Antibody-mediated rejection (AMR) is one of the main causes of premature renal allograft failure.1-3 Because the established treatment with plasmapheresis (PPH) and intravenous immunoglobulin preparations (IVIG) is obviously not sufficiently effective,4,5 protocols directly targeting B cells and plasma cells with rituximab6-8 and bortezomib9-11 have been investigated. In one of the first clinical studies in this field, we observed a trend toward a better efficacy of bortezomib-based treatment in combination with PPH and IVIG as compared to fixed dose rituximab-based treatment.12 However, even bortezomib-based treatment had only limited efficacy. To improve treatment efficacy without increasing substance-specific side effects, we recently investigated the use of rituximab in combination with bortezomib, PPH, and high-dose IVIG. Compared with patients treated with the same regimen without rituximab combined administration of bortezomib and rituximab did not further improve graft survival or function.13 Instead, we observed an increased incidence of adverse events, especially bacterial infections.
For several decades, cyclophosphamide has been successfully used to treat autoimmune diseases associated with autoantibodies. According to current guidelines, cyclophosphamide is recommended for the treatment of systemic anti-neutrophil cytoplasmatic antibody (ANCA)-associated vasculitis14-16 and certain classes of lupus nephritis.17 In case of ANCA-associated vasculitis, cyclophosphamide applied as intravenous (i.v.) pulse treatment was shown to be equally effective compared with daily oral treatment at a reduced cumulative dose and fewer side effects.18 According to current evidence the recommended dose of i.v. pulse treatment in patients with ANCA vasculitis is 15 mg/kg with dose adjustments for age and renal function.19 The use of cyclophosphamide in human organ transplantation was described by Starzl et al20 in 1971. In the 1980s Vangelista et al21 and Bonomini et al22 reported on the treatment of patients with renal allograft rejection and anti-HLA antibodies with PPH and cyclophosphamide. Cyclophosphamide was also used to treat AMR after liver23 and heart transplantation.24
Here, we report on the efficacy and safety of i.v. cyclophosphamide pulse treatment in combination with PPH and high-dose IVIG in 13 consecutive patients with acute AMR after renal transplantation. To our knowledge, this is the first systematic report on the efficacy of i.v. cyclophosphamide treatment of acute AMR in kidney transplant patients since the implementation of a pathology-based definition for AMR and the introduction of solid-phase immunoassays to detect circulating donor-specific HLA antibodies (DSA).
MATERIALS AND METHODS
Between March 2013 and November 2015, we initiated treatment of 13 consecutive patients with biopsy-proven acute AMR with 6 pulses of i.v. cyclophosphamide (15 mg/kg, adjusted for age and renal function) at 3-week intervals, 6 sessions of PPH (2.5 L/session, 4% albumin), high-dose (1.5 g/kg) polyvalent human IVIG (KIOVIG), and a 3-day pulse of methylprednisolone (500 mg/d i.v.). Starting at the day after the last PPH each patient received a total of 1.5 g/kg polyvalent human IVIG on 2 to 3 consecutive days with a maximum daily dose of 60 g. Cyclophosphamide was reduced by 2.5 mg/kg per pulse for patients with a serum creatinine greater than 300 μmol/L, by 2.5 mg/kg for patients older than 60 years, and by 5 mg/kg for patients with a serum creatinine greater than 300 μmol/L and older than 60 years. Cyclophosphamide pulses were accompanied by prophylaxis with mesna (400 mg i.v. at 0, 4, and 8 hours), ondansetron (8 mg i.v. at 0 hour) as well as NaCl (0.9% i.v., 500 mL before infusion, 1000 mL during infusion, and 500 mL after infusion). Treatment was halted before completion of 6 cyclophosphamide pulses when serum creatinine and proteinuria returned to baseline, and DSA decreased to below 500 mean fluorescence intensity (MFI). Blood counts were checked at 7 to 14 days after each pulse and immediately before the next pulse. The dose of the subsequent pulse was reduced by 20% for patients with a leukocyte nadir less than 3000/mm3. After diagnosis, patients received triple maintenance immunosuppression including steroids, tacrolimus, and mycophenolate. One patient was converted from tacrolimus to cyclosporine A in an attempt to reduce proteinuria. During the treatment period, we applied 1000 mg/d of mycophenolate mofetil or 720 mg/d of enteric-coated mycophenolate sodium, respectively. The tacrolimus dose was adjusted to target levels of 5 to 8 ng/mL. All patients received prophylaxis with trimethoprim-sulfamethoxazole (80/400 mg daily), valganciclovir (adapted to estimated glomerular filtration rate [eGFR]), and oral amphotericin B suspension (1 mL, 4× daily) until the end of the treatment period, that is, 4 weeks after the last cyclophosphamide pulse. All patients were regularly monitored in our outpatient clinic.
Renal transplantation was performed at the Charité hospital based on a negative complement-dependent cytotoxicity crossmatch (CDC-XM) with and without dithiothreitol using T and B lymphocytes with current and historical serum. In addition, graft allocation was based on a negative virtual crossmatch by considering current and historical unacceptable antigens as defined by Luminex-based single antigen bead assays. Consequently, only patients with de novo DSA were included.
Renal biopsies were taken on indication only. All patients presented with clinically relevant allograft dysfunction posttransplant manifesting as an otherwise unexplained increase of serum creatinine (≥0.3 mg/dL), proteinuria (≥1 g/d), or primary nonfunction in the early phase after transplantation. Renal allograft pathology was carried out by 2 experienced nephropathologists (B.R., K.W.). The diagnosis of AMR was based on the presence of circulating DSA and significant allograft pathology according to the definitions of the Banff classification.25 C4d staining was done by indirect immunofluorescence on paraffin sections using a polyclonal rabbit anti-human C4d IgG antibody (Biomedica, Vienna, Austria). Only patients who gave their written informed consent were considered eligible for enrollment.
Serum samples before and after treatment were screened for HLA antibodies (HLAab) by the Luminex bead-based assay LABScreen Mixed (One Lambda, Canoga Park, CA, USA). In addition, HLAab specificities were determined by LABScreen Single Antigen beads assay (One Lambda). As an indicator for the antibody level, the normalized MFI was used. HLAab were considered positive when exceeding an MFI value of 500. The DSA showing the highest MFI at the time of AMR diagnosis (DSAmax) and the MFI sum of all DSA (DSAsum) were tracked to indicate the effectiveness of treatment.
End of follow-up was November 10, 2016. Data were assessed based on intention-to-treat analysis. Renal allograft survival was defined as the interval between diagnosis of AMR and return to maintenance dialysis treatment or end of follow-up. The eGFR was calculated according to the chronic kidney disease epidemiology collaboration formula.26 All adverse events, abnormal laboratory values and hospitalizations were tracked from our web-based electronic patient record system ‘TBase’,27 and graded according to the Common Terminology Criteria for Adverse Events (CTCAE) version 4.0.28 Comparison between groups was carried out using Fisher exact test for categorical variables and Mann-Whitney U test for continuous variables. Wilcoxon signed-rank test was used for pairwise comparison between different time points. A probability of less than 0.05 was considered as statistically significant. Statistical analysis was carried out using IBM SPSS Statistics for Windows, Version 22.0 (IBM Corp., Armonk, NY) and STATA 11 IC software (StataCorp., College Station, TX).
The relevant patient characteristics are shown in Table 1. At time of transplantation, none of the patients had DSA. All 13 patients had received induction therapy with basiliximab together with maintenance immunosuppression consisting of steroids, calcineurin inhibitors, and mycophenolate. The interval between transplantation and diagnosis was 41 (0-124) months. Early AMR occurring less than 1 year after transplantation was observed in 4 of 13 patients. These 4 patients had received triple immunosuppression including steroids before diagnosis. In 9 of 13 patients AMR occurred at more than 1 year after transplantation. All of these patients were on double maintenance immunosuppression without steroids before diagnosis. All patients had a diagnosis of acute AMR (Table 2). The minimum follow-up period was 12 months after diagnosis. Median observation time after diagnosis was 18 (12-44) months (Table 3). Four patients received less than 6 cyclophosphamide pulses. In 2 of these patients (patients 1 and 11), cyclophosphamide treatment was regularly halted after 1 and 3 pulses of cyclophosphamide, respectively, because of complete recovery of renal function and decrease of DSAmax to below 500 MFI. In the remaining 2 patients (patients 7 and 12) cyclophosphamide treatment was prematurely stopped. In 1 of these patients (patient 7) treatment was stopped after 4 pulses of cyclophosphamide because of a newly established diagnosis of parapsoriasis en petits plaques. The other patient (patient 12) explicitly refused any further immunosuppressive pulse treatment after 2 pulses of cyclophosphamide for personal reasons. In this patient, AMR was caused by a period of several weeks of `drug holiday´ before diagnosis, which the patient frankly conceded. Over a 3-month period, serum creatinine had risen from 1.0 to 6.9 mg/dL. Due to oliguria and volume overload the patient had to be placed on hemodialysis treatment at the time of diagnosis. Despite partial recovery and temporal freedom from dialysis, the patient refused further treatment after 2 cyclophosphamide pulses, and returned to maintenance hemodialysis at 1 month after diagnosis. One patient (patient 3) received an additional methylprednisolone pulse (500 mg, 3×) together with PPH (18×) because of biopsy-proven evidence for acute AMR at 8 months after the initial diagnosis and 4 months after completion of cyclophosphamide treatment.
Graft survival at the end of follow-up was 77% (10/13) in all patients (Figure 1), and 91% (10/11) in all patients with complete treatment. Three patients resumed maintenance dialysis treatment during the observation period. Besides patient 12, patients 2 and 7 returned to maintenance dialysis at 34 and 12 months after diagnosis, respectively. Patient 2 had completed all 6 pulses of cyclophosphamide treatment. However, he had an enduring history of insufficient medication adherence, which did not substantially improve after diagnosis despite intense and repeated education on regular medication intake. After diagnosis, his tacrolimus levels remained below our target range of 5 to 8 ng/mL in one third of measurements. Notably, 2 times the tacrolimus level was even below the lower limit of detection (<2.0 ng/mL). In patient 7, treatment had been prematurely stopped after 4 doses. He had to undergo aortic and mitral valve replacement at 12 months after diagnosis because of severe aortic and mitral valve stenosis. After the operation, he developed acute renal failure from which renal function did not recover.
Mean serum creatinine increased from 1.6±0.5 mg/dL at 6 months before diagnosis to 3.7±2.4 mg/dL at diagnosis (P = 0.01) (Figure 2A). Correspondingly, eGFR decreased from 54.2±19.9 mL/min per 1.73 m2 to 25.8±16.4 mL/min per 1.73 m2 (P = 0.01) (Figure 2B). During the same period, urinary protein to creatinine ratio increased from 318±279 mg/g to 1034±916 mg/g (P = 0.02) (Figure 2C). At 3 months after diagnosis serum creatinine decreased to 2.1±0.7 mg/dL (P = 0.01), and eGFR increased to 37.3±18.7 mL/min per 1.73 m2 (P = 0.01). Urinary protein to creatinine ratio remained relatively stable at 968±1175 mg/g (P = 0.92).
HLA mismatches and HLAab are summarized in Table 2. The number of HLA mismatches for class I and II was 3.1±1.2 and 3.0±0.8 (P = 0.4), respectively. However, HLA class II antibodies were more frequently detected than class I antibodies as indicated by the percentage of HLA antibody panel reactivity (%PRA) (59% vs. 19%, P = 0.001). Similarly, DSA were mainly detected against HLA class II antigens (85% vs. 31%, P = 0.008), preferably HLA-DQ in 11 (85%) patients. Importantly, all DSA were de novo DSA. Concerning the response to treatment HLAab responders were defined by a minimum decrease of DSAmax and/or DSAsum of 5% or greater after treatment together with a minimum MFI level of 500 MFI or greater at diagnosis. Because DSA levels at diagnosis were less than 500 MFI, patient 1 was excluded from the analyses on HLAab responders. After treatment, DSAsum and DSAmax MFI decreased in 7 (70%) of 10 and 6 (60%) of 10 patients, who completed treatment, respectively. The MFI levels of DSAmax at diagnosis stratified between HLAab responders (n=7) and nonresponders (n=5) were different with 3879 MFI (interquartile range [IQR], 2187-4749) versus 13 131 MFI (IQR, 9434-15 433) (P = 0.04). In a univariate logistic regression analysis, a DSAmax level of less than 8000 MFI (n=7) was predictive for response (6/7, 86%) to cyclophosphamide treatment (odds ratio, 0.04; 95%confidence interval, 0.002-0.88; P = 0.04). DSAsum at diagnosis was 4749 MFI (IQR, 2187-7754) in HLAab responders as compared to 14 213 MFI (IQR, 13 131-16 933) in nonresponders (P = 0.04). A DSAsum of less than 10 000 MFI at the time of diagnosis perfectly predicted the response to treatment (logistic regression analysis omitted). Altogether, 100% (6/6) patients with a DSAsum of less than 10 000 MFI but only 17% (1/6) with a DSAsum of more than 10 000 MFI responded to treatment (P = 0.015). Notably, graft survival among these 7 HLAab responders was 100%. In 3 (30%) of 10 patients DSA levels even decreased to below the cutoff of 500 MFI after treatment. The median level of DSAsum and DSAmax MFI for all patients (n=13) did not decrease (9412 vs 11 270 and 4809 vs 5960). In 5 (38%) of 13 patients neither DSAmax nor DSAsum decreased. Two of these patients (patients 7 and 12 described above) did not complete treatment according to our projected protocol. One of these patients (patient 2 described above) had an enduring history of nonadherence. The reason for the nonresponse of the remaining 2 patients is unknown.
Graft survival was 100% (4/4) in patients with early AMR as compared with 67% (6/9) in patients with late AMR (P = 0.17). No significant differences between patients with early and late AMR were found concerning serum creatinine, eGFR and the observed adverse events (data not shown). Proteinuria at 3 months after diagnosis was significantly lower in patients with early AMR as compared to patients with late AMR (148±52 mg/g vs 1378±1261 mg/g; P = 0.004). Concerning the HLAab response, cyclophosphamide treatment was most effective among patients with early AMR, as 3 (100%) of 3 patients experienced a decrease in DSAmax and/or DSAsum. In comparison, DSAmax and/or DSAsum decreased in 4 (44%) of 9 patients with late AMR (P = 0.2).
The MFI reduction among class I (n=3) versus class II (n=20) DSA was not statistically different with −1083 MFI (IQR, −3234 to −456) and −254 MFI (IQR, −1094 to −2541) (P = 0.17), respectively. In contrast, the reduction in the breadth of total HLAab indicated by %PRA was significantly different between class I and class II with −4.9±18.8 versus −21.5±28.7 percent points (P = 0.045), respectively.
The observed adverse events are shown in Table 4. During and after treatment all patients received close-meshed follow-up in our outpatient clinic including complete blood count monitoring. In 1 patient (patient 7), treatment was stopped after 4 pulses of cyclophosphamide because of a diagnosis of parapsoriasis en petits plaques. Four months later, all skin lesions disappeared. In 3 (23%) of 13 patients, the cyclophosphamide dose was reduced according to our protocol because of a leukopenia less than 3000/mm3 at nadir. Anemia was treated by adaption of erythropoietin doses in 12 (92%) of 13 patients. In 3 (23%) of 13 patients blood transfusions were administered. In 1 of these patients anemia was caused by gastric ulcer bleeding. Allergic reactions during IVIG administration were observed in 5 (38%) of 13 patients and treated with antihistamines and prednisolone. We noticed 14 hospitalizations for 9 of 13 patients. The leading cause for hospitalization was infection (n=8). Six patients experienced a single hospitalization because of an infectious event. The same patient (patient 3), who received a second treatment course due to ongoing acute AMR was hospitalized 6 times, 5 of these hospitalizations occurred after the second treatment course. Reasons for hospitalization of this patient were: (i) pain in the left lower abdomen of unknown origin with spontaneous resolution, (ii) herpes zoster treated with i.v. aciclovir, (iii) symptomatic anemia with need for blood transfusions, (iv) gastric ulcer diagnosed by gastroscopy, (v) prerenal acute renal failure with need for i.v. fluids, and (vi) pneumonia with need for i.v. antibiotic treatment. Currently, the patient is in good health with a serum creatinine of 1.8 mg/dL (eGFR 29 mL/min per 1.73 m2) at 3 months after the last hospitalization. Importantly, none of our patients died and none of our patients developed malignancy during follow-up.
To date, the available treatment options for AMR after renal transplantation have not been proven to be sufficiently effective. Some reports indicate that treatment with rituximab may be effective.6-8 However, our own results12,13 as well as the RITUX ERAH study,29 a recent randomized controlled study, could not confirm sufficient efficacy. On the other hand, the existing evidence on bortezomib is still preliminary. Although bortezomib treatment seems to be helpful in certain cases of AMR, it has meanwhile become clear that bortezomib-based treatment is not a long-lasting cure for all patients.9 The BORTEJECT study, an ongoing randomized controlled trial investigating the effect of 2 cycles of bortezomib on late AMR will hopefully expand our knowledge on the efficacy of bortezomib treatment.30,31
Starting in 2005 we have treated all of our patients with biopsy-proven AMR with standardized protocols. Because AMR as well as ANCA-associated vasculitis are both mediated via pathogenic antibodies, and because cyclophosphamide has been proven to be effective in the treatment of ANCA-associated vasculitis for decades, we decided to use cyclophosphamide treatment in patients with biopsy-proven acute AMR. We chose to apply i.v. pulse treatment according to the recommended dose for patients with ANCA-associated vasculitis, because it was shown to be equally effective compared to daily oral treatment at a reduced cumulative dose and fewer side effects.18,19 Since March 2013, we applied this regimen to all patients in our institution with acute AMR and aimed to prospectively assess efficacy and safety.
Our results indicate that cyclophosphamide-based treatment might become a valuable alternative for the treatment of AMR in the future. Graft survival was not different (P = 0.67) as compared with our previous group (Figure 1) treated with a combination of bortezomib and rituximab in addition to the same adjunct therapy consisting of high-dose IVIG and PPH.13 During follow-up, we observed 3 graft losses. One of these patients (patient 12) refused further treatment so that only 2 of 6 cyclophosphamide pulses were administered. Although histology at diagnosis seemed to be promising inasmuch as mainly acute lesions were evident, the patient refused further treatment for personal reasons. Treatment was also prematurely stopped in patient 7, because of an unclear dermatological disease. He finally resumed maintenance hemodialysis treatment after double heart valve replacement. In the third patient (patient 2), graft loss was obviously caused by continued nonadherence before and after diagnosis despite repeated and intense education on the importance of regular medication intake. Therefore, the 3 graft losses can be attributed at least in part to premature termination of treatment and nonadherence. The fact that DSA levels could not be decreased in these three patients reflects the clinical course.
Importantly, the decline of renal function assessed by serum creatinine and eGFR could be reversed after treatment. The fact that renal function did not recover completely suggests that a certain degree of irreversible chronic organ damage, as indicated by the transplant glomerulopathy (cg) score, had already occurred, and could not be reversed.
Interestingly, we observed more HLA class II antibodies than class I antibodies, although the number of HLA mismatches for class I and II was not different. We assume that this phenomenon may at least in part be due to the fact that class I antibodies are preferentially adsorbed by constitutively expressed HLA class I molecules on the allograft.32 Notably, DSAmax MFI levels could be reduced to less than 500 MFI for 3 patients, and DSAsum as well as DSAmax decreased by 43% and 51% in the majority of patients, respectively. On the other hand, DSA could not be reduced in 5 patients. Premature termination of treatment (patients 7 and 12) and nonadherence (patient 2) explain the failure to respond of 3 of 5 patients. Concerning the remaining 2 patients, it is unclear, why their DSA levels did not decrease during follow-up. Interestingly, our data indicate that cyclophosphamide treatment was most effective among patients with an early AMR, relatively low DSAmax of less than 8,000 MFI and a DSAsum of less than 10 000 MFI.
The side effects of cyclophosphamide treatment are known to be dose dependent. According to the British Society for Rheumatology guideline for the management of ANCA-associated vasculitis lifetime exposure to cyclophosphamide should be 25 g or less.14 In our present study, the applied dosages were clearly below this threshold. To date, all patients are in good health, and none of the patients developed any kind of malignancy. In 1 patient (patient 7), treatment was halted after 4 cyclophosphamide pulses because of a diagnosis of parapsoriasis en petits plaques. Whether this diagnosis was caused by cyclophosphamide treatment cannot be excluded. In addition, it is currently not clear, whether parapsoriasis en petits plaques does have the potential to transform into cutaneous T cell lymphoma.33 Generally, cyclophosphamide treatment is not known to provoke cutaneous T cell lymphoma. Nevertheless, to minimize the risk for this patient, we halted cyclophosphamide treatment at this stage. Fortunately, all skin lesions disappeared 4 months later. In 3 patients, the cyclophosphamide dose was reduced according to our protocol because of leukopenia less than 3000/mm3 at nadir. After dose reduction, leukocyte counts recovered in these patients. In our opinion, the overall frequency and severity of the observed adverse events was acceptable.
The main limitation of our study is the fact that it is a retrospective study with a limited number of patients and a limited observation period. To clearly discriminate the effect attributable to cyclophosphamide alone, it would have been necessary either to treat a group of patients exclusively with cyclophosphamide, or to treat a group of patients exclusively with PPH and IVIG. In our view, both approaches would end up in a dilemma as treatment of AMR with cyclophosphamide alone would mean to exclude patients from the established treatment, that is, PPH and IVIG, and to completely rely on the efficacy of a substance, which has not yet been sufficiently investigated. On the other hand, our own experience in this field12,13 makes clear that treatment with IVIG and PPH alone would not be sufficient. Meanwhile, we have investigated several combinations of PPH and IVIG together with rituximab or bortezomib or both. None of these combinations was completely satisfactory concerning graft outcome. Therefore, treatment of acute AMR with PPH and IVIG alone would mean less immunosuppression and consequently a higher risk for graft failure. For comparison, we included the results of our latest group of patients in Figure 1, which has been described in detail recently.13 This group had been treated with rituximab and bortezomib in combination with the same protocol of PPH and IVIG as the group presented here. Thus, the difference between both groups is that rituximab and bortezomib were substituted by cyclophosphamide. We are also well aware of the fact that the follow-up period is limited. However, as we observed a considerable amount of graft losses during the first year after diagnosis in our previous groups,12,13 and as the adverse events attributable to treatment mainly occur during the first year after treatment, we think that these data are important to provide a valuable information on the efficacy and safety of the presented protocol during the early period after treatment. Because protocol biopsies are not performed at our center, follow-up histology surveillance was not available, and we are not able to comment on potential subclinical rejections and the histological response to treatment.
Taken together, the obtained results are preliminary and must be regarded with caution. Nevertheless, it is the first study investigating the efficacy and safety of cyclophosphamide treatment in renal allograft recipients with acute AMR based on modern diagnostics. The observed graft survival and the fact that the decline of renal function could be reversed indicate that cyclophosphamide-based treatment may be an additional therapeutic option. On the other hand, the fact that DSA levels could not be decreased in all patients leaves open questions. In the future, randomized controlled studies including long-term follow-up are necessary to further evaluate the efficacy and safety of cyclophosphamide treatment in patients with AMR.
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