Twenty-five out of 26 patients achieved complete or partial response to a bortezomib regimen. The median time to response was 1.5 months. Eighteen patients had a complete response. An analysis of the degree and time of DSAmax response for the patients revealed a partial response was achieved by 1 month in 11 patients, between 1 and 2 months in 3 patients, and between 2 and 3 months in 10 patients. One additional patient achieved a partial response beyond 3 months. One patient remained refractory at last follow-up.
When comparing bortezomib-alone to bortezomib-based combination therapy, rates of partial and complete response were similar. Time to any response (partial or complete) was slightly faster in the bortezomib-based combination group, but this was not statistically different from the bortezomib-alone group (median 29 vs. 88 days, respectively). Bortezomib-alone-treated patients achieved a partial response by 1 month in 36% of patients, between 1 and 2 months in 9% of patients, and between 2 and 3 months in 45% of patients. Bortezomib-based combination patients achieved a partial response by 1 month in 46% of patients, between 1 and 2 months in 13% of patients, and between 2 and 3 months in 33% of patients. The one refractory patient was treated with bortezomib-based combination therapy.
Durability of DSA Remission Following Bortezomib
The goal of treatment in the 18 patients who achieved complete response was remission. At last follow-up, eight patients remain in remission. The median time of remission in these patients is 14 months. The remaining 10 patients (56%) who achieved a complete response relapsed. The median time to relapse was 3.8 months. The rate and median time to relapse did not differ based on the use of bortezomib-alone or bortezomib-based combination therapy (Fig. 2).
Achievement of DSA remission was correlated with better allograft function at last follow-up. For those patients in remission, the median time of DSA freedom (after achievement of complete remission) is 14.4 months (2.3–33.2 months). In those who relapsed, the median time of DSA freedom was 3.8 months (0–12.5 months; Fig. 3). The baseline mean serum creatinine in the remission, relapsed, and noncomplete response groups was 1.32±0.20, 1.20±0.22, and 1.01±0.13, respectively. At last follow-up, the mean serum creatinine was 1.42±0.24, 1.58±0.38, and 1.68±0.69, respectively. The median change in serum creatinine (from treatment to last follow-up) in the remission patients is +6.5% (range −14% to +20%), compared with +41% (−29% to +108%) in the relapsed patients (Fig. 3, Mann-Whitney P=0.023).
A complete response to bortezomib therapy was not influenced by age at transplant, degree of HLA mismatch, pretreatment immunosuppression, concomitant therapy, posttreatment immunosuppression, time from transplant to antibody appearance, time from antibody appearance to treatment, serum creatinine at time of treatment, initial DSAmax mean fluorescence intensity (MFI), peak DSAmax MFI, and DSA class. A rapid partial response (achieving 50% reduction within 1 month of treatment) was the one variable that was loosely associated with a complete response (odds ratio 8.75, 95% confidence interval 0.88–86.6). Conversely, a high starting DSAmax MFI (>5000 MFI) was found to loosely associate with a lack of complete response (odds ratio 0.3, 95% confidence interval 0.05–1.70).
As with the complete response analysis, age at transplant, degree of HLA mismatch, pretreatment immunosuppression, concomitant therapy, posttreatment immunosuppression, time from transplant to antibody appearance, time from antibody appearance to treatment, serum creatinine at time of treatment, initial DSAmax MFI, peak DSAmax MFI, DSA class, and partial response at 1 month were not found to predict remission. The only factor found to predict continued remission on univariate analysis was the addition of a calcineurin inhibitor (CNI) or mycophenolate mofetil (MMF) at the time of bortezomib therapy. The seven patients treated with a MMF or CNI addition benefited increasing their days of DSA-free survival by 91% (relapse hazard ratio 0.09, 95% confidence interval 0.01–0.76).
In this study, we evaluated the efficacy of bortezomib-based therapy in patients with DSA before evidence of allograft dysfunction. The overall response rate (partial plus complete response) was high (96%). Sixty-nine percent of patients achieved a complete response and this response lasted for an average of 9 months. At last follow-up, the ultimate goal of remission following complete response was achieved in only eight patients. Five have maintained a DSA-free period for over 2 years following complete response. Remission was most likely in patients who had addition of a CNI or MMF at the time of bortezomib therapy. Additionally, in those patients who achieved remission, the change in creatinine from treatment start to last follow-up was significantly better.
The data in this article demonstrate that bortezomib therapy does have a high rate of response. Achievement of a partial or complete response with therapies such as IVIg, rituximab, and plasmapheresis has been shown to occur at a rate of 33% to 60% in acute rejection (17–19) and complete response has been shown to be as high as 64% with IVIg+rituximab in preemptive treatment of DSA in lung transplant patients (10). In this study, bortezomib therapy resulted in partial or complete response in 96% of patients and complete response in 69% of patients. This shows that improved or at least equivalent efficacy can be achieved with bortezomib, although comparative studies are warranted to confirm this finding. This is an important preliminary finding given that bortezomib is lower in cost per treatment than IVIg therapy.
However, despite completely removing antibodies in approximately two thirds of patients, not all patients achieved complete response. This indicates that further studies are needed to find the optimal strategy for using bortezomib. One cycle as used in these patients may not be enough. Based on the result from a desensitization trial from the Mayo Clinic, as many as four cycles may be needed to decrease multiple antibodies (14). Also, high DSAmax MFI or class II antibodies may be more resistant to therapy (20).
Our finding that patients with relapse have a greater increase in serum creatinine than those in remission indicates that in addition to removal, sustaining remission may also be important to long-term posttransplant success. From this study, one factor that may improve remission was the use of increased immunosuppression (MMF or CNI) after bortezomib. Bortezomib leads to response in nearly all patients and a complete removal in many, yet its durability varies. In patients with no immunosuppression change after treatment, the period of remission was short. Only those who had a concomitant increase in baseline immunosuppression (MMF or CNI) had persistent remission beyond 1 year. The one exception was a patient with low MFI at baseline who never rebounded despite the lack of immunosuppression change. This durable removal and remission resulted in stability of allograft function. Conversely, in those patients who relapsed, allograft function worsened. This is the same result Hachem et al. found (10), which reinforces the fact that removal, not reduction, should be the goal in treating antibodies.
Several limitations of this trial should be noted. First, comparisons between bortezomib-alone versus bortezomib-combination therapy patients are not statistically powered to show a difference. A larger trial is warranted to prove or disprove these preliminary findings. Second, these patients are preemptively treated with bortezomib and therefore the results of this report may differ from that seen in the acute rejection setting.
In conclusion, the proteasome inhibitor bortezomib induces clinically significant responses, with minimal side effects (11, 12, 21–23). Several randomized trials to confirm these findings are ongoing. The results shown in this report along with the future clinical studies should provide guidance on how to manage patients with de novo DSA following transplant.
MATERIALS AND METHODS
Study patients were at least 18 years of age and were preemptively treated with bortezomib to treat DSA. Measurable (positive) DSA was defined as at least one DSA specificity above 1000 MFI. In all cases, allograft dysfunction (i.e., serum creatinine increase) was not present at time of HLA antibody detection and therefore biopsies were not conducted to detect subclinical rejection.
All patients were recipients of a living-donor kidney transplant between January 2008 and June 2009 at the Institute of Kidney Diseases and Research Centre-Institute of Transplantation Sciences (IKDRC-ITS), Ahmedabad, India. All patients underwent transplantation under clonal stimulation-deletion protocol described in a previous publication (22). Within 1 week of transplantation, patients were on prednisone alone, or off all immunosuppression. Prednisone dosing ranged from 0 to 20 mg per day. At the time of transplant, all patients were compliment-dependent cytotoxicity, crossmatch, and flow cytometry T- and B-cell crossmatch negative.
All patients were consented to the use of bortezomib. Institutional review board approval was obtained for use of their data.
Study Design and Treatment
All patients received bortezomib (1.3 mg/m2 of body surface area) as an intravenous bolus (taking 3–5 sec to administer) twice a week for 2 weeks, on days 1, 4, 8, and 11 of a 21-day cycle. All bortezomib doses were accompanied by a single dose of intravenous methylprednisone, 125 mg. In those patients who concomitantly received plasmapheresis (n=14), two to four sessions were conducted during the bortezomib cycle.
Anti-HLA-Specific IgG Antibody Testing
Anti-HLA antibodies were monitored weekly using single-antigen bead panels by Luminex assay (LABScreen, One Lambda, Canoga Park, CA). LABScreen assay was performed according to the manufacturer's protocol. Briefly, 20 μL of test serum was incubated with the beads for 30 min at room temperature in the dark. All samples were diluted 1:3 in 1× phosphate-buffered saline. Next, samples were washed and 100 μL of 1:100 anti-human-IgG-PE was added. After a second incubation step, samples were washed twice and the samples were read on the LABScan100 flow analyzer (One Lambda). Trimmed mean fluorescence values were obtained from the output file generated by the flow analyzer and normalized using the formula described in the LABScreen single antigen product insert: ([sample-specific florescent value for bead #N−sample-specific fluorescent value for negative control bead]/[background negative control serum fluorescent value for bead #N−background negative control serum fluorescent value for the negative control bead]).
DSAmax is defined as the antibody specificity that arises first posttransplantation. If multiple antibody specificities arise at the same sample point, the antibody with the highest MFI is considered the DSAmax. All other antibodies that arise later or at lower MFI values are considered secondary. DSA removal is defined as decreasing the DSAmax MFI to less than 1000. Complete response is defined as obtaining DSA removal. Partial response is defined as a 50% reduction in DSAmax but not DSA removal. DSA remission (DSA freedom) is defined as the time when all DSA (DSAmax and secondary DSA) are below 1000 MFI. The DSA remission time begins when a complete response is achieved and ends when any DSA (DSAmax, secondary) reappears or a new DSA specificity appears. This DSAmax/secondary DSA reappearance or new DSA appearance is termed DSA relapse. Refractory DSA is defined as a DSAmax that has no change or increase in MFI.
Assessment of Efficacy
The endpoints evaluated in this study were the DSAmax progression following the use of bortezomib-based therapy. Specifically, the study assessed the overall rate of partial and complete response following bortezomib, the duration of response (i.e., remission), and DSA relapse rate. Evaluation of the endpoints occurred at the end of the cycle and at 1, 3, 6, 9, 12, 18, and 24 months after treatment.
Time-to-event analysis was performed according to the Kaplan-Meier method. The time to any response (partial or complete) was defined as the time from the initial administration of bortezomib to the first time point that the DSAmax MFI dropped by 50% in intensity from its day 0 value. Duration of remission was defined as the time from the achievement of complete response to the time of DSA relapse.
All statistical analyses were performed using Stata/MP version 10.1 (College Station, TX). The P values presented are two-sided and a P value of less than 0.05 was considered statistically significant. Wilcoxon signed-rank test was used to compare median MFI values from baseline to year 1. The comparative analyses between bortezomib-alone versus bortezomib-based combination therapy were summarized descriptively. A univariate analysis to assess the factors associated with remission was performed using logistic regression. The small sample size prohibited use of multivariate logistic regression analysis. Cox proportional hazards modeling was used to evaluate variables as they related to time to relapse (from time of complete response).
1. Terasaki PI, Ozawa M. Predicting kidney graft failure by HLA antibodies: A prospective trial. Am J Transplant 2004; 4: 438.
2. Lee PC, Terasaki PI, Takemoto SK, et al.. All chronic rejection failures of kidney transplants were preceded by the development of HLA antibodies. Transplantation
2002; 74: 1192.
3. Mizutani K, Terasaki P, Bignon JD, et al.. Association of kidney transplant failure and antibodies against MICA. Hum Immunol 2006; 67: 683.
4. Ozawa M, Rebellato LM, Terasaki PI, et al.. Longitudinal testing of 266 renal allograft patients for HLA and MICA antibodies: Greenville experience. Clin Transpl 2006: 265.
5. van den Berg-Loonen EM, Terasaki P, Kohanof S, et al.. Longitudinal testing of seventy-six renal allograft patients for HLA antibodies: Maastricht experience. Clin Transpl 2006: 305.
6. Worthington JE, Martin S, Al-Husseini DM, et al.. Posttransplantation production of donor HLA-specific antibodies as a predictor of renal transplant outcome. Transplantation
2003; 75: 1034.
7. Terasaki PI. Humoral theory of transplantation
. Am J Transplant 2003; 3: 665.
8. Terasaki PI, Cai J. Human leukocyte antigen antibodies and chronic rejection: from association to causation. Transplantation
2008; 86: 377.
9. Stegall MD, Park WD, Dean PG, Cosio FG. Improving long-term renal allograft survival via a road less traveled by. Am J Transplant 2011; 11: 1382.
10. Hachem RR, Yusen RD, Meyers BF, et al.. Anti-human leukocyte antigen antibodies and preemptive antibody-directed therapy after lung transplantation
. J Heart Lung Transplant 2010; 29: 973.
11. Everly MJ, Everly JJ, Susskind B, et al.. Bortezomib
provides effective therapy for antibody- and cell-mediated acute rejection. Transplantation
2008; 86: 1754.
12. Perry DK, Burns JM, Pollinger HS, et al.. Proteasome
inhibition causes apoptosis of normal human plasma cells preventing alloantibody production. Am J Transplant 2009; 9: 201.
13. Diwan TS, Raghavaiah S, Burns JM, et al.. The impact of proteasome
inhibition on alloantibody-producing plasma cells in vivo. Transplantation
2011; 91: 536.
14. Raghavaiah SR, Daiwan TS, Burns JM, et al.. Depletion of alloantibody-secreting plasma cells by proteasome
inhibition improves desensitization. Am J Transplant 2010; 10(suppl 4): 44.
15. Everly MJ, Terasaki PI, Hopfield J, et al.. Protective immunity remains intact after antibody removal by means of proteasome
2010; 90: 1493.
16. Everly MJ. A summary of bortezomib
use in transplantation
across 29 centers. Clin Transpl 2009: 323.
17. Everly MJ, Everly JJ, Arend LJ, et al.. Reducing de novo donor-specific antibody levels during acute rejection diminishes renal allograft loss. Am J Transplant 2009; 9: 1063.
18. Everly MJ, Rebellato LM, Ozawa M, et al.. Beyond histology: Lowering human leukocyte antigen antibody to improve renal allograft survival in acute rejection. Transplantation
2010; 89: 962.
19. Lefaucheur C, Nochy D, Andrade J, et al.. Comparison of combination plasmapheresis/IVIg/anti-CD20 versus high-dose IVIg in the treatment of antibody-mediated rejection. Am J Transplant 2009; 9: 1099.
20. Walker J, Lubetzky M, Aull M, et al.. Bortezomib
therapy and reversal of antibody-mediated rejection: Differential sensitivity of HLA class I and class II antibodies. Am J Transplant 2011; 11(suppl 2): 160.
21. Everly JJ, Walsh RC, Alloway RR, et al.. Proteasome
inhibition for antibody-mediated rejection. Curr Opin Organ Transplant 2009; 14: 662.
22. Trivedi HL, Terasaki PI, Feroz A, et al.. Abrogation of anti-HLA antibodies via proteasome
2009; 87: 1555.
23. Walsh RC, Everly JJ, Brailey P, et al.. Proteasome
inhibitor-based primary therapy for antibody-mediated renal allograft rejection. Transplantation
2010; 89: 277.
Keywords:© 2012 Lippincott Williams & Wilkins, Inc.
Proteasome; Alloantibodies; Transplantation; Bortezomib