (ABMR). The main focus of kidney sessions was on how to address biopsies meeting criteria for CA TCMR plus borderline or acute TCMR. Recent studies on the clinical impact of borderline infiltrates were also presented to clarify whether the threshold for interstitial inflammation in diagnosis of borderline should be i0 or i1. Sessions on ABMR focused on biopsies showing microvascular inflammation in the absence of C4d staining or detectable donor‐specific antibodies; the potential value of molecular diagnostics in such cases and recommendations for use of the latter in the setting of solid organ transplantation are presented in the accompanying meeting report. Finally, several speakers discussed the capabilities of artificial intelligence and the potential for use of machine learning algorithms in diagnosis and personalized therapeutics in solid organ transplantation.     

Disappearance of T Cell-Mediated Rejection Despite Continued Antibody-Mediated Rejection in Late Kidney Transplant Recipients

The prevalent renal transplant population presents an opportunity to observe the adaptive changes in the alloimmune response over time, but such studies have been limited by uncertainties in the conventional biopsy diagnosis of T cell-mediated rejection (TCMR) and antibody-mediated rejection (ABMR). To circumvent these limitations, we used microarrays and conventional methods to investigate rejection in 703 unselected biopsies taken 3 days to 35 years post-transplant from North American and European centers. Using conventional methods, we diagnosed rejection in 205 biopsy specimens (28%): 67 pure TCMR, 110 pure ABMR, and 28 mixed (89 designated borderline). Using microarrays, we diagnosed rejection in 228 biopsy specimens (32%): 76 pure TCMR, 124 pure ABMR, and 28 mixed (no borderline). Molecular assessment confirmed most conventional diagnoses (agreement was 90% for TCMR and 83% for ABMR) but revealed some errors, particularly in mixed rejection, and improved prediction of failure. ABMR was strongly associated with increased graft loss, but TCMR was not. ABMR became common in biopsy specimens obtained >1 year post-transplant and continued to appear in all subsequent intervals. TCMR was common early but progressively disappeared over time. In 108 biopsy specimens obtained 10.2–35 years post-transplant, TCMR defined by molecular and conventional features was never observed. We conclude that the main cause of kidney transplant failure is ABMR, which can present even decades after transplantation. In contrast, TCMR disappears by 10 years post-transplant, implying that a state of partial adaptive tolerance emerges over time in the kidney transplant population.     

Generating automated kidney transplant biopsy reports combining molecular measurements with ensembles of machine learning classifiers

We previously reported a system for assessing rejection in kidney transplant biopsies using microarray‐based gene expression data, the Molecular Microscope^® Diagnostic System (MMDx). The present study was designed to optimize the accuracy and stability of MMDx diagnoses by replacing single machine learning classifiers with ensembles of diverse classifier methods. We also examined the use of automated report sign‐outs and the agreement between multiple human interpreters of the molecular results. Ensembles generated diagnoses that were both more accurate than the best individual classifiers, and nearly as stable as the best, consistent with expectations from the machine learning literature. Human experts had ≈93% agreement (balanced accuracy) signing out the reports, and random forest‐based automated sign‐outs showed similar levels of agreement with the human experts (92% and 94% for predicting the expert MMDx sign‐outs for T cell–mediated (TCMR) and antibody‐mediated rejection (ABMR), respectively). In most cases disagreements, whether between experts or between experts and automated sign‐outs, were in biopsies near diagnostic thresholds. Considerable disagreement with histology persisted. The balanced accuracies of MMDx sign‐outs for histology diagnoses of TCMR and ABMR were 73% and 78%, respectively. Disagreement with histology is largely due to the known noise in histology assessments (ClinicalTrials.gov NCT01299168).     

Anti‐C1s monoclonal antibody BIVV009 in late antibody‐mediated kidney allograft rejection—results from a first‐in‐patient phase 1 trial

The classical pathway (CP) of complement may contribute to the pathogenesis of antibody‐mediated rejection (ABMR). Selective CP blockade may be a promising strategy to counteract rejection. The objective of this first‐in‐patient phase 1b trial was to evaluate the safety/tolerability and CP‐blocking potential of 4 weekly doses (60 mg/kg) of the anti‐C1s antibody BIVV009 in complement‐mediated disorders. Here we describe the results in a cohort of 10 stable kidney transplant recipients (median of 4.3 years posttransplantation) with late active ABMR and features of CP activation, such as capillary C4d or complement‐fixing donor‐specific antibodies (DSA). During 7 weeks follow‐up, no severe adverse events were reported, and BIVV009 profoundly inhibited overall and DSA‐triggered CP activation in serum. Five of 8 C4d‐positive recipients turned C4d‐negative in 5‐week follow‐up biopsies, while another 2 recipients showed a substantial decrease in C4d scores. There was, however, no change in microcirculation inflammation, gene expression patterns, DSA levels, or kidney function. In conclusion, we demonstrate that BIVV009 effectively blocks alloantibody‐triggered CP activation, even though short‐course treatment had no effect on indices of activity in late ABMR. This initial trial provides a valuable basis for future studies designed to clarify the therapeutic value of CP blockade in transplantation. ClinicalTrials.gov NCT#02502903.     

Using Molecular Phenotyping to Guide Improvements in the Histologic Diagnosis of T Cell–Mediated Rejection

Recognition that some lesions typical of T cell–mediated rejection (TCMR) also occur in antibody‐mediated rejection requires revision of the histologic TCMR definition. To guide this process, we assessed the relative importance of various lesions and the performance of new histology diagnostic algorithms, using molecular TCMR scores as histology‐independent estimates of true TCMR. In 703 indication biopsies, random forest analysis and logistic regression indicated that interstitial infiltrate (i‐lesions) and tubulitis (t‐lesions) were the key histologic predictors of molecular TCMR, with arteritis (v‐lesions) having less importance. Histology predicted molecular TCMR more accurately when diagnoses were assigned by strictly applying the Banff rules to the lesion scores and redefining isolated v‐lesion TCMR. This improved prediction from area under the curve (AUC) 0.70 with existing rules to AUC 0.80. Further improvements were achieved by introducing more categories to reflect inflammation (AUC 0.84), by summing the lesion scores (AUC 0.85) and by logistic regression (AUC 0.90). We concluded that histologic assessment of TCMR can be improved by placing more emphasis on i‐ and t‐lesions and incorporating new algorithms for diagnosis. Nevertheless, some discrepancies between histologic and molecular diagnoses persist, partially due to the inherent nonspecificity of i‐ and t‐lesions, and molecular methods will be required to help resolve these cases.     

Kidney Intragraft Donor‐Specific Antibodies as Determinant of Antibody‐Mediated Lesions and Poor Graft Outcome

Allograft pathology, antibody–tissue interaction as demonstrated by C4d deposition and serological evidence of donor‐specific antibodies (DSA) are the cardinal diagnostic features of antibody‐mediated lesions (AML) in kidney transplantation. However, discrepancy between histological and serological findings is common, and more reliable diagnostic tools are called for. Here, we asked whether the in situ detection of DSA could serve as marker for AML. To that end, we applied the anti‐HLA single antigen flow bead assay to eluates from 51 needle core graft biopsies performed for cause. Intragraft antibody profiles were correlated to serum DSA (sDSA), histological data and transplant outcome. The prevalence and the mean number of intragraft DSA (gDSA) were lower than that of sDSA (15/51 gDSA+ vs. 37/51 sDSA+ patients; 1.64 gDSA vs. 2.24 sDSA per patient). DSA were detected in all anti‐HLA antibody‐positive biopsies (15/15). The presence of gDSA was significantly associated with (1) microcirculation lesions taken individually (g, cg) and analyzed in functional clusters (ptc + g + cg > 0, cg + mm > 0), (2) C4d positivity and (3) a worse short‐term transplant outcome (p = 0.05). These associations were not found for patients presenting only sDSA. Taken together, these results indicate that gDSA is a severity marker of antibody‐mediated pathogenic process.     

Histological picture of antibody‐mediated rejection without donor‐specific anti‐HLA antibodies: Clinical presentation and implications for outcome

In this cohort study (n = 935 transplantations), we investigated the phenotype and risk of graft failure in patients with histological criteria for antibody‐mediated rejection (ABMR) in the absence of circulating donor‐specific anti‐human leukocyte antigen (HLA) antibodies (DSA), and compared this to patients with definite ABMR and HLA‐DSA‐positivity. The histological picture did not differ between HLA‐DSA‐positive (n = 85) and HLA‐DSA‐negative (n = 123) cases of ABMR histology, apart from increased complement split product 4d (C4d) deposition in the peritubular capillaries in HLA‐DSA‐positive cases. Histology of ABMR without HLA‐DSA was more transient than DSA‐positive ABMR, and patients with ABMR histology without HLA‐DSA had graft survival superior to that of HLA‐DSA‐positive patients, independent of concomitant T cell–mediated rejection (38.2%) or borderline changes (17.9%). Multivariate analysis showed that the risk of graft failure was not higher in patients with histological picture of ABMR (ABMR_h) in the absence of HLA‐DSA, compared to patients without ABMR_h. Despite an association between C4d deposition and HLA‐DSA‐positivity, using C4d deposition as alternative for the DSA criterion in the diagnosis of ABMR, as proposed in Banff 2017, did not contribute to the prognosis of graft function and graft failure. We concluded that biopsies with ABMR_h but without detectable HLA‐DSA represent a distinct, often transient phenotype with superior allograft survival.