Renal transplantation in patients with end-stage kidney disease improves both duration and quality of life.1-3 Using advanced immunosuppressive therapeutics, the incidence of T cell–mediated rejection can be reduced, and graft survival rates increased.4 However, recipients with donor-specific antibodies (DSA) before renal transplantation (ie, sensitized renal transplantation) show higher rates of antibody-mediated rejection (AMR).5-7 AMR is difficult to prevent with conventional immunosuppressive drugs in DSA-positive patients and is a major cause of renal allograft loss.8 Recently, development in desensitization protocol gives DSA-positive patients more opportunities to receive successful renal transplantation.9,10
In general, 2 protocols are implemented for desensitization before renal transplantation: either high-dose IVIG (2 g/kg) alone or low-dose IVIG (100 mg/kg) in combination with plasma exchange (PE).11-14 Although it is unknown which of these 2 protocols is superior, IVIG plays a key role in desensitization protocols. Rituximab (anti-CD20 antibody) has also proved beneficial in such protocols.15,16 A 4-month regimen of IVIG (2 g/kg per month) is advocated by guidelines for clinical use of IVIG published by public agencies of countries.17-21 IVIG has similarly helped in resolving posttransplantation episodes of steroid-resistant rejection.10 Such rescue therapies generally involve high-dose IVIG (2 g/kg) given as a single dose22 or over the course of several days.23
At present, preferred protocols for preemptive desensitization appear to be transplant center-specific, with limited or no randomized prospective studies to compare efficacies.9 Moreover, there have been no definitive studies on dosages and timing of IVIG administration. In this study, we monitored changes in T cell flow cytometry crossmatch (FCXM) as part of a desensitization protocol in which recipients demonstrating positive T cell FCXM received IVIG (1 g/kg) for 4 days within a 1-week period (total of 4 g/kg) after double-filtration plasmapheresis (DFPP) in preparation for living-donor transplantation. T cell FCXM was measured after each IVIG dose and up to 4 weeks after initiating IVIG administration, allowing us to better establish the timing of conversion and the effectiveness of cumulative IVIG dosing. This super high-dose IVIG-based desensitization protocol may be an alternative to conventional protocols for recipients with DSA.
MATERIALS AND METHODS
A phase II/III, open-label, single-arm multicenter prospective trial was performed in Japan between November 2013 and September 2015. The study protocol was approved by the institutional review board of each participating institution and conducted in accordance with the Good Clinical Practice described in the Helsinki Declaration. Patients provided written informed consent before registering in this study. ClinicalTrials.gov Identifier: NCT02032095.
Patient Demographics and Characteristics
A total of 17 patients were enrolled for study in preparation for living-donor renal transplantation. Each participant displayed the following: (1) negative T cell complement-dependent cytotoxicity, (2) positive T cell FCXM, and (3) stage-5 chronic kidney disease. Relevant patient characteristics were collected for analysis.
Patients showing positive T cell FCXM after 2 rounds of DFPP were administered 1 g/kg per day IVIG (Venoglobulin IH 5%; Japan Blood Products Organization, Tokyo, Japan) for 4 days over a 1-week period. T cell and B cell FCXM were measured the day after each of the 4 IVIG doses. For patients showing positive T cell FCXM after the fourth administration, FCXM was measured weekly until negative conversion was confirmed, for up to 4 weeks after initiating IVIG. The primary study endpoint was percentage of patients converting to T cell FCXM-negative status after the fourth IVIG dosage. Patients who prematurely discontinued IVIG dosing were evaluated according to last observation carried forward. Secondary study endpoints were percentage of patients showing conversion to T cell and B cell FCXM-negative status (last observation carried forward) 4 weeks after initiating IVIG administration and percentage of patients converting to T cell and B cell FCXM-negative status after each dose of IVIG (1 g/kg). This study plan aimed to confirm the potential of IVIG as a desensitization therapy and did not require renal transplantation, but planned for transplantation to be performed in cases in which FCXM-T became negative.
Donor-specific Crossmatching Analysis
FCXM determinations were conducted at a single clinical laboratory (ReproCELL Inc., Yokohama, Japan) equipped with a cell analyzer (FACSCalibur; Becton, Dickinson and Co., Franklin Lakes, NJ). Class I- and II-positive control sera (One Lambda, Inc., Canoga Park, CA) were purchased, whereas prospective donor plasma served as a negative control. Median ratio was calculated by dividing median fluorescence intensity of patient specimens by median fluorescence intensity of the negative control. Median ratio less than 1.4 for T cells and less than 1.7 for B cells defined negative. These cutoff values of FCXM were determined by +2SD of the measurements of 34 cases which negative Flow panel-reactive antibody (PRA) screening outcomes.
Monitoring of Adverse Events
Adverse events occurring within 4 weeks after initiating the IVIG regimen were recorded and assessed for relevance to treatment, applying codes stipulated in the Medical Dictionary for Regulatory Activities (v18.1). Adverse events having a causal association with IVIG administration were considered side effects.
All computations relied on standard software (SAS v9.3; SAS Institute, Cary, NC). To assess IVIG efficacy in this protocol, a full-analysis set was applied. A 95% confidence interval (CI) is shown for percentage of patients showing conversion to T cell or B cell FCXM-negative status. In Kaplan-Meier estimates, the endpoint was projecting duration (days) required for conversion of 25%, 50%, and 75% of patients to FCXM-negative status and 95% CI. In addition, the cumulative conversion rates (with 95% CI) for conversion to FCXM-negative status 4 weeks after initiating IVIG were calculated.
Patient Demographics and Characteristics
Demographics and characteristics of the patients studied are summarized in Table 1. Four men and 13 women (mean age, 50.5 ± 8.6 years) were enrolled in this study. Pertinent historical exposures included the following: pregnancy (n = 12), blood transfusion (n = 6), and transplantation (n = 4). At baseline, 6 patients had never undergone dialysis, and 16 patients showed positive B cell FCXM. T cell FCXM median values before and after DFPP (before IVIG dosing) were 3.1 (range, 1.4-21.8) and 2.7 (range, 1.4-14.0), respectively. Median values of B cell FCXM were 4.3 (range, 1.6-26.0) and 3.6 (range, 1.6-41.1), respectively.
In Figures 1 and 2, T cell and B cell FCXM changes in each patient studied and in specific patient subsets (FCXM ≤6 or ≤10 before first DFPP) are plotted. Successive conversion rates for IVIG doses 1 to 4 were as follows: 29.4% (5/17) after the first dose; 35.3% (6/17) after the second dose; 56.3% (9/16) after the third dose; and 46.7% (7/15) after the fourth dose (Table 2).
As primary endpoint, 8 of 17 patients became T cell FCXM negative after the fourth dose of IVIG or at discontinuation, for a conversion rate of 47.1% (95% CI, 23.0-72.2%). Two of 8 patients who were T cell FCXM positive after the fourth dose of IVIG converted to negative T cell FCXM 1 week later. As a result, 10 of 17 patients became T cell FCXM negative within 4 weeks after initiating IVIG administration (58.8% conversion rate; 95% CI, 32.9-81.6%). On the other hand, 8 of 15 patients given 4 doses of IVIG did not become T cell FCXM-negative after the fourth dose; and of these patients, 4 had T cell FCXM median ratio of 1.4, falling short of the criterion set (<1.4). One week later, median ratios in 2 of these 4 patients eventually met the criterion for negative T cell FCXM. The mean values of the T cell FCXM median ratio decreased to 74.7%, 60.0%, 54.6%, and 50.5% after the first, second, third, and fourth administration of IVIG, respectively, compared with the values after DFPP (before IVIG administration) (Figure 3A).
In Kaplan-Meier estimates, the cumulative rate of conversion to T cell FCXM-negative status 4 weeks after initiating IVIG administration was 60.3% (95% CI, 38.2-83.0%), with 25% and 50% points reached on day 4 (95% CI, 1-6) and day 13 (95% CI inestimable due to so few patients), respectively. The 75% point could not be estimated (Figure 4A). Of 15 patients with T cell FCXM median ratio of 6.0 or less before desensitization, 10 became T cell FCXM-negative 4 weeks after initiating IVIG administration, for a conversion rate of 66.7%. In the other 5 patients, median ratios declined to 1.4 for 3 patients and to 1.7 and 1.9 for the 2 remaining. Two patients with particularly high median ratios (19.3 and 21.8) did not convert to negative T cell FCXM, but these values did fall substantially (to 2.8 and 4.7, respectively).
Nine of 16 patients became B cell FCXM-negative 4 weeks after initiating IVIG administration, for an overall B cell FCXM-negative conversion rate of 56.3% (95% CI, 29.9-80.2%). Also, negative B cell FCXM was observed in 6 of 10 patients who eventually converted to T cell FCXM-negative status. After IVIG administrations 1 to 4, B cell FCXM conversion rates were 25.0% (4/16 patients), 18.8% (3/16 patients), 60.0% (9/15 patients), and 60.0% (9/15 patients), respectively (Table 2). In Kaplan-Meier estimates, the cumulative rate of conversion to B cell FCXM-negative status 4 weeks after the first IVIG dose was 66.7% (95% CI, 37.4-92.4%). The 25% and 50% points were reached on day 5 (95% CI, 1-6) and day 17 (95% CI inestimable due to so few patients), respectively; and the 75% point could not be estimated (Figure 4B). Mean values of B cell FCXM median ratio declined to 72.8%, 59.3%, 49.6%, and 42.5% after first, second, third, and fourth administrations of IVIG, respectively, compared with values after DFPP (before IVIG dosing) (Figure 3B). Overall, the mean serum IgG levels after DFPP (before IVIG administration), after completing the IVIG regimen, and 4 weeks after initiating IVIG administration were 547.9 ± 205.0 mg/dL, 4923.3 ± 635.7 mg/dL, and 2,733.3 ± 455.7 mg/dL, respectively. Among those 10 patients who eventually converted to T cell FCXM-negative status after desensitization therapy, 4 received living-donor renal transplants within the time frame of this study, and none of them experienced AMR. In 2 of 6 remaining cases, living donor kidney transplant was done after the end of the time frame of this study, and there was no AMR observed. In the remaining 4 cases, kidney transplant was not done due to nonmedical reasons.
No deaths occurred during the study period. Adverse events, moderate or mild (total of 52), were observed in 13 patients (76.5%), and side effects (total of 38) were recorded in 11 patients (64.7%). Table 3 lists the spectrum of side effects. The chief side effects included headache (29.4%), hepatic dysfunction (17.6%), rash (17.6%), and nausea (11.8%).
Four patients (23.5%) suffered serious adverse events (total of 6), and serious side effects (total of 5) were recorded in 3 patients (17.6%). Serious side effects included leukopenia (5.9%), neutropenia (5.9%), thrombocytopenia (5.9%), headache (5.9%), and aggravation of renal function (5.9%), all of which were predictable. The protocol was discontinued in 2 patients after the third dose of IVIG. One became cytopenic (leukopenia, neutropenia, thrombocytopenia), without need of granulocyte colony-stimulating factor or platelet transfusions, and the other experienced headaches. In another patient, hemodialysis was initiated for the first time and continued until the point of transplantation to address deteriorating renal function after IVIG administration. There was no thromboembolism which is a concern due to the large dose of IVIG.
In renal transplantation recipients with DSA, AMR occurs at a high rate, and the posttransplantation prognosis is poor.8 Desensitization protocols developed over the past several years to overcome this immunologic barrier generally have entailed high-dose IVIG alone or low-dose IVIG combined with PE.11-14 Recently, regimens incorporating rituximab16 or bortezomib24 have been tested as well. Although IVIG plays a key role in desensitization protocols, it is unknown whether high-dose IVIG alone or low-dose IVIG combined with PE is superior. Furthermore, there is no protocol to date where the number of IVIG administrations and intervals have proven optimal. In this study, we measured changes in T cell and B cell FCXM after each dose of IVIG (1 g/kg × 4) administered to sensitized renal transplant recipients within 1 week before transplantation to determine the effects of such treatment.
Administration of IVIG as a monthly dose of 2 g/kg is commonly considered high-dose therapy. Glotz et al11 have reported that renal transplantation was possible in 13 of 15 recipients who were desensitized using 3 courses of IVIG (2 g/kg each) given at 4-week intervals. In 11 of these patients, all recipients of deceased-donor renal transplants, the PRA rate had declined from 64% (50-77%) to 14% (0-24%), representing a mean decrease of 80% through desensitization. The remaining 2 patients were crossmatch-negative and underwent living-donor renal transplantation. Jordan et al13 likewise reported a placebo-controlled, randomized, double-blind controlled trial in patients showing high sensitization (PRA of at least 50% for 3 months). After receiving of 2 g/kg IVIG per month for 4 months, the level of PRA was significantly lower in patients given IVIG (vs placebo), and the rate of transplantation was significantly higher (IVIG, 16/46, 35%; placebo, 8/46, 17%). In our study, the rate of conversion to T cell FCXM-negative status after cumulative administration of 1, 2, 3, and 4 g/kg IVIG generally increased in a dose-related manner, culminating in a 60.3% cumulative conversion rate 4 weeks after initiating IVIG. In contrast, the rate of conversion to T cell and B cell FCXM-negative states after standard IVIG dosing of 2 g/kg has not been high, and repeated IVIG administration seemed to be required at or after renal transplantation. Using our IVIG regimen, the FCXM-negative conversion rate increased by 20% or greater through cumulative IVIG administration of 3 g/kg or more (ie, cumulative dose >2 g/kg). Thus, effective, short-term desensitization is likely responsible for the extent of patient conversion achieved.
FCXM can be expressed as a median channel shift (MCS) derived through linear scale calculation or as a ratio calculated via log scale. According to the Laboratory Manual of the American Society for Histocompatibility and Immunogenetics,25 each institution is obliged to define a standard FCXM cutoff value. In the Laboratory Manual of the American Society for Histocompatibility and Immunogenetics, cutoff values in studies of T cells are at 10 to 15 for MCS 256 scale and ratio of 1.0. For B cells, cutoff values are at 25 to 30 for MCS 256 scale and ratio of 1.2. An abundance of previous research has focused on MCS values of FCXM (FXMCS), graft survival rates, and the incidence of AMR in anti-HLA antigen-positive renal transplantation. In these investigations, clinical cutoff values have varied, as have the mean fluorescence intensities of DSA, and no consensus has yet been reached in establishing associations with clinical events such as AMR.
However, Gloor et al26 have reported good results (37% incidence of AMR and ≥90% graft survival for ~5 years) by desensitizing patients with negative T cell antihuman globulin-enhanced complement-dependent cytotoxicity and FXMCS values ≥300, lowering FXMCS below 300 (cutoff value of FXMCS-T, 52; FXMCS-B, 104). Jordan et al27 have also claimed a lowering of AMR to less than 16% within 1 year after renal transplantation, using a DSA-relative intensity scale score of 17 or less (scored according to mean fluorescence intensity), and a FXMCS less than 225 (cutoff value of FXMCS-T: 130, FXMCS-B: 70) to gauge desensitization. Hence, renal transplantation may not be prohibitive, even if negative FCXM is not achieved. The cutoff values used herein less than 1.4 for T cells and less than 1.7 for B cells. When this value of 1.4 or less was defined as negative, the subsequent T cell FCXM conversion rate after cumulative administration of 4 g/kg of IVIG was 73.3%. Neither PRA nor Luminex assays were explored for this study, and we acknowledge that an additional study will be needed.
Rebound of DSA and accompanying AMR have proven to be problematic for desensitization protocols involving PE and IVIG administration, the rate of AMR being as high as 30% to 50%.9,28 Recent studies have shown better long-term graft survival rates in recipients desensitized through high-dose IVIG plus rituximab, compared with those subjected to high-dose IVIG alone.29 Transplantation was planned for patients of ours who were FCXM-negative after cumulative administration of 4 g/kg IVIG. Four of such patients received living-donor renal transplants after treatment with steroids, calcineurin inhibitors, mycophenolate mofetil, plasmapheresis, and rituximab. All of them did well posttransplantation, with no signs of AMR.
Planned desensitization and immunosuppressive therapy in the short term are important for living-donor renal transplantation in DSA-positive recipients. Evaluating a desensitization protocol based on FCXM and PRA determinants is therefore critical. In this study, we have shown that cumulative doses of IVIG ≥3 g/kg may boost the FCXM-negative conversion rate beyond that attained through conventional IVIG administration (2 g/kg). Although negative conversions of T cell FCXM peaked at IVIG doses of 3 g/kg, there are some cases in which it becomes negative after the fourth administration or 1 week after the fourth administration. Therefore, in this study, we conclude that IVIG ≥3 g/kg (3-time administration) is effective.
The primary goal of this study was to monitor changes in T cell and B cell FCXM during an IVIG-based desensitization protocol that differed from the high-dose IVIG alone or low-dose IVIG plus PE protocols typically used. Of the 17 patients studied, only 4 received living-donor renal transplants; and this patient sampling is smaller than that of other published studies.29 Nonetheless, the results of this limited trial were encouraging. A broader evaluation of our desensitization regimen in greater numbers of transplant recipients is needed to properly assess patient and graft survival at 1 year and longer. This short-term high-dose IVIG desensitization protocol may be an alternative to conventional protocols for recipients with DSA.
The authors thank all participating patients and their family members as well as the investigators and study coordinators at the 13 centers involved in the trial. They also thank Japan Blood Products Organization for study monitoring, data collection and management, and statistical analysis. Editorial support, in the form of medical writing, was provided by Cactus Communications, and funded by Japan Blood Products Organization, Tokyo, Japan.
GB-0998 Study Group principal investigators are as follows.
Sapporo City General Hospital, Sapporo: Hiroshi Harada; Akita University Hospital, Akita: Shigeru Sato; Japan Community Health Care Organization Sendai Hospital, Sendai: Noritoshi Amada; Jichi Medical University, Shimotsuke: Takashi Yagisawa; Chiba-East National Hospital, Chiba: Naotake Akutsu; Tokyo Women's Medical University, Tokyo: Kazunari Tanabe; Toho University Faculty of Medicine, Tokyo: Atsushi Aikawa; Nagoya Daini Red Cross Hospital, Nagoya: Yoshihiko Watarai; Japan Community Health Care Organization Chukyo Hospital, Nagoya: Tomonori Komatsu; Fujita Health University, School of Medicine, Toyoake: Takashi Kenmochi; Osaka University Graduate School of Medicine, Suita: Yoichi Kakuta; Institute of Biomedical and Health Sciences, Hiroshima University, Hiroshima: Hideki Ohdan; Graduate School of Medical Sciences, Kyusyu University, Fukuoka: Kei Kurihara
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