Antibody-mediated rejection (AMR) is a major cause of graft loss after kidney transplantation (KT)1 and is mainly associated with preformed anti-human leukocyte antigen (HLA) donor-specific antibodies (DSAs) (phenotype 1) or de novo DSA (dnDSA) (phenotype 2).2 Recently, Aubert et al3 reported that preexisting DSA AMR had superior graft survival compared with dnDSA, which could partly be explained by the fact that patients with dnDSA AMR are biopsied later, when graft dysfunction and/or proteinuria is already present, associated with chronic active lesions.
AMR is a process with an early stage named called “subclinical AMR” (sAMR), in which histologic lesions are present in the kidney graft without clinical graft dysfunction.4 The impact of such lesions is now well recognized on the occurrence of transplant glomerulopathy5 and graft survival at 5 years post-biopsy6 in phenotype 1 AMR. The impact of sAMR associated with dnDSA at any time posttransplant has been less studied and reported. The monitoring of dnDSA post-KT paired with a systematic biopsy in case of appearance, even in the absence of graft dysfunction, is not part of a routine clinical practice in all KT centers. This strategy could be a valuable option, to begin treatment of AMR before graft dysfunction occurs, and therefore to attempt to improve prognosis associated with phenotype 2 AMR. Recently, Parajuli et al7 suggested that early diagnosis and treatment of sAMR using DSA monitoring may improve outcomes after KT, but in this study, DSAs were mainly preformed DSA and the follow-up was relatively short to detect graft loss in the subclinical group.
In order to demonstrate the interest of such a strategy and the impact of sAMR on graft function and survival, we designed a multicentric retrospective study including patients with dnDSA after KT, biopsied for the appearance of DSA in the absence of graft dysfunction. We reported histologic characteristics associated with sAMR and compared outcomes between patients with and without a specific treatment for sAMR.
MATERIALS AND METHODS
Patients from 9 French KT centers from the Spiesser group were retrospectively enrolled in this study, based on the following inclusion criteria:
- – Kidney transplant recipients over 18 years old
- – dnDSA (mean fluorescence intensity [MFI], >1000) at any time posttransplantation
- – Kidney graft biopsy performed between 2008 and 2016 within 6 months of DSA detection, without graft dysfunction (serum creatinine variation <20% above baseline between DSA occurrence and DSA detection and proteinuria/creatininuria ratio <0.5 g/g).
- – No specific treatment for dnDSA detection begun before kidney biopsy.
Nevertheless, during this period, some patients with subclinical dnDSA did not get a protocolar biopsy and were not included.
Basic demographic information, date of KT, and immunosuppressive therapy were recorded from medical charts and from the ASTRE database from the Spiesser group (final agreement from the French commission of the Commission nationale de l'informatique et des libertés (CNIL), decision DR-2012-518, October 29, 2012). We recorded the histology of kidney biopsies, DSA information, as well as patient and graft survival. We calculated estimated glomerular filtration rate (eGFR) using the Modification of Diet in Renal Disease equation8 at 1, 3, and 5 years after biopsy, when patients reached this point. We defined graft failure as eGFR <5 mL/min/1.73 m2 and/or return to chronic dialysis.
Assessment of dnDSA
In all centers, DSAs were monitored yearly after KT with a Luminex screening test and confirmed with a single antigen beads solid phase assay (Labscreen; One Lambda) in case of positivity. When a dnDSA was detected, sera between KT and DSA detection were tested again with a single antigen beads to date exactly the occurrence of this DSA if screening test was only performed during this period. The single antigen beads assay was used according to the manufacturer’s instructions in each center. The definition of dnDSA was any DSA identified after transplant that reached MFI >1000 and was not detected before transplant. The intensity of DSA used in the present study was related to the serum on the day of the biopsy. Immunodominant DSA (iDSA) was defined as the dnDSA with the highest intensity. The sum of the DSA (sDSA) was defined as the sum of the MFI of all dnDSA in the patient.
Kidney biopsy tissue was processed for light microscopy and C4d by immunofluorescence and immunoperoxidase on paraffin sections. Peritubular C4d was considered positive if C4d staining was in at least 10% of peritubular capillaries (C4d2 or C4d3) by immunofluorescence on frozen sections or in any peritubular capillaries by immunoperoxidase on paraffin sections (C4d score, >0).9 All transplant biopsies for dnDSA were reviewed for the study and scored according to the 2017 Banff classification10 by 1 experienced pathologist (A.F.).
Immunosuppression and Treatments Protocols
Doses and drug levels were individually adjusted at physician’s discretion based on the patient’s clinical condition and comorbidities. There was no standardized protocol for sAMR treatment in the study and we considered as “specific treatment” for sAMR a modification of the immunosuppressive regimen including at least 1 of the following: intravenous immunoglobulins, rituximab, plasmapheresis.
Statistics were performed using Statview version 5.0 (SAS Institute Inc., Brie Comte Robert, France). Quantitative variables were expressed as mean ± SD, whereas qualitative variables were expressed in numbers and percentages. Categorical variables were compared using the chi-square test, and the Student test was used for continuous variables. A 2-sided P value < 0.05 was considered to be statistically significant. Patient and graft survival data were assessed by Kaplan–Meier analysis. Log rank test was used to compare survival between groups. Logistic regression analysis was used to evaluate potential factors associated with the diagnosis of active sAMR and chronic active sAMR and to determine risk factors for graft dysfunction in patients without sAMR lesions at 5 years. All factors with P < 0.1 in the univariate analysis were included in the multivariate model. A P < 0.05 was considered statistically significant. Results were presented as hazard ratio and 95% confidence interval. To determine factors associated with active sAMR, 2 models were used: the first one (model 1) including iDSA as and the second (model 2) including sDSA: both factors could not be included in the same model because these 2 parameters depend one on the other.
One hundred twenty-three kidney transplant recipients were included in the study. General characteristics of the population are reported in Table 1. Patients had a dnDSA detected after a mean time of 5.7 ± 5.1 years after KT. Mean time between DSA detection and DSA occurrence was 12.8 ± 21.2 months (<12 months: 70%; >60 months: 4.9%). At the time of biopsy, eGFR was stable since DSA occurrence (55.3 ± 18.9 mL/min/1.73 m2) and proteinuria was 0.17 ± 0.15 g/g of creatininuria. Immunosuppressive therapy used at the time of DSA de novo detection is summarized in Table 2. The mean follow-up was 53.4 ± 21.5 months after the protocol biopsy. Ninety patients reached at least 5 years of follow-up.
Characteristics of dnDSA
DSAs were mainly class II HLA antibodies and mainly anti-DQ (71.5% of dnDSA) (Table 3). On the day of the biopsy, mean MFI of iDSA was 6247 ± 4016 (1017–20 141) and the mean sum of the MFI of all DSA was 7786 ± 5562 (1017–31 261).
Histologic Lesions of Protocol Biopsies
All biopsy lesions are reported and depicted in Figure 1. According to Banff 2017 classification 51, sAMR (41.4%) was diagnosed, of which 32 active (26%) and 19 chronic active sAMRs (15.5%). Seventy-two biopsies found no AMR (58.5%). Peritubular C4d deposition was positive in 53.1% of active sAMR cases and in 31.6% of the biopsies with chronic active sAMR.
General characteristics of the patients, DSA and histology, according to the sAMR status are reported in Table 4.
Factors Associated With the Diagnosis of Active sAMR and Chronic Active AMR
In multivariate analysis (Tables 5 and 6), predictive factors associated with the diagnosis of active sAMR were MFI of iDSA >4000 and age of the recipient <45 years old according to model 1, and sum of the MFI of all DSA >6300 and the absence of steroids on the day of the biopsy according to model 2. The cut-off values of MFI for iDSA and sDSA were determined using receiver operating characteristic (ROC) curve (Figure S1, SDC,http://links.lww.com/TP/B845). The area under the curve for the combination of multivariate factors associated with active sAMR in the presence of dnDSA was 0.783 (95% CI, 0.691-0.857) for model 1 and 0.795 (95% CI, 0.704-0.868) for model 2.
In multivariate analysis (Table 7), the only predictive factor associated with the diagnosis of chronic active sAMR was a proteinuria >200 mg/g at biopsy. The cut-off value for proteinuria was also determined using ROC curve (Figure S2, SDC, http://links.lww.com/TP/B845).
Evolution of eGFR at 1, 3, and 5 Years Post-Biopsy
According to the nature of the lesions found on the biopsy, the evolution of eGFR was significantly different, as reported in Figure 2. The decrease of eGFR between the biopsy and 5 years post-biopsy was significantly higher in patients with active sAMR (−25.2 ± 28.3 mL/min/1.73 m2) and in patients with chronic active sAMR (−17.7 ± 17.9 mL/min/1.73 m2), compared with patients without lesions of AMR in which eGFR was stable (−0.8 ± 14.4 mL/min/1.73 m2). At 3 years postbiopsy, the drop of eGFR was already significant in patients with active and chronic active sAMR, more important in case of active sAMR.
Graft and Patient Survival
During the follow-up, we noticed 13 graft losses, after a mean time of 40.0 ± 16.6 months—7 of 32 patients in patients with active sAMR (21.8%); 4 of 19 patients in patients with chronic active sAMR (21%); and 2 of 72 patients in patients without AMR. The causes of graft loss were from chronic AMR in the sAMR group and 1 from chronic active AMR and 1 from calcineurin inhibitor toxicity in group without sAMR. Death-censored graft survival (Figure 3) was significantly higher in patients without sAMR compared with the 2 other groups but was not different between active and chronic active sAMR. We presented in Figure 4 the death-censored graft survival in patients with sAMR according to the peritubular c4d staining status. We did not show any significant difference between the 2 groups (P = 0.08).
During the follow-up, 6 deaths were observed after a mean time of 54.4 ± 28.3 months. Two patients died from a posttransplant lymphoproliferative disease, 2 patients from infectious disease (1 bacterial infection, 1 pneumocystosis), 1 patient from cardiovascular disease, and 1 from an undetermined cause. Five deaths occurred in the sAMR group (2 in patients with active sAMR, 3 in patients with chronic active sAMR) and 1 in a patient without AMR. Survival was significantly higher in patients without sAMR compared with patients of the sAMR group (Figure 5).
Occurrence of sAMR, C4d staining, and graft loss according to different MFI cut-off values of sDSA are presented in Figure 6.
Evolution and Treatment of Active sAMR
We attempted to determine the efficacy of a treatment targeting active sAMR on the evolution of eGFR and on graft loss. Table 8 reports the characteristics of patients with active sAMR according to the presence or not of a specific treatment. Nineteen patients over 32 (59.4%) received a treatment for AMR. Notably, the group of patients receiving a treatment for sAMR did not differ from the group of untreated patients regarding eGFR, proteinuria, and chronic histologic lesions. Patients receiving a treatment were mainly male (P = 0.0004) and had a higher MFI of iDSA and sDSA (P = 0.04 and P = 0.03).
Graft survival (Figure 7) was not significantly different between the 2 groups (P = 0.69). In the subgroup of C4d-positive sAMR (n = 17), graft survival was nonsignificantly higher in group who received a specific treatment (n = 11; P = 0.21). In the subset of patients who reached 5 years of follow-up, evolution of eGFR after the biopsy was not significantly different between the 2 groups defined according to treatment, even when considering the subgroup of C4d-positive active sAMR.
Among patients with active sAMR (n = 32), 22 (68.7%) had a control biopsy after a mean time of 21.3 ± 9.8 months—14 of 22 patients (63.6%) developed chronic active AMR lesions on the biopsy. Only 4 of 22 patients (18.2%) had no more AMR lesions. There was no difference between the group receiving specific therapy for AMR and the group without treatment.
Evolution of Patients Without sAMR
In order to determine risk factors for graft dysfunction in patients without sAMR lesions, we selected a group of 53 of 72 (73.6%) patients who reached 5 years of follow-up after the initial biopsy (Table 9). We considered a group of patients without graft dysfunction (delta of eGFR < 20%; n = 42) and a group with dysfunction (delta of eGFR > 20%; n = 11). The only risk factor associated with dysfunction was the eGFR at the time of the protocol biopsy. Evolution of eGFR according to initial eGFR is depicted in Figure 8.
Among patients without AMR lesions (n = 72), 21 (29.2%) had a control biopsy after a mean time of 31.3 ± 14.9 months—2 patients developed active AMR, 4 patients developed chronic active AMR, and the other 15 patients had no AMR lesions on the biopsy.
We report the first multicentric study on the value of kidney graft biopsy when a subclinical dnDSA is found, at any time after KT; the use of protocol biopsy in the presence of dnDSA leads to the diagnosis of sAMR in over 40% of cases. Despite studies on the occurrence of dnDSA and the risk of rejection or allograft loss,10,11 little is known about the association between dnDSA and lesions of sAMR. Wiebe et al12 demonstrated that recipients with subclinical dnDSA experience eGFR decline and progress to graft loss, albeit at a slower rate than those who initially present with a clinical dnDSA phenotype. Our data confirm results reported by Yamamoto et al13 in a smaller sample, in which they found approximately 40% of sAMR in the same context. Schinstock et al14 previously proposed this strategy and reported a small monocentric cohort of 54 patients biopsied for dnDSA. Parajuli et al15 reported up to 50% of sAMR in 29 patients who developed dnDSA with stable function. This is an important and practical question, associated with the usefulness to monitor DSA after KT.
Our study shows that the MFI of dnDSA on the day of the biopsy has some prognostic value: the higher the MFI at the time of biopsy (iDSA, >4000; sDSA, >6300), the higher the incidence of active sAMR. These data are perfectly comparable to those depicted by Schinstock et al14 with a particular risk of active sAMR associated with a sum MFI of dnDSA over 3000. In the context of preformed DSA, Lefaucheur et al16 reported that the relative risk for graft loss in patients who underwent transplantation with peak HLA–DSAs >3000 was 3.8. Wiebe et al12 found that dnDSA MFI sum at the time of dnDSA detection predicted the risk of post-dnDSA graft loss. This suggests that performing protocol biopsy for dnDSA could be guided by the MFI of the DSA. Regarding the diagnosis of chronic active AMR, which is a more advanced stage of AMR, proteinuria, even low, is the best predictive item which could help to consider biopsy. MFI of dnDSA appeared not to be determinant in this context. We speculate that MFI was presumably higher at the beginning of the AMR process and then decreased along with the evolution to chronic lesions. Monitoring of DSA was performed yearly, and a variation between 2 points in subclinical condition could, therefore, have been missed. Finally, not all DSAs are equal: C3d or C1q fixing DSA17-19 or IgG 3 subtype20 are described as more deleterious, but we unfortunately did not perform these specific tests in the present study. In a recent study, Schinstock et al21 confirmed that IgG 3 positivity at the time of dnDSA detection was strongly associated with early allograft loss.
We demonstrated that sAMR associated with dnDSA has an impact on graft function and allograft survival. Loupy et al5 demonstrated similar impact of sAMR in the specific context of preformed DSA, with the progression to chronic AMR lesions between 3 and 12 months posttransplantation and an impact on graft function and survival at 5 years posttransplant.6 Yamamoto et al13 and Schinstock et al14 reported the same impact on renal function in a context of dnDSA. A major asset in our study is the longer follow-up after biopsy, allowing us to determine graft loss after subclinical dnDSA appearance and the impact of sAMR on graft survival. This had an impact on clinical trials testing new drugs for active sAMR because it suggested that the primary endpoint could not be the graft survival in studies with usual follow-ups.4,22
Monitoring of dnDSA after KT can help to detect early stage AMR. But little is known about the utility to begin a specific treatment for AMR in these conditions. In almost all studies dealing with the risk of allograft loss after a clinical episode of AMR, lower eGFR is associated with worst survival.3,23 Regarding subclinical T-cell–mediated rejection, the early detection and treatment may improve structural and functional outcomes leading to better long-term graft survival,24,25 but this still remains a matter of debate. In a context of sAMR associated with preformed DSA, Loupy et al5 confirmed that when AMR is not treated, it can result in chronic AMR along with impaired graft function. Parajuli et al7 suggested that early diagnosis and treatment of sAMR are associated with improved outcomes compared with clinical AMR and are associated with similar outcomes when compared with patients without rejection (dnDSA without sAMR). Once again, the follow-up in the subgroup of patients with sAMR in the latter study was too short to conclude (29.5 ± 16.8 mo). Yamamoto et al13 failed to demonstrate the efficacy of a treatment in their population. With a longer follow-up, we demonstrated that patients with sAMR do not have the same outcomes compared with patients without sAMR. In the subgroup of patients with active sAMR, we failed to determine whether a specific treatment in these conditions could improve the outcomes of AMR, graft function, and graft survival. This could partly be explained by the small number of patients but also by the heterogeneity of this subgroup of patients with sAMR. A prospective randomized trial for new strategies for the treatment of active sAMR26 is necessary to answer this question.
A majority of patients did not have sAMR on the first protocol biopsy performed. Do they have a greater risk to experience graft dysfunction, graft loss, or sAMR later? In their study, Schinstock et al14 propose to biopsy patients with dnDSA at the time of detection and 1 year after because they reported that when dnDSA was detected, only 25% had histological of active sAMR but the incidence increased to 52.9% 1 year later. In our study, 21 (29.2%) patients without AMR had a control biopsy after a mean time of 31.3 ± 14.9 months; among these, only 2 patients developed active AMR, 4 developed chronic AMR, while the other 15 patients had no AMR lesions on the control biopsy. The only factor in this subgroup of having an impact on graft function at 5 years was the eGFR at baseline, and there were only 2 graft loss. In patients with excellent graft function without sAMR and presumed good adherence, evolution is generally good as depicted by Parajuli et al7 for patients with dnDSA without sAMR. Specific treatment based on dnDSA appearance, without sAMR lesions, cannot be recommended because it is probably not efficient. Indeed, as reported by Matignon et al27 in a pilot study including kidney allograft recipients with early dnDSA, preemptive treatment with high-dose IVIG alone did not prevent active AMR and had minimal effects on DSA outcome and B-cell phenotype.
The prevalence of chronic lesions was striking in our study, sometimes at an advanced stage, with good and stable and graft function and very low proteinuria. The process of AMR seems sometimes to begin before DSA appearance, and without clinical or biological warning, is very difficult to detect. Wiebe et al12 report that in recipients with dnDSA, the rate eGFR decline was significantly increased before dnDSA detection (–2.89 versus –0.65 mL/min/1.73 m2/y; P < 0.0001), suggesting that dnDSA is both a marker and contributor to ongoing alloimmunity. We need more robust biomarkers before DSA appearance to guide us to detect earlier stage of AMR.28 Recently, Luque et al29 reported that monitoring donor-reactive memory B-cells may be useful to further characterize humoral rejection after KT.
Although our study is multicentric, with a relatively important sample, this is a retrospective study with potential bias. First, not all patients with subclinical dnDSA have had a biopsy during the period and were not included in the study; this could have overestimated the number of sAMR. Second, time between DSA detection and DSA occurrence varies importantly and could impact on the diagnosis or not of sAMR lesions. Nevertheless, this time was not predictive of sAMR in multivariate analysis. Furthermore, all transplant biopsies for dnDSA were reviewed for the study and scored according to the 2017 Banff classification9 by 1 experienced pathologist, a very important point to avoid any interindividual variability, but we did not do the same for dnDSA assessment, while interlaboratory variability exists and could be important.30 A prospective multicentric study on dnDSA and protocol biopsy could help to minimize these biases and to draw solid conclusions in this context.
In conclusion, in this relatively large multicentric study, we reported that a systematic biopsy performed for dnDSA in the absence of graft dysfunction leads to a diagnosis of sAMR in over 40% of cases. Patients with dnDSA but without sAMR seem to have a better graft function and survival compared with patients with sAMR. This screening strategy, guided by the MFI of dnDSA and clinical conditions, could help to detect early stage of AMR process even if we did not observe any effect of specific therapy on the evolution of humoral rejection. Nevertheless, clinical trials are necessary to evaluate the benefit of immunosuppressive regimen (standard of care versus new drugs) on graft function and survival in active sAMR.
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