Early clinical experience using donor‐derived cell‐free DNA to detect rejection in kidney transplant recipients

Donor‐derived cell‐free DNA (dd‐cfDNA) became Medicare reimbursable in the United States in October 2017 for the detection of rejection in kidney transplant recipients based on results from its pivotal validation trial, but it has not yet been externally validated. We assessed 63 adult kidney transplant recipients with suspicion of rejection with dd‐cfDNA and allograft biopsy. Of these, 27 (43%) patients had donor–specific antibodies and 34 (54%) were found to have rejection by biopsy. The percentage of dd‐cfDNA was higher among patients with antibody–mediated rejection (ABMR; median 1.35%; interquartile range [IQR]: 1.10%‐1.90%) compared to those with no rejection (median 0.38%, IQR: 0.26%‐1.10%; P < .001) and cell–mediated rejection (CMR; median: 0.27%, IQR: 0.19%‐1.30%; P = .01). The dd‐cfDNA test did not discriminate patients with CMR from those without rejection. The area under the ROC curve (AUC) for CMR was 0.42 (95% CI: 0.17‐0.66). For ABMR, the AUC was 0.82 (95% CI: 0.71‐0.93) and a dd‐cfDNA ≥0.74% yielded a sensitivity of 100%, specificity 71.8%, PPV 68.6%, and NPV 100%. The dd‐cfDNA test did not discriminate CMR from no rejection among kidney transplant recipients, although performance characteristics were stronger for the discrimination of ABMR.     

Long‐term outcomes of eculizumab‐treated positive crossmatch recipients: Allograft survival,histologic findings,and natural history of the donor‐specific antibodies

We aimed to determine the long‐term outcomes of eculizumab‐treated, positive crossmatch (+XM) kidney transplant recipients compared with +XM and age‐matched negative crossmatch (?XM) controls. We performed an observational retrospective study and examined allograft survival, histologic findings, long‐term B‐cell flow cytometric XM (BFXM), and allograft‐loss–associated factors. The mean (SD) posttransplant follow‐up was 6.3 (2.5) years in the eculizumab group; 7.6 (3.5), +XM control group; 7.9 (2.5), ?XM control group. The overall and death‐censored allograft survival rates were similar in +XM groups (P = .73, P = .48) but reduced compared with ?XM control patients (P < .001, P < .001). In the eculizumab‐treated group, 57.9% (11/19) of the allografts had chronic antibody‐mediated rejection, but death‐censored allograft survival was 76.6%, 5 years; 75.4%, 7 years. Baseline IgG3 positivity and BFXM ≥300 were associated with allograft loss. C1q positivity was also associated with allograft loss but did not reach statistical significance. Donor‐specific antibodies appeared to decrease in eculizumab‐treated patients. After excluding patients with posttransplant plasmapheresis, 42.3% (9/21) had negative BFXMs; 31.8% (7/22), completely negative single‐antigen beads 1 year posttransplant. Eculizumab‐treated +XM patients had reduced allograft survival compared with ?XM controls but similar survival to +XM controls. BFXM and complement‐activating donor‐specific antibodies (by IgG3 and C1q testing) may be used for risk stratification in +XM transplantation.     

Utility of protocol kidney biopsies for de novo donor‐specific antibodies

There is limited information about the role of protocol kidney biopsies for de novo donor‐specific antibodies (dnDSA) in kidney transplant recipients, especially in those with stable graft function. We initiated a routine posttransplant DSA monitoring and surveillance biopsy program for dnDSA since 2014. We identified 45 kidney transplant recipients with dnDSA detected between January 2014 and February 2017 who underwent kidney biopsy within 60 days of detection of dnDSA. Twenty‐nine (64%) had stable graft function and 16 (36%) had impaired graft function at the time of dnDSA detection. Even in the group with stable graft function, we found a high rate of rejection (53%) on biopsy. Eighty‐eight percent of patients with impaired graft function had rejection. Those patients with impaired graft function had significantly lower estimated glomerular filtration rate at 12 months postbiopsy and at last follow‐up. Those with impaired graft function had more graft failures; however, this result was not statistically significant. The high rate of asymptomatic rejection, and the fact that outcomes in asymptomatic patients are poor, is in support of the utility of surveillance biopsies in patients with dnDSA.     

Response to treatment and long‐term outcomes in kidney transplant recipients with acute T cell–mediated rejection

The recent recognition of complex and chronic phenotypes of T cell–mediated rejection (TCMR) has fostered the need to better evaluate the response of acute TCMR—a condition previously considered to lack relevant consequences for allograft survival—to the standard of care. In a prospective cohort of kidney recipients (n = 256) with biopsy‐proven acute TCMR receiving corticosteroids, we investigated clinical, histological, and immunological phenotypes at the time of acute TCMR diagnosis and 3 months posttreatment. Independent posttreatment determinants of allograft loss included the glomerular filtration rate (GFR) (HR = 0.94; 95% CI = 0.92‐0.96; P < .001), proteinuria (HR = 1.40; 95% CI = 1.10‐1.79; P = .007), time since transplantation (HR = 1.02; 95% CI = 1.00‐1.03; P = .016), peritubular capillaritis (HR = 2.27; 95% CI = 1.13‐4.55; P = .022), interstitial inflammation in sclerotic cortical parenchyma (i‐IF/TA) (HR = 1.87; 95% CI = 1.08‐3.25; P = .025), and donor‐specific anti‐HLA antibodies (DSAs) (HR = 2.67; 95% CI = 1.46‐4.88; P = .001). Prognostic value was improved using a composite evaluation of response to treatment versus clinical parameters only (cNRI = 0.68; 95% CI = 0.41‐0.95; P < .001). A classification tree for allograft loss identified five patterns of response to treatment based on the posttreatment GFR, i‐IF/TA, and anti‐HLA DSAs (cross‐validated accuracy = 0.80). Compared with responders (n = 155, 60.5%), nonresponders (n = 101, 39.5%) had a higher incidence of de novo DSAs, antibody‐mediated rejection, and allograft loss at 10 years (P < .001 for all comparisons). Thus, clinical, histological, and immunological assessment of response to treatment of acute TCMR revealed different profiles of the response to treatment with distinct outcomes.     

Reassessment of the clinical impact of preformed donor‐specific anti‐HLA‐Cw antibodies in kidney transplantation

Anti‐denatured HLA‐Cw antibodies are highly prevalent, whereas anti‐native HLA‐Cw antibodies seem to lead to random flow cytometry crossmatch results. We aimed to reassess crossmatch prediction for anti‐HLA‐Cw using 2 types of single antigen flow beads (classical beads and beads with diminished expression of denatured HLA), and to compare the pathogenicity of preformed anti‐denatured and anti‐native HLA‐Cw antibodies in kidney transplantation. We performed 135 crossmatches with sera reacting against donor HLA‐Cw (classical beads fluorescence ≥500); only 20.6% were positive. Forty‐three (31.6%) were anti‐denatured HLA antibodies (beads with diminished expression of denatured HLA fluorescence <300); all were crossmatch negative. The correlation between classical beads fluorescence and the crossmatch ratio was low (ρ = 0.178), and slightly higher with beads with diminished expression of denatured HLA (ρ = 0.289). We studied 52 kidney recipients with preformed anti‐HLA‐Cw donor‐specific antibodies. Those with anti‐native HLA antibodies experienced more acute and chronic antibody‐mediated rejections (P = .006 and .03, respectively), and displayed a lower graft survival (P = .04). Patients with anti‐native HLA‐Cw antibodies more frequently had previous sensitizing events (P < .000001) or plausibility of their antibody profile according to known anti‐native HLA‐Cw eplets (P = .0001). Anti‐native but not anti‐denatured HLA‐Cw antibodies are deleterious, which underscores the need for reagents with diminished expression of denatured HLA.     

Effect of recipient gender and donor‐specific antibodies on antibody‐mediated rejection after heart transplantation

Gender‐difference regarding antibody‐mediated rejection (AMR) after heart transplantation has been described. However, no study accounted for the presence of preformed donor‐specific antibodies (pfDSA), a known risk factor of AMR, more common among women than men. In a single‐institution 6‐year cohort (2010‐2015), time to AMR was assessed, comparing men with women by survival analysis with a 1‐year death‐censored follow‐up. All AMRs were biopsy proven. Confounding variables that were accounted for included mean intensity fluorescence (MFI) of pfDSA, recipient age, HLA‐, size‐ and sex‐mismatch. 463 patients were included. Overall incidence of AMR was 10.3% at 1 year. After adjusting for confounding variables, independent risk factors of AMR were female recipient gender (adjusted hazard‐ratio [adj. HR] = 1.78 [1.06‐2.99]), P = .03) and the presence of pfDSA (adj. HR = 3.20 [1.80‐5.70], P < .001). This association remained significant when considering pfDSA by their MFI; female recipient gender had an adj. HR = 2.2 (P = .026) and MFI of pfDSA (per 1 MFI‐increase) adj. HR = 1.0002 (P < .0001). In this cohort, women were at higher risk of AMR than men and this risk increase was additive to that of pfDSA. These findings may suggest a gender‐related difference in the severity of pfDSA.     

Isolated v‐lesion in kidney transplant recipients: Characteristics,association with DSA,and histological follow‐up

Isolated v‐lesion (IvL) represents a rare and challenging situation in renal allograft biopsies because it is unknown whether IvL truly represents rejection, antibody‐ or T cell–mediated, or not. This multicentric retrospective study describes the clinicopathological features of IvL with an emphasis on the donor‐specific antibody (DSA) status, histological follow‐up, and graft survival. Inclusion criteria were the presence of v‐lesion with minimal interstitial (i ≤ 1) and microvascular inflammation (g + ptc≤1). C4d‐positive biopsies were excluded. We retrospectively found 33 IvL biopsies in 33 patients, mainly performed in the early posttransplantation period (median time 27 days) and clinically indicated in 66.7%. A minority of recipients (5/33, 15.2%) had DSA at the time of biopsy. IvL was treated by anti‐rejection therapy in 21 cases (63.6%), whereas 12 (36.4%) were untreated. Seventy percent of untreated patients and 66% of treated patients showed favorable histological evolution on subsequent biopsy. Kidney graft survival in IvL was significantly higher than in a matched cohort of antibody‐mediated rejection with arteritis. In conclusion, IvL is not primarily antibody‐mediated and may show a favorable evolution. The heterogeneity of IvL pathophysiology on early biopsies should prompt DSA testing as well as close clinical and histological follow‐up in all patients with IvL.     

Differential effects of donor‐specific HLA antibodies in living versus deceased donor transplant

The presence of donor‐specific anti‐HLA antibodies (DSAs) is associated with increased risk of graft failure after kidney transplant. We hypothesized that DSAs against HLA class I, class II, or both classes indicate a different risk for graft loss between deceased and living donor transplant. In this study, we investigated the impact of pretransplant DSAs, by using single antigen bead assays, on long‐term graft survival in 3237 deceased and 1487 living donor kidney transplants with a negative complement‐dependent crossmatch. In living donor transplants, we found a limited effect on graft survival of DSAs against class I or II antigens after transplant. Class I and II DSAs combined resulted in decreased 10‐year graft survival (84% to 75%). In contrast, after deceased donor transplant, patients with class I or class II DSAs had a 10‐year graft survival of 59% and 60%, respectively, both significantly lower than the survival for patients without DSAs (76%). The combination of class I and II DSAs resulted in a 10‐year survival of 54% in deceased donor transplants. In conclusion, class I and II DSAs are a clear risk factor for graft loss in deceased donor transplants, while in living donor transplants, class I and II DSAs seem to be associated with an increased risk for graft failure, but this could not be assessed due to their low prevalence.     

Meeting report of the STAR—Sensitization in Transplantation Assessment of Risk: Naïve Abdominal Transplant Organ subgroup focus on kidney transplantation

The development of de novo donor‐specific HLA antibody (dnDSA) is a critical feature contributing to late allograft failure. The complexity of the issue is further complicated by organ‐specific differences, detection techniques, reliance of tissue histopathology and changing diagnostic criteria, ineffective therapies, and lack of consensus. To tackle these issues, the Sensitization in Transplantation Assessment of Risk (STAR) 2017 was initiated as a collaboration of the American Society of Transplantation and American Society of Histocompatibility and Immunogenetics consisting of 8 working groups with the goal to provide guidelines on how to assess risk and risk stratify patients based on their potential alloimmune and DSA status. Herein is a summary of discussions by the Naïve Abdominal Working Group, highlighting currently available data and identifying gaps in our knowledge on the development and impact of dnDSA following kidney transplantation.     

Lower tacrolimus exposure and time in therapeutic range increase the risk of de novo donor‐specific antibodies in the first year of kidney transplantation

De novo donor‐specific antibodies (dnDSAs) have been associated with reduced graft survival. Tacrolimus (TAC)–based regimens are the most common among immunosuppressive approaches used in in clinical practice today, yet an optimal therapeutic dose to prevent dnDSAs has not been established. We evaluated mean TAC C₀ (tacrolimus trough concentration) and TAC time in therapeutic range for the risk of dnDSAs in a cohort of 538 patients in the first year after kidney transplantation. A mean TAC C₀ < 8 ng/mL was associated with dnDSAs by 6 months (odds ratio [OR] 2.51, 95% confidence interval [CI] 1.32–4.79, P = .005) and by 12 months (OR 2.32, 95% CI 1.30–4.15, P = .004), and there was a graded increase in risk with lower mean TAC C₀. TAC time in the therapeutic range of <60% was associated with dnDSAs (OR 2.05, 95% CI 1.28‐3.30, P = .003) and acute rejection (hazard ratio [HR] 4.18, 95% CI 2.31–7.58, P < .001) by 12 months and death‐censored graft loss by 5 years (HR 3.12, 95% CI 1.53–6.37, P = .002). TAC minimization may come at a cost of higher rates of dnDSAs, and TAC time in therapeutic range may be a valuable strategy to stratify patients at increased risk of adverse outcomes.     

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.     

Plasma‐Derived C1 Esterase Inhibitor for Acute Antibody‐Mediated Rejection Following Kidney Transplantation: Results of a Randomized Double‐Blind Placebo‐Controlled Pilot Study

Antibody‐mediated rejection (AMR) is typically treated with plasmapheresis (PP) and intravenous immunoglobulin (standard of care; SOC); however, there is an unmet need for more effective therapy. We report a phase 2b, multicenter double‐blind randomized placebo‐controlled pilot study to evaluate the use of human plasma‐derived C1 esterase inhibitor (C1 INH) as add‐on therapy to SOC for AMR. Eighteen patients received 20 000 units of C1 INH or placebo (C1 INH n = 9, placebo n = 9) in divided doses every other day for 2 weeks. No discontinuations, graft losses, deaths, or study drug‐related serious adverse events occurred. While the study's primary end point, a difference between groups in day 20 pathology or graft survival, was not achieved, the C1 INH group demonstrated a trend toward sustained improvement in renal function. Six‐month biopsies performed in 14 subjects (C1 INH = 7, placebo = 7) showed no transplant glomerulopathy (TG) (PTC+cg≥1b) in the C1 INH group, whereas 3 of 7 placebo subjects had TG. Endogenous C1 INH measured before and after PP demonstrated decreased functional C1 INH serum concentration by 43.3% (p < 0.05) for both cohorts (C1 INH and placebo) associated with PP, although exogenous C1 INH–treated patients achieved supraphysiological levels throughout. This new finding suggests that C1 INH replacement may be useful in the treatment of AMR.     

Impact of pretransplant donor‐specific antibodies on kidney allograft recipients with negative flow cytometry cross‐matches

The Luminex test can detect low levels of donor‐specific antibody (DSA) that cannot be detected by flow‐cytometric cross‐matching (FCXM) in kidney transplantation (KT). This study evaluated the impact of DSA on clinical outcomes in KT recipients negative on FCXM. Of 575 consecutive patients who underwent living donor KT between January 2013 and July 2016, 494 (85.9%) were DSA‐negative and 81 (14.1%) were DSA‐positive. Although rates of acute cellular rejection (ACR) at 1 year were similar in the 2 groups (P = .54), the incidence of antibody‐mediated rejection (ABMR) was significantly higher in the DSA‐positive group (P < .01). There was no statistically significant association between rejection‐free graft survival (RFGS) rates and pretransplant class I DSA. However, evaluation of pretransplant class II DSA showed that RFGS rates were significantly lower in patients with mean fluorescence intensity (MFI) >3000 than in patients with DSA‐negative (P < .01). On multivariate analyses, class II DSA MFI ≥5000 was a significant risk factor for acute rejection (hazard ratio, 7.48; P < .01). These findings suggested that pretransplant DSA alone did not affect graft survival in KT recipients without desensitization. However, class II DSA MFI >5000 was an independent predictor of acute rejection in DSA‐positive patients.     

Markers of graft microvascular endothelial injury may identify harmful donor‐specific anti‐HLA antibodies and predict kidney allograft loss

Graft microvasculature is a major target of donor‐specific antibodies (DSA) and endothelial damage is direct evidence of antibody‐mediated rejection (ABMR). Using immunohistochemistry, we analyzed the expression of three microvascular endothelial activation markers (fascin, vimentin, and hsp47), suggestive of endothelial‐to‐mesenchymal transition (EndMT) in 351 graft biopsies from 248 kidney recipients, with concomitant screening of circulating antihuman leukocyte antigen (HLA) DSA at the time of the biopsy. The factors associated with EndMT marker expression were DSA and the presence of microvascular inflammation (MI). EndMT expressing grafts had significantly more allograft loss compared to EndMT negative grafts (P < .0001). The expression of EndMT markers positively correlated with anti‐HLA DSA class II mean fluorescence intensity (MFI) levels and especially identified DQ and DR antibodies as being more closely associated with microvascular injury. Moreover, only DSA linked to positive EndMT score affected allograft survival, regardless of DSA MFI levels or presence of C4d deposition. Thus, EndMT markers could represent a clinically relevant tool for early identification of ongoing endothelial injury, harmful DSA, and patients at high risk for allograft failure.     

Safety and efficacy of eculizumab for the prevention of antibody‐mediated rejection after deceased‐donor kidney transplantation in patients with preformed donor‐specific antibodies

The presence of preformed donor‐specific antibodies in transplant recipients increases the risk of acute antibody‐mediated rejection (AMR). Results of an open‐label single‐arm trial to evaluate the safety and efficacy of eculizumab in preventing acute AMR in recipients of deceased‐donor kidney transplants with preformed donor‐specific antibodies are reported. Participants received eculizumab as follows: 1200 mg immediately before reperfusion; 900 mg on posttransplant days 1, 7, 14, 21, and 28; and 1200 mg at weeks 5, 7, and 9. All patients received thymoglobulin induction therapy and standard maintenance immunosuppression including steroids. The primary end point was treatment failure rate, a composite of biopsy‐proved grade II/III AMR (Banff 2007 criteria), graft loss, death, or loss to follow‐up, within 9 weeks posttransplant. Eighty patients received transplants (48 women); the median age was 52 years (range 24‐70 years). Observed treatment failure rate (8.8%) was significantly lower than expected for standard care (40%; P < .001). By 9 weeks, 3 of 80 patients had experienced AMR, and 4 of 80 had experienced graft loss. At 36 months, graft and patient survival rates were 83.4% and 91.5%, respectively. Eculizumab was well tolerated and no new safety concerns were identified. Eculizumab has the potential to provide prophylaxis against injury caused by acute AMR in such patients (EudraCT 2010‐019631‐35).     

Pre‐existing donor‐specific antibodies are detrimental to kidney allograft only when persistent after transplantation

Donor‐specific antibodies (DSA) increase the risk of allograft rejection and graft failure. They may be present before transplant or develop de novo after transplantation. Here, we studied the evolution of preformed DSA and their impact on graft outcome in kidney transplant recipients. Using the Luminex Single Antigen assay, we analyzed the sera on the day of transplantation of 239 patients who received a kidney transplant. Thirty‐seven patients (15.5%) had pre‐existing DSA detected the day of transplantation. After 5 years, the pre‐existing DSA disappeared in 22 patients whereas they persisted in 12. Variables associated with DSA persistence were age <50 years (P = 0.009), a history of previous transplantation (P = 0.039), the presence of class II DSA (P = 0.009), an MFI of preformed DSA >3500 (P < 0.001), and the presence of two or more DSA (P < 0.001). DSA persistence was associated with a higher risk of graft loss and antibody‐mediated rejection. Previously undetected preformed DSA are deleterious to graft survival only when they persist after transplantation.     

Anti‐Donor HLA Antibody Response After Pancreatic Islet Grafting: Characteristics,Risk Factors,and Impact on Graft Function

Pancreatic islet grafting restores endogenous insulin production in type 1 diabetic patients, but long‐term outcomes remain disappointing as a result of immunological destruction of allogeneic islets. In solid organ transplantation, donor‐specific anti‐HLA antibodies (DSA) are the first cause of organ failure. This retrospective multicentric study aimed at providing in‐depth characterization of DSA response after pancreatic islet grafting, identifying the risk factor for DSA generation and determining the impact of DSA on graft function. Forty‐two pancreatic islet graft recipients from the Groupe Rhin‐Rhône‐Alpes‐Genève pour la Greffe d'Ilots de Langerhans consortium were enrolled. Pre‐ and postgrafting sera were screened for the presence of DSA and their ability to activate complement. Prevalence of DSA was 25% at 3 years postgrafting. The risk of sensitization increased steeply after immunosuppressive drug withdrawal. DSA repertoire diversity correlated with the number of HLA and eplet mismatches. DSA titer was significantly lower from that observed in solid organ transplantation. No detected DSA bound the complement fraction C3d. Finally, in contrast with solid organ transplantation, DSA did not seem to negatively affect pancreatic islet graft survival. This might be due to the low DSA titers, specific features of IgG limiting their ability to activate the complement and/or the lack of allogenic endothelial targets in pancreatic islet grafts.     

Specificity,strength, and evolution of pretransplant donor‐specific HLA antibodies determine outcome after kidney transplantation

In this cohort study (N = 924), we investigated the evolution and clinical significance of pretransplant donor‐specific HLA antibodies (preDSA), detected in the single‐antigen beads assay but complement‐dependent cytotoxicity crossmatch‐negative. Donor specificity of the preDSA (N = 107) was determined by high‐resolution genotyping of donor‐recipient pairs. We found that in 52% of the patients with preDSA, preDSA spontaneously resolved within the first 3 months posttransplant. PreDSA that persisted posttransplant had higher pretransplant median fluorescence intensity values and more specificity against DQ. Patients with both resolved and persistent DSA had a high incidence of histological picture of antibody‐mediated rejection (ABMR_h; 54% and 59% respectively). Patients with preDSA that persisted posttransplant had worse 10‐year graft survival compared to resolved DSA and preDSA‐negative patients. Compared to cases without preDSA, Cox modeling revealed an increased risk of graft failure only in the patients with persistent DSA, in the presence (hazard ratio [HR] = 8.3) but also in the absence (HR = 4.3) of ABMR_h. In contrast, no increased risk of graft failure was seen in patients with resolved DSA. We conclude that persistence of preDSA posttransplant has a negative impact on graft survival, beyond ABMR_h. Even in the absence of antibody‐targeting therapy, low median fluorescence intensity DSA and non‐DQ preDSA often disappear early posttransplantation and are not deleterious for graft outcome.