The diffuse extent of peritubular capillaritis in renal allograft rejection is an independent risk factor for graft loss

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1 International Society of Nephrology see commentary on page 218 The diffuse extent of peritubular capillaritis in renal allograft rejection is an independent risk factor for graft loss Nicolas Kozakowski 1, Harald Herkner 2, Georg A. Böhmig 3, Heinz Regele 1, Christoph Kornauth 1, Gregor Bond 3 and Željko Kikić 3 1 Institute of Clinical Pathology, Medical University of Vienna, Vienna, Austria; 2 Department of Emergency Medicine, Medical University of Vienna, Vienna, Austria and 3 Department of Medicine III, Division of Nephrology and Dialysis, Medical University of Vienna, Vienna, Austria By the Banff classification, the score of peritubular capillaritis, its extent, and its cellular composition should normally be reported in renal allograft pathology. While the score represents an important diagnostic and prognostic variable, the clinical value of capillaritis extent or composition has yet to be resolved. In a retrospective study of 749 renal transplant recipients subjected to 1322 indication biopsies, we found that prevalence scores of 1, 2, or 3 in the biopsy specimens were 10.7, 11.6, and 2.6%, respectively. Focal and diffuse peritubular capillaritis (inflammation over 50% of cortical peritubular capillaries) was diagnosed in 10.5 or 14.4% of cases, respectively. Mononuclear, granulocytic, and mixed peritubular capillaritis was present in 13.1, 3.3, and 8.5%, respectively. While peritubular capillaritis without further subclassification was not related to higher allograft loss rates, a score of 3 (hazard ratio 2.57 (CI: )) and diffuse peritubular capillaritis (1.67 ( )) were significant impartial risk factors for allograft loss. Diffuse peritubular capillaritis was independently associated with features of chronic antibody-mediated rejection and greater egfr decline after 3 years. In contrast, detailed report of leukocytic composition in peritubular capillaritis did not confer additional prognostic information. Thus, in contrast to typing the infiltrating inflammatory cells, the score and extent of peritubular capillaritis in kidney allograft pathology is essential to assess transplant prognosis. Kidney International (2015) 88, ; doi: /ki ; published online 4 March 2015 KEYWORDS: allograft loss; C4d; diffuse capillaritis; GFR; peritubular capillaritis; ptc score Correspondence: Željko Kikić, Department of Medicine III, Division of Nephrology and Dialysis, Medical University of Vienna, Währinger Gürtel 18-20, Vienna A-1090, Austria. zeljko.kikic@meduniwien.ac.at Received 18 August 2014; revised 5 January 2015; accepted 22 January 2015; published online 4 March 2015 In recent years, the peritubular capillaries (PTC) have been defined as a crucial compartment in renal allograft pathology. 1 4 Leukocytic engorgement of those, i.e., peritubular capillaritis (ptc), has emerged as an important histological feature of acute antibody-mediated rejection (AMR), leading to the incorporation of ptc in the Banff classification of renal allograft pathology. 2 Ptc is also of substantial clinical relevance as a feature of subclinical rejection 5,6 and a precursor lesion for chronic graft injury, 7,8 especially leading to chronic microvascular injury, 2,5,8,9 including transplant glomerulopathy. 10 Beyond this clinical relevance, several issues remain unanswered. In a later Banff consensus conference, it has been recommended that more detailed information should be given about ptc in the pathology report. 4 Subcharacterizations of ptc including its extent (focal or diffuse), its intensity (as first given in the ptc score proposed in the Banff consensus meeting 2007), 11 and its leukocytic composition were introduced. This recommendation represented the basis for a study by Gibson and colleagues, 12 which primarily focused on diagnostic issues of ptc. In this study, clinical outcomes such as estimated glomerular filtration rate (egfr) were reported only for a small subcohort of recipients, without information on graft survival results. Apart from this study, precise reporting of leukocyte subpopulations in ptc has only been evaluated in far smaller case series. 7,13,14 From a clinician s point of view, histopathology reporting should be primarily based on clinical relevance, yet to our knowledge no adequately designed study has examined associations of specific leukocyte subpopulations in ptc or the extent of ptc with hard end points such as graft survival rates and renal function. In the present study, we primarily hypothesized that the clinical impact of ptc on allograft outcomes depends on its extent, namely diffuse ptc, and that determining leukocytic subpopulations in ptc might be of comparably lower clinical relevance. To test these hypotheses, we performed a large retrospective cohort study of 1322 indication biopsies and investigated clinical outcomes in univariate and multivariate 332 Kidney International (2015) 88,

2 models in relationship with the different qualities of ptc, namely ptc score, ptc extent, and ptc cellular composition. RESULTS Histomorphology Ptc was diagnosed in 329 of the 1322 (24.9%) studied biopsies. The prevalence of Banff ptc scores 1, 2, or 3 in the biopsy specimens was 10.7%, 11.6%, and 2.6%, respectively. Focal and diffuse ptc (inflammation of 450% of cortical PTC in the biopsy core) were found in 10.5% (n = 139) vs. 14.4% (n = 190) of the cases, whereas mononuclear, granulocytic, and mixed ptc were observed in 13.1% (n = 173), 3.3% (n = 43), and 8.5% (n = 113), respectively (Table 1). Figure 1 demonstrates the study algorithm at the patient level, whereas Figure 2 illustrates typical histomorphology of the subtypes of ptc. The inter-observer concordance in 50 selected specimens for the subclassification of ptc was substantial for extent (kappa = 0.64), moderate for score (kappa = 0.53), and moderate for leukocytic composition (kappa = 0.51 vs by Gibson et al. 12 ). Ptc and features of acute and chronic rejection When analyzing the association of ptc on histological rejection features at a biopsy level, all available biopsies were included, and we used random coefficient logistic regression models enabling correlation between multiple biopsies per patient. In this analysis, features of T cell mediated rejection (TCMR = Banff 1a) were observed for both diffuse (odds ratio, OR = 6.18 (confidence interval, CI: ), Po0.001) and, to a slighter extent, focal ptc (OR = 1.68 (CI: ), P = 0.037). Moreover, higher ptc scores were associated with an increased risk for TCMR (for ptc1, 2, and 3, respectively: OR = 3.38, 3.45, and 4.12 with P 0.002). The rate of C4d-positive graft dysfunction was significantly higher with concomitant ptc (25.4 vs. 9%, Po0.001). This association was mainly driven by increasing ptc scores, with the highest prevalence in ptc3, whereas extent and leukocytic composition were not related to C4d-positive graft dysfunction (Table 1). Features of chronic cellular allograft injury (ci, ct, or cv) and chronic AMR, i.e., transplant glomerulopathy (cg), were both related to ptc and higher ptc scores, whereas diffuse ptc showed a significant association only with chronic cellular rejection. For chronic AMR, a trend for increased prevalence of diffuse ptc was calculated. Further details are given in Table 1. Ptc score and allograft outcome Baseline variables overall and in relationship with the presence of ptc in the first indication biopsy are presented in Table 2. For calculation of death-censored graft survival (mean follow-up after indication biopsy: ± months), only information obtained starting from the first indication biopsy (n = 749) was used. Proportions of included recipients according to the predefined subclassifications of Table 1 Peritubular capillaritis score, extent, cellular composition in relation to single Banff criteria and immunohistochemical positivity for C4d Ptc P-value Ptc score P-value Ptc extent P-value Ptcleukocyticcomposition P-value Focal Diffuse Mononuclear Granulocytic Mixed 993 (75.11) 329 (24.88) 141 (10.66) 154 (11.65) 34 (2.57) 139 (10.5) 190 (14.4) 173 (13.1) 43 (3.3) 113 (8.5) Number of cases, (%) g40 (%) o o cg40 (%) o i40 (%) o o ci40 (%) o * t40 (%) o o ct40 (%) o o v40 (%) o o cv40 (%) ah40 (%) C4d o o Abbreviations: ptc, peritubular capillaritis; P-values according to Pearson s test. Percentages in the first line are in relation to the total number of cases in each category. Percentages in every other line are in relation to the total number of cases of the corresponding row. Kidney International (2015) 88,

3 ptc Score: ptc 0, n = 568 ptc 1, n = 81 ptc 2, n = 82 ptc 3, n =18 Renal TX ( ) 1248 recipients Indication biopsy: 885 recipients Adequate material for evaluation of peritubular capillaries: 749 recipients ptc Extent: ptc Composition: No ptc, n =568 No ptc, n =568 Focal(<50%), n =80 Monocytic ptc, n =89 Diffuse(>50%), n =101 Mixed ptc, n =65 Granulocytic ptc, n =27 Figure 1 Study flowchart. ptc, peritubular capillaritis; TX, transplantation. ptc (score, extent, and cellular composition) are demonstrated in the study flowchart (Figure 1). Although Kaplan Meier analysis (Figure 3a) showed no significant association of ptc without further subclassification with graft loss (hazard ratio, HR = 1.3 (CI: ), P = 0.1), we observed a significant relationship of Banff ptc scores with long-term allograft survival (log-rank test, P = 0.04; Figure 3b). Compared with no ptc, the ptc score showed a stepwise increase of graft loss, being highest for ptc3 [ptc1 (OR = 1.01 (CI: ), P = 0.95), ptc2 (OR = 1.36 (CI: ), P = 0.16), and ptc3 (OR = 2.37 (CI: ), P = 0.013)]. Even after adjustment for confounding variables (baseline immunosuppression, C4d-positive graft dysfunction, TCMR = Banff Ia, retransplantation, human leukocyte antigen (HLA) mismatch and presensitization (CDC PRA 410%), ptc3 remained a strong independent risk factor for graft loss (HR = 2.57 (CI: ), P = 0.01; Table 3). Extent of ptc and allograft outcome Diffuse ptc (inflammation of 450% of cortical PTC in the biopsy core), in comparison with focal ptc (HR = 0.72 (CI: ), P = 0.25), emerged as a highly significant risk factor for graft loss (HR = 1.86 (CI: ), P = 0.001; Figure 4a), displaying 8-year censored graft loss rates for none, focal, and diffuse ptc of 75% vs. 79% vs. 58%, P = 0.001, respectively. Moreover, in Cox regression analysis adjusted for the aforementioned confounders, diffuse ptc was independently associated with higher allograft loss rates (HR = 1.67 (CI: ), P = 0.015). This independent association remained significant (HR = 2.84 (CI: ), P = 0.005) even after adjustment for ptc score. In the adjusted model, ptc score and ptc extent were predictive independently of each other (P for interaction = 0.44). Leukocytic composition of ptc and allograft outcome In contrast to its score and extent, cellular composition of ptc quantified according to the predominant leukocytic subpopulation did not reveal monocytic, mixed, or granulocytic ptc as significant risk factors for graft loss (Figure 4b, P = 0.37), in both univariate and multivariate analysis (Table 3). Follow-up biopsies; effect of multiple biopsies on outcome As multiple rejection episodes may represent an important confounder potentially indicating worse outcome, we a b 10 µm 10 µm c d 5 µm 5 µm Figure 2 Histological features of peritubular capillaritis. Histological features of peritubular capillaritis: diffuse (a), focal (b), granulocytic (c), and mononuclear ptc (d). Original magnification: a b: 100, c d: 400. Empty arrows: ptc, full arrows: neutrophilic granulocytes, and arrowheads: mononuclear cells. ptc, peritubular capillaritis. 334 Kidney International (2015) 88,

4 Table 2 Demographics for the study population and for the cases with or without peritubular capillaritis Study population (749) Ptc = 0 (568) Ptc 40 (181) P-value Donor related Donor age, years, mean ± s.d ± ± ± Living donor (%) 95 (12.7) 69 (12.1) 26 (14.4) 0.44 HLA MM, mean ± s.d. 2.9 ± ± ± CIT, hours, mean ± s.d ± ± ± Recipient related Female (%) 254 (33.9) 187 (32.9) 67 (37) 0.31 Biopsy time post TX, months, mean ± s.d. 2.4 ± ± ± Number of biopsy, mean ± s.d. 1.8 ± ± ± Age at biopsy, years, mean ± s.d ± ± ± 14 o0.001 TCMR = Banff 1 (%) 224 (29.9) 137 (24.1) 87 (48.1) o0.001 C4d-positive graft dysfunction (%) 77 (10.3) 41 (7.2) 36 (19.9) o0.001 Presensitization (CDC PRA410%) (%) 156 (20.8) 109 (19.2) 47 (26) Retransplantation 130 (17.4) 85 (15) 45 (24.9) Graft loss (%) 167 (22.3) 115 (20.2) 52 (28.7) Serum creatinine, mg/dl, at 3 years, mean ± s.d. 2.2 ± ± ± Estimated GFR- Mayo at 3 years, ml/min/m 2, mean ± s.d ± ± ± Baseline immunosuppression Cyclosporine A (%) 556 (74.2) 405 (71.3) 151 (83.4) Tacrolimus (%) 112 (15) 96 (16.9) 16 (8.8) mtor inhibitor (%) 20 (2.7) 15 (2.6) 5 (2.8) 0.93 Depleting antibodies (%) 52 (6.9) 45 (7.9) 7 (3.9) 0.06 IL-2 inhibitor (%) 9 (1.2) 7 (1.2) 2 (1.1) 0.88 Abbreviations: CDC, complement-dependent cytotoxicity; CIT, cold ischemia time; GFR, glomerular filtration rate; HLA, human leukocyte antigen; IL, interleukin; MM, mismatch; mtor, mammalian target of rapamycin; P-values according to the Student s t-test; PRA, panel reactive antibodies; presensitization, complement-dependent cytotoxicity panel reactive antibodies 410%; TCMR, T cell mediated rejection; TX, transplantation Cumulative survival (%) P =0.1 No ptc ptc Cumulative survival (%) P =0.04 No ptc ptc1 ptc2 ptc Graft survival (months) Graft survival (months) No ptc ptc ptc ptc ptc 2 ptc Figure 3 Kaplan Meier allograft survival curves for the presence of peritubular capillaritis and Banff ptc score. (a) Kaplan Meier analysis comparing the influence of no ptc (solid line) versus ptc (dashed line) on death-censored graft survival, and (b) Kaplan Meier analysis comparing the influence of Banff ptc scores 0 (solid line), 1 (dashed line), 2 (dotted line), and 3 (semi-dashed line) on death-censored graft loss, P according to log-rank test. performed another multivariate Cox regression model to correct for the number of biopsies per patient. Indication biopsies were performed two, three, four, five, and six times in 180, 97, 36, 13, and 7 recipients, respectively. Intraclass correlation coefficient showed a rho of 0.99 for ptc extent, indicating that the observation of focal or diffuse ptc in a biopsy reliably predicts the same feature of ptc in the following biopsy. After correction for multiple rejection episodes (a strong risk factor for unfavorable outcome (HR = 1.64 (CI: ), Po0.001)), diffuse ptc remained a strong independent risk factor for graft loss (HR = 1.62 (CI: ), P = 0.025). In contrast, the inclusion of multiple rejection episodes in the model reduced the previously observed independent statistical association of ptc score 3, Kidney International (2015) 88,

5 Table 3 Peritubular capillaritis score, extent, cellular composition, and their relationship with death-censored graft loss, Cox regression analysis Ptc score HR P-value Ptc extent HR P-value Ptc leukocytic composition HR P-value 0 Reference 0 Reference 0 Reference ( ) 0.73 Focal 0.65 ( ) 0.15 Monocytic 1.06 ( ) ( ) 0.58 Diffuse 1.67 ( ) Mixed 1.31 ( ) ( ) 0.01 Granulocytic 1.22 ( ) 0.47 TCMR (Banff 1) 1.02 ( ) ( ) ( ) 0.53 C4d+ dysfunction 1.42 ( ) ( ) ( ) 0.18 Retransplantation 1.58 ( ) ( ) ( ) 0.93 HLA MM 1.19 ( ) ( ) ( ) Presensitization 1.06 ( ) ( ) ( ) 0.68 Cyclosporine A Reference Reference Reference Tacrolimus 0.95 ( ) ( ) ( ) 0.79 mtor inhibitor 1.88 ( ) ( ) ( ) 0.13 Depleting Ab 1.05 ( ) ( ) ( ) 0.93 IL-2 inhibitor 0.44 ( ) ( ) ( ) 0.4 Abbreviations: Ab, antibody; HLA, human leukocyte antigen; HR, hazard ratio; IL, interleukin; MM, mismatch; mtor, mammalian target of rapamycin; presensitization, complement-dependent cytotoxicity panel reactive antibodies 410%; TCMR, T cell mediated rejection Cumulative survival (%) No ptc Focal ptc P =0.001 Diffuse ptc Graft survival (months) Graft survival (months) Cumulative survival (%) No ptc Mixed ptc 20 Mononuclear ptc Granulocytic ptc P =0.37 No ptc No ptc Focal ptc Diffuse ptc Mononuclear ptc Mixed ptc Granulocytic ptc Figure 4 Kaplan Meier allograft survival curves for extent and cellular composition of peritubular capillaritis. (a) Kaplan Meier analysis comparing the influence of the extent of ptc, namely no (solid line) vs. focal ptc (dashed line) and diffuse (semi-dashed line) on death-censored graft survival, and (b) Kaplan Meier analysis comparing the influence of leukocytic composition in ptc, namely no (solid line), mononuclear (semi-dashed line), granulocytic (dotted line), and mixed (dashed line) on death-censored graft loss, P according to log-rank test. yet still displaying a clinically relevant trend with a twofold increased risk for graft loss (HR = 1.93 (CI: ), P = 0.07)). There was no significant association of ptc score and multiple biopsies (P = 0.13) Ptc in DSA-positive patients For only a subgroup (n = 85) of our study population, donorspecific antibody (DSA) data were available (because of their inclusion in previous studies). This subcohort, however, showed low comparability with the overall cohort in terms of relevant confounders with a significant association toward a higher risk cohort for AMR (data not shown) and could not be included in further multivariate models. Forty out of the 85 patients tested for DSA were classified as positive. In those patients, diffuse ptc was observed in 3/40 (vs. DSA negative cases 5/45, P = 0.4) and ptc 2 in 10/40 (vs. DSA negative cases 8/45, P = 0.2). Ptc, glomerular lesions, and graft loss To overcome the lack of DSA data for the whole study population, we first performed a subanalysis of patients suspicious for humoral rejection, given compatible histomorphology (g40, in combination with ptc, a strong surrogate of antibody antigen binding 15 ), disregarding C4d status, thus including probable C4d-negative humoral rejections. Including glomerulitis in our multivariate risk model did not change the observed independent association of diffuse ptc with graft loss (HR = 1.65 (CI: ), P = 0.036). In contrast, ptc score 3 lost the previously significant association with graft loss, only displaying a clinically relevant trend for increased 336 Kidney International (2015) 88,

6 Table 4 The association of peritubular capillaritis score, extent, and leukocytic composition with the slope of estimated glomerular filtration rate (Mayo Clinic) variation between 1 and 3 years ΔeGFR, ml/min per 1.73 m 2 /year 1 3 year Ptc Ptc extent 40 P-value Focal P-value Diffuse P-value Coef.+s.e ± ± ± Ptc score Coef.+s.e ± ± ± Ptcleukocyticcomposition Mononuclear Granulocytic Mixed Coef.+s.e ± ± ± Abbreviations: Coef., coefficient; egfr, estimated glomerular filteration rate; ptc, peritubular capillaritis. P-values according to Pearson s test (compared with ptc0 for leukocytic composition). graft loss (ptc1: HR = 0.92 (CI: ), P = 0.78, ptc2: HR = 1.29 (CI: ), P = 0.34, ptc3: HR = 2.08 (CI: ), P = 0.13, respectively). Second, thirty-four biopsy specimens displayed features suggestive of chronic AMR (camr) with C4d-positive dysfunction and transplant glomerulopathy. Although the prevalence of camr was low, multivariate analysis, as previously described, revealed that diffuse ptc (OR = 4.48 (CI: ), P = 0.003), presensitization (OR = 2.34 (CI: ), P = 0.04), and retransplantation (OR = 2.62 (CI: ), P = 0.02) were strong independent risk factors for the development of lesions suspicious of camr. Ptc and renal function As a secondary end point, egfr slope (ΔeGFR) between the first and third year after transplantation was calculated. First, we could observe that ptc per se showed a significant relationship with greater ΔeGFR of 1.91 ml/min per 1.73 m 2 /year (P = 0.023). The analysis of renal function according to ptc extent and ptc score revealed a significantly greater ΔeGFR of 2.2 ml/min per 1.73 m 2 /year (P = 0.033) for diffuse ptc and ptc 2, with ΔeGFR of 3.22 ml/min per 1.73 m 2 /year (P = 0.005). Finally, we analyzed the effect of the cellular composition on ΔeGFR (Table 4), where only granulocytic ptc showed a significant egfr decrease after 3 years of 3.06 ml/min per 1.73 m 2 /year (P = 0.02). Further details are shown in Table 4. DISCUSSION The major findings of this study are the hitherto unknown prognostic relevance of diffuse ptc in renal allograft biopsies in contrast to the relatively low prognostic significance of its cellular composition. An extended follow-up allowed us to determine both Banff ptc score 3 and diffuse ptc as strong independent risk factors for censored graft loss, even after consideration of important baseline confounders and additional histological risk factors such as ptc score, glomerulitis, or multiple rejection episodes. Moreover, diffuse ptc also turned out to be an independent risk factor for the development of camr (defined as transplant glomerulopathy+c4d), 3,7,9 a previously unpublished finding. The reported prevalence of ptc varies (17 46%) mostly owing to the inclusion of indication or protocol biopsies 7,12 14 in published cohorts. Accordingly, in our cohort of indication biopsies, we observed an intermediate prevalence of 25%. The inclusion of a large sample size of 749 recipients with 1322 indication biopsies allowed us to correct for potential limitations of former published studies, specifically addressing or marginally dealing with ptc and involving smaller sample sizes (ranging from 86 to 154 ptc-positive cases). 7,13,14 The largest study published so far by Gibson et al., 12 especially focusing on diagnostic ptc features, included overall 742 biopsies and reported precise results of 163 ptc-positive cases, with only limited ability for applying multivariate models. The evaluation of the extent of ptc in transplant pathology reports has been finely addressed in the second largest study to this topic, 12 showing moderate inter-observer variability for reporting this feature. In our study, we have also demonstrated the best concordance in reporting ptc extent compared with other features of ptc. Even if Gibson et al. 12 principally aimed at demonstrating feasibility of scoring ptc, their data indicated a potentially deleterious impact of diffuse ptc on future allograft function (significant loss of egfr at 2 years). Our study confirms this observation and reports for the first time a major independent association of diffuse ptc with graft loss and a greater egfr slope after 3 years. Moreover, although in our cohort the simple dichotomous classification of ptc (positive vs. negative) showed a 30% increased risk of allograft loss, yet failed to reach statistical significance, a more detailed study revealed significantly worse allograft outcome with high-grade ptc. Reinforcing this fact, we could document that this finding was independent of various confounding variables. However, when trying to correct for other important confounders such as glomerulitis or multiple rejection episodes, the observed independent association for ptc3 lost its statistical significance. Still, ptc3 displayed a trend for a twofold increased graft loss risk, which may be considered clinically relevant. Our results furthermore re-emphasized the negative role of ptc by its association with both features of AMR and TCMR, as previously described. 1,12 Ptc was not only associated with acute rejection features, but also appeared to be Kidney International (2015) 88,

7 highly associated with lesions of chronic injury i.e., chronic tubulointerstitial damage and transplant glomerulopathy. 3,7,9,16 In contrast, when studying only the cellular composition of ptc, we found no significant association with important clinical end points. Moreover, even a subanalysis (data not shown) derived from the recommendation of the BANFF classification 2005 (use asterisk to indicate only mononuclear cells and absence of neutrophils) 4 was not associated with clinical end points. Somewhat surprisingly, even predominantly granulocytic ptc did not negatively influence outcome in our cohort. Traditionally, infiltration of PTC with polymorphonuclear leukocytes in acute rejection is seen as worrisome and has been previously identified in hyperacute rejection, in the early descriptions of AMR, 17,18 or in patients with high immunological risk. 19,20 Particularly in highly sensitized recipients, the identification of a high number of polymorphonuclear leukocytes may be associated with a higher graft loss risk, 17,18 and also in our cohort we observed a significantly higher ΔeGFR in case of granulocytic ptc. However, the relatively low prevalence of this lesion in the previously published cohorts, i.e., 8/21 AMR cases with more polymorphonuclear leukocyte in biopsy, 20 or in our set (n = 27), and the lack of information on ptc extent in the majority of previous publications 1 make predominantly granulocytic ptc only cautiously interpretable, not allowing definitive conclusions regarding its true biological and diagnostic impact. We are well aware of the retrospective nature of our study and its potential drawbacks. Our results derived from indication biopsies may differ from findings in protocol biopsies, which is not part of our clinical routine. We tried to correct for this potential selection bias by including a large sample size of consecutive transplant recipients. More importantly, the main aim of the current study was to test the independent effect of different features of ptc on outcome. Whether this effect may have been more pronounced in the presence of de novo development of HLA antibodies 21 cannot be answered in the course of this study, as these data were not assessed routinely. The presence of DSA, however, is a crucial diagnostic feature for the diagnosis of AMR, 15 which makes the presented results not completely generalizable according to current definitions of AMR. Unfortunately, we had DSA data only for a small subpopulation, in which DSA-positive recipients did not show an increased proportion of diffuse ptc. However, the small sample size did not allow inclusion of this feature as a probable confounding variable. We have, however, tried to correct this potential bias by several ways and provided further arguments that the association of diffuse ptc with graft loss may not merely reflect another surrogate of DSA. First, we could clearly demonstrate that even in the setting of intragraft complement activation detected by C4d immunohistochemistry in PTC, a strong marker for circulating DSA, 22 and also indirect evidence of antigen antibody binding on the endothelium, high-grade and diffuse ptc remained independent risk factors for allograft loss and impaired renal function. Second, we expanded the analyzed cohort at risk for AMR by adding glomerulitis in our Cox regression model and therefore including another surrogate of antigen antibody binding [microvascular inflammation score 2(g 1+ptc 1)], in addition to C4d-positive dysfunction, as previously described. 23 The inclusion of two surrogates of antigen antibody binding, however, reduced the significant association of ptc score in terms of graft loss, whereas diffuse ptc remained a strong predictor of graft loss. Third, the inclusion of TCMR as another confounder, where ptc can also be frequently observed, 12 allowed large exclusion of a potential interaction of our findings with mixed rejection cases (AMR+TCMR). 12 This largely scaled study provides strong evidence of the usefulness of a clear-cut scoring system of ptc, with diffuse ptc and ptc score 3 as strong independent risk factors for impaired graft outcomes. In contrast, leukocytic composition in ptc does not add significant prognostic information. The association of diffuse ptc with outcome is independent of ptc score and may represent a novel risk factor for the development of camr, higher graft loss rates, and impaired renal function. Hence, in contrast to typing its infiltrating inflammatory cells, it seems that indicating score and extent of ptc in routine kidney allograft pathology report is compulsory for the assessment of transplant prognosis. MATERIALS AND METHODS Patients In the current retrospective study, 885 of 1248 consecutive adult kidney allograft recipients with indication biopsy, who had been transplanted at the Medical University of Vienna between January 1999 and April 2006, were eligible for study. Of those subjects, 749 recipients fulfilled inclusion criteria, which were as follows: (i) availability of at least one indication biopsy performed for unexplained graft dysfunction and/or proteinuria and (ii) availability of adequate material for a comprehensive re-evaluation and subclassification of ptc. For the primary end point, i.e., deathcensored graft loss (defined as the date of initiation of either form of renal replacement therapy after first indication biopsy), solely histological information starting from the point of first indication biopsy was used for all included recipients (n = 749). The secondary end point was egfr slope (ΔeGFR) after 3 years according to the Mayo Equation 24 (n = 476 recipients). Patients on dialysis were considered as having an egfr of 5 ml/min per 1.73 m 2. 15,25 A study flowchart is provided in Figure 1. Baseline immunosuppression is detailed in Table 2. As a result of a prospective C4d stain at our institution since 1999, 72% of recipients with C4d-positive graft dysfunction received one or a combination of the below listed antihumoral therapies: 7 were switched to tacrolimus and/or received a steroid bolus; 14 had thymoglobulin (anti-thymocyte globulin)-based rejection or induction therapy; immunoadsorption-based induction or rejection therapy was performed in 25 patients, with or without intravenous immunoglobulins and/or anti-thymocyte globulin; 1 increased immunosuppression (initiation of mycophenolate mofetil and steroids); 13 did not receive any treatment; and 5 received steroid pulse therapy alone. 338 Kidney International (2015) 88,

8 Histology For 1322 indication biopsies (mean time after transplantation 2.4 months ± 8 s.d.), adequate material for a detailed evaluation and subclassification of ptc was available. C4d staining was performed via immunohistochemistry on deparafinized sections, as previously described, 26,27 and interpreted according to the recommendations of the Banff classification. One pathologist (NK), who was blinded to clinical data, performed the re-evaluation of ptc, using periodic acid Schiff and/or hematoxylin and eosin staining when available. Ptc has been assessed for its extent (10 50% of the PTC of renal cortex: focal, 450%: diffuse) and its intensity/score according to the ptc score (referring to infiltrating cells per capillary), as defined by the Banff classification, 4 excluding areas of pyelonephritis, infarction, or subcapsular regions. Unlike the recommendation briefly described in the Banff 05 meeting report ( Use asterisk (*) to indicate only mononuclear cells and absence of neutrophils ) 4 or the count used in a previous study ( presence of mononuclear cells only, proportion of polymorphonuclear leukocytes 50% vs. 450% of intraluminal cells ), 12 we decided to assess the composition of ptc in a different semiquantitative manner. The quantification of leukocytic composition of ptc was as follows: (i) predominantly mononuclear (475% mononuclear cells), when mononuclear cells were at least three times as many as granulocytes; (ii) granulocytic dominated (475% granulocytes), when granulocytes were at least three times as many as mononuclear cells; or (iii) mixed, if no dominant population was identified. The main aim behind this deviation from the only previously published scoring 12 was to provide a clear-cut scoring scheme for leukocyte composition with potential prognostic benefits. For reproducibility studies, three nephropathologists (NK, HR, and CK) scored ptc according to the criteria used by Gibson et al. 12 and according to the criteria mentioned above in 50 cases from the cohort, in a blinded manner. HLA Serology Complement-dependent cytotoxicity (CDC) panel reactive antibody (PRA) reactivity was reported for all included subjects, and recipient presensitization was defined when a threshold 410% CDC PRA reactivity was reached. Posttransplant DSA values were not routinely performed in the respective time period in our center. For a small subcohort of previously published recipients, however, posttransplant serology was available for further study. 28,29 HLA singleantigen reactivities were assessed on a Luminex platform by applying LABScreen kits (One Lambda, Canoga Park, CA) according to the manufacturer's protocol. Test thresholds were defined according to mean fluorescence intensity above 500. All study procedures have been approved by the institutional ethics committee adhering assurance to the Declaration of Helsinki and Istanbul. Statistics We present our data as follows: frequencies as count and percentage. For univariate analyses, we used the Fisher's exact test or χ 2 -test as specified; ptc was the risk factor of interest, which we analyzed separately according to score, extent, and cellular composition, as categorized variable. Reproducibility studies were conducted by calculating a mean quadratic weighed Cohen s kappa coefficient. For the primary end point, i.e., death-censored graft loss, the unit of analysis was individual patients, considering risk factor information from the first available biopsy only. We assessed the effect of ptc on graft survival using the Kaplan Meier method, the observation beginning from the point of indication biopsy. The effect of ptc and its qualities on graft loss was estimated using proportional hazards Cox regression models. Potential confounders for multivariate analysis were as follows: baseline immunosuppression, C4dpositive graft dysfunction, acute TCMR = Banff Ia, retransplantation, HLA mismatch, and presensitization (CDC PRA 410%). As a secondary end point, we assessed the effect of ptc qualities on ΔeGFR between one and three years post transplantation, using linear regression and used t-tests for hypothesis testing. Other outcome parameters were C4d-positive transplant dysfunction and TCMR as dichotomized variables (yes vs. no). For these analyses, all available biopsies were used, and biopsy was the unit of analysis. For estimation of the effect of ptc on graft rejection, we used random coefficient logistic regression models to allow for a correlation between multiple biopsies per patient. We used the likelihood ratio test to assess linear effects from exposures with ordered categories, and to test for first-order interaction. We used MS Excel for Mac 2011 and Stata 11 for Mac (Stata, College Station, TX) for data management and calculations. Generally, we considered a two-sided P-value o0.05 as statistically significant. DISCLOSURE All the authors declared no competing interests. ACKNOWLEDGMENTS The authors thank Karin Priessner for excellent technical assistance. REFERENCES 1. Fahim T, Bohmig GA, Exner M et al. The cellular lesion of humoral rejection: predominant recruitment of monocytes to peritubular and glomerular capillaries. Am J Transplant 2007; 7: Racusen LC, Colvin RB, Solez K et al. Antibody-mediated rejection criteria-an addition to the Banff 97 classification of renal allograft rejection. Am J Transplant 2003; 3: Shimizu A, Yamada K, Sachs DH et al. Persistent rejection of peritubular capillaries and tubules is associated with progressive interstitial fibrosis. Kidney Int 2002; 61: Solez K, Colvin RB, Racusen LC et al. 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