Successful kidney transplantation in highly sensitized patients

Abstract

Highly sensitized patients experience an increased number of rejection episodes and have poorer graft survival rates; hence, sensitization is a significant barrier to both access to and the success of organ transplantation. This study reports our experience in kidney transplantation in highly sensitized patients. Fourteen patients with sensitization or high levels of panel-reactive antibodies (PRA) were studied. All patients were desensitized with pre-transplant intravenous immunoglobulin (IVIG)/plasmapheresis (PP) with or without rituximab and thymoglobulin induction therapy, combined with a Prograf/MMF/Pred immunosuppressive regimen. Of 14 patients, 10 showed good graft functions without acute rejection (AR) episodes. Acute cellular rejection in two patients was reversed by methylprednisolone. Two patients underwent antibody-mediated rejection; one was treated with PP/IVIG successfully, whereas the other lost graft functions due to the de novo production of donor-specific antibodies (DSA). Graft functions were stable, and there were no AR episodes in other patients. Conclusively, desensitization using PP/IVIG with or without rituximab increases the likelihood of successful live-donor kidney transplantation in sensitized recipients.

Keywords

Kidney transplantation / desensitization

Introduction

The presence of antibodies against both class I and II human leukocyte antigens (HLAs) that cause hyperacute rejection and graft loss was recognized in the early years of clinical transplantation. Graft survival rates in high-risk sensitized patients with HLA antibodies from previous transplantation, transfusion, or pregnancy are inferior to that in unsensitized recipients partly because of antibody-mediated rejection (AMR) resulting from undetected or de novo donor-specific HLA antibodies [1,2]. Currently, 34.4% of patients in the waiting list in the United States are sensitized to HLAs, and 14.4% have a peak percentage of panel-reactive antibody (PRA) greater than 80%. These patients are less likely to find compatible deceased donors and have a significantly longer waiting time. Meanwhile, different centers have published research results on various desensitization protocols. Several desensitization protocols have been developed to prevent the AMR of kidney allografts, and they have increased the success rate of transplantation in sensitized recipients [3–5]. However, protocols differ across centers and have different clinical outcomes. This study reports our experience in live-donor renal transplantation in highly sensitized patients. The basic desensitization protocol included pretransplant plasmapheresis (PP), anti-CD20 antibody, induction therapy with low dose intravenous immunoglobulin (IVIG) and rabbit antithymocyte globulin (RATG), immunosuppression with tacrolimus, mycophenolate mofetil, and corticosteroids. We evaluated the graft outcomes of kidney transplantation from living donors after desensitization in highly sensitized patients.

Materials and methods

Patients

A retrospective review was performed of 14 highly sensitized patients treated with PP/IVIG or PP/IVIG/rituximab prior to live-donor renal transplantation between January 2008 and September 2010. Highly sensitized patients are those who had previous or current positive cross-match tests either by antihuman globulin-enhanced, complement-dependent cytotoxicity (AHG-CDC) or by flow cytometry (FCM), or patients who underwent re-transplants and had PRA levels≥50%.

Cross-matching

Histocompatibility techniques were used for HLA typing. Cross-match tests were performed using the AHG-CDC assay and FCM. A positive cross-match by FCM was defined as a displacement of the mean channel fluorescence (MCF) by more than 10 channels relative to a negative control and a donor autologous control. PRA levels were determined by enzyme-linked immunosorbent assays (ELISA) (LAT, One Lambda Inc., CA, USA). Antibody screening was performed for all recipients on pre-transplant serum using ELISA IgG class I (QUIKSCREEN I GTI, Waukesha, WI), Flow-PRA single-antigen HLA class I and II antigen antibody detection assay (One Lamda Inc., Canoga Park, CA), and Flow-PRA class I and II screening assay (One Lamda Inc.). The identification of HLA antibodies was performed using single antigen beads testing. The determination of a positive result was based on a significant shift in bead population to the right of the negative control bead. Donor-specific antibodies were detected by Luminex single antigen beads, We currently use mean fluorescence intensity (MFI)≥1000 as a positive cutoff.

Desensitization and immunosuppressive regimen

Transplant candidates with positive cross-match results underwent a desensitization protocol consisting of a course of alternate-day total PP followed by IVIG infusion (100 mg/kg). Plasma volume was replaced with 5% albumin solution, and fresh frozen plasma was administered on the preoperative day. Two recipients received an additional intravenous rituximab (Genentech, San Francisco, CA) and an anti-CD20 antibody at 375 mg/m² body surface area on transplant at 7 days and 1 day. After each treatment, cross-matching was repeated. The goal of the desensitization protocol was to achieve a negative cross-match test or a PRA level of<20% and no DSA, transplant surgery was scheduled for the following day. This protocol produces a rapid reduction in anti-HLA titers that allows for transplantation after 4-5 plasmapheresis treatments. Patients did not have additional PP/IVIG after surgery and did not receive rituximab, except when they developed biopsy-proven acute AMR. All patients received antibody induction therapy and a seven-day course of thymoglobulin (Genzyme, Cambridge, MA) (rATG) at 25-50 mg/day, initiated intraoperatively. Dosing was adjusted for leukopenia or thrombocytopenia with target CD₃ count<50 cells/mm³. A follow-up PRA test was performed three days after transplantation. Additional tests were performed when clinically indicated.

Seven days prior to transplantation, patients were initiated on tacrolimus (Prograf, Astellas, Deerfield, IL) (TAC) at 0.1 mg/kg/day with target levels 9-12 ng/mL (by immunoassay) in the first three months and 7-10 ng/mL thereafter. Mycophenolate mofetil (CellCept, Roche, Nutley, NJ) (MMF) was given at 1.5 g/day, even after transplantation. Dosing was adjusted for side effects, such as diarrhea and leucopenia. Following an intraoperative dose of 500 mg intravenous methylprednisolone, additional daily doses of 500 mg were administered, and a tapering dose of oral prednisone was initiated. The daily dose was tapered to 10 mg in three months and 5 mg in one year.

Clinical follow-up

Patients with delayed graft function (DGF), defined as the need for dialysis during the first week after surgery, and slow graft function (SGF), defined as serum creatinine (SCr)>3.0 mg/dL (265 μmol/L) on day 5 post-transplant, underwent allograft biopsies every 7-10 days until SCr was stabilized. Protocol biopsies on predefined time points were not performed. After initial hospital confinement, patients were followed-up in the outpatient transplant clinic at regular intervals. Glomerular filtration rate (GFR) was measured and a 24-h creatinine clearance was obtained from all patients. An unexplained increase in SCr was followed by transplant ultrasound and biopsy. Biopsies were evaluated using Banff 97 criteria for evidence of acute cellular rejection (ACR), AMR, transplant glomerulopathy (TG), thrombotic microangiopathy (TMA), and BK nephropathy. Patients with AMR were treated with intravenous steroids, a thrice-weekly course of PP/IVIG, and (in two cases) rituximab in addition to optimizing the maintenance regimen. Follow-up biopsies were performed as needed to evaluate response to therapy.

Clinical data and statistics

The following data were analyzed: (1) demographic and immunologic characteristics of patients, (2) effect of desensitization, (3) post-transplant allograft function and DSA, (4) histological findings on allograft biopsies, and (5) rate of AR and allograft loss. Descriptive values were presented as mean±SD or median and range.

Results

Patient demographics and immunologic status

All 14 highly sensitized patients who underwent desensitization during the study period were ABO-compatible. Ten patients underwent re-transplants, nine had previous transfusions, and three had more than three HLA mismatches. Four female recipients had previous pregnancy history. Two patients were cross-match-positive, and others were cross-match-negative but had high PRA levels (≥50%). Of the cross-match-negative patients, five had previous or current DSA detected by PRA, and four underwent re-transplants (Table 1).

Effect of desensitization

All 14 patients completed the desensitization protocol and were able to undergo transplantation. Two patients who were cross-match-positive became cross-match-negative by both AHG-CDC and FCM after PP/IVIG treatments. Of the two patients who received rituximab, peripheral CD19⁺cells—which represent the pool of B cells—were selectively depleted after rituximab infusion. Meanwhile, CD4⁺ and CD8⁺ cells were not affected. In three patients with negative cross-match tests, previous or current DSA detected by PRA became undetectable after desensitization. All 14 patients who had pre-transplant PRA levels≥50% showed a decrease in PRA levels. After desensitization therapy and prior to operation, the mean decreases in PRA class I and class II levels were 43.6% and 38.5% respectively (Fig. 1).

Post-transplant allograft function

Ten patients showed immediate graft function recovery, demonstrating active diuresis and rapid falls in serum creatinine concentration. Four patients showed DGF, but two patients did not reach the normal serum creatinine concentration. Histological findings on allograft biopsies showed AMR in two patients, and subsequent PP/IVIG and rituximab therapies were needed. Post-transplant DSA monitoring revealed a reemergence of DSA in these two patients.

Kidney graft function was evaluated using SCr levels and GFR. Mean serum creatinine concentration and calculated GFR in patients after desensitization were significantly decreased during the first month of follow-up and progressively improved during the 12-month follow-up period. During the 12-month follow-up, mean SCr stabilized at 98.3±10.7 μmol/L and mean GFR was maintained at 43.5±6.6 mL/min. Serum creatinine and GFR at various time points are summarized in Fig. 2.

Acute rejection and graft survival

Four patients exhibited AR episodes in the early post-transplantation period. Two patients showed a slow decline in urine output and an increase in SCr levels with no circulating DSA, and were cross-match-negative either by AHG-CDC or FCM, and histological findings on allograft biopsies showed ACR. ACR was reversed by 500 mg of methylprednisolone for three consecutive days, and one patient demonstrated acute rejection on the seventh post-transplant day with circulating DSA. Graft function improved with subsequent PP/IVIG and rituximab therapy. Meanwhile, the other patient demonstrated AMR on the tenth post-transplant day with circulating DSA. AMR resulted in graft failure despite subsequent PP/IVIG and rituximab treatment. Graft nephrectomy was performed with signs of disseminated intravascular coagulation. Ten patients showed good graft function after a 12-month follow-up, and there had been no more AR episodes. The patient survival rate was 100%.

Discussion

The benefits of kidney transplantation are proven by prolonged survival and improved quality of life for both children and adults. Despite these well-documented benefits, transplant frequency remains low due to limited organ availability. In patients with high levels of pre-formed anti-HLA antibodies, transplant rates are extremely low because of the additional immunologic barrier that increases rejection risks [6,7]. Highly sensitized patients are destined to remain in the waiting list for extended periods of time for dialysis, which is an added risk factor for patient and graft survival [8]. Thus, sensitization is a significant barrier to both access to and the success of organ transplantation.

Currently, two primary protocols have emerged: plasmapheresis and high-dose (IVIG) protocol. Significant success has been realized with both protocols [9,10]. In addition, the use of other techniques, such as B cell elimination with rituximab, newer immunosuppressive agents, protein A absorption, plasma exchange with concomitant Prograf (Astellas Pharmaceuticals, Deerfield, IL), and Cellcept (Roche Inc., Nutley, NJ) therapy, are being investigated. Alternative approaches to desensitizing patients are also necessary.

Jordan et al. reported the successful use of high-dose IVIG for desensitization. The removal of donor-specific antibodies seems effective in allowing transplantation in highly sensitized patients and in treating AMR episodes [11–13]. Akalin et al. reported that IVIG, combined with plasmapheresis, is extremely effective in reversing AMR episodes. Although many variations in the theme of IVIG and plasmapheresis have been presented, both therapies can be effective in the treatment of highly sensitized patients [14,15]. Stegall et al. [15] reported that IVIG decreased DSA activity in all treated patients, yet only 38% (5/13) achieved a negative crossmatch. In contrast, a negative crossmatch was achieved in 84% in PP group and 88% in the PP/monitoring group. Even with a negative crossmatch, the rejection rates were 80% (IVIG), 37% (PP) and 29% (PP/monitoring), respectively. So they concluded that multiple PP treatments led to more reproducible desensitization and lower humoral rejection rates than a single high-dose of IVIG, but that no regimen was completely effective in preventing humoral rejection.

Rituximab, a chimeric anti-CD20 mAb, which is approved for the treatment of lymphoma, eliminates B cells efficiently. Rituximab eliminates B cells through three potential mechanisms: antibody dependent, cell-mediated cytotoxicity, complement-dependent cytotoxicity, and apoptosis [16,17]. Many possible therapeutic combinations might be considered but should be undertaken with great caution because the risks for this patient population are high, and the possibility of repeat transplantation is low.

There is no standardized protocol for desensitization strategies, but the basic principles include the elimination of pre-formed DSA, inhibition of the production of de novo DSA, and strong maintenance immunosuppression considering the interaction between T and B cells. In our study, patients progressed through various treatment options, including PP/IVIG, rituximab, thymoglobulin induction therapy, and combined Prograf/MMF/Pred immunosuppressive regimen, until successful transplantation was achieved. We believe that IVIG, combined with PP, has an important role in the treatment of highly sensitized patients awaiting transplantation. All functioning allografts have maintained stable graft function during follow-up. This finding demonstrates that the desensitization strategy is safe and effective in achieving successful transplantation in sensitized patients. We chose patients who underwent live-donor kidney transplantation and with previously positive cross-match tests or re-transplants (PRA levels≥50%). All patients had living donors, and 50% were haploidentical for HLA. Moreover, biopsies and regular PRA and DSA monitoring provided more precise information on the status of allografts. This explains excellent outcomes in our center.

Four patients had AR episodes in the post-transplantation follow-up period. Two patients exhibited ACR and such was reversed by methylprednisolone. Meanwhile, the other two exhibited AMR. One graft function that underwent AMR improved with subsequent PP/IVIG and rituximab therapies, whereas the other graft that underwent AMR failed to reverse. Graft nephrectomy was performed with the occurrence of disseminated intravascular coagulation. This finding suggests that the early post-transplant period is important in determining successful transplantation in sensitized patients.

Long-term graft and patient survival rates were excellent among our patients. Ten patients are currently dialysis free after a median follow-up of 12 months. Moreover, AR episodes are no longer observed, and the patient survival rate is 100%. This finding suggests that graft functions can be maintained for a long time if sensitization treatment is successful.

In our study, the use of surveillance biopsies allows a more precise assessment of allograft function and the early detection of histological changes indicating AMR. Buehrig et al. suggested that surveillance biopsy is an important adjunct for monitoring allograft functions in sensitized patients [18]. Results suggest that desensitization is successful in preventing and treating subclinical AMR in these patients through surveillance biopsy.

In summary, desensitization protocols should both eliminate PRA levels and prevent the de novo production of alloantibodies. The desensitization strategy using PP/IVIG with or without rituximab, plus thymoglobulin induction therapy and combined Prograf/MMF/Pred immunosuppressive regimen, increases the likelihood of successful living-donor kidney transplantation in highly sensitized recipients.

References

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