Objectives: Antibody-mediated rejection is a main cause of long-term kidney allograft loss. Nonad-herence and tacrolimus intrapatient variability have been identified as risk factors for developing de novo donor-specific antibodies. Tacrolimus, given once daily, can improve adherence and reduce variabilities among patients. The aim of this retrospective obser-vational study was to compare the incidences of donor-specific antibodies at 2 years posttransplant in de novo kidney transplant recipients given tacrolimus either once or twice daily.

Materials and Methods: Non-HLA sensitized de novo kidney-transplant recipients given tacrolimus either once daily (n = 82) or twice daily (n = 168), combined with mycophenolic acid with or without steroids, were included in the study. All patients were screened for anti-HLA antibodies before transplant, at 6, 12, and 24 months posttransplant, and each time the patient presented with impaired kidney function.

Results: The 2-year incidence of donor-specific antibodies was 2.8%. During the follow-up period, 6 patients (3.6%) receiving tacrolimus twice daily and one patient (1.2%) receiving tacrolimus once daily developed a donor-specific antibody (P = .43). The incidence of antibody-mediated rejection was 4.8% under tacrolimus once daily and 2.7% under tacro-limus twice daily (P = .5). Tacrolimus intrapatient variability was similar with both formulations and was not associated with development of donor-specific antibodies.

Conclusions: The use of tacrolimus-based immunosup-pression associated with mycophenolic acid was associated with a low risk of de novo donor-specific antibodies. After 2 years, the incidence of de novo donor-specific antibodies did not differ significantly between patients treated with tacrolimus once daily versus those treated with the twice-daily formulation.

Key words : Acute rejection, Formulation, Renal transplantation, Variability

Introduction

Recent studies have shown that antibody-mediated rejection is a main cause of long-term kidney allograft loss.^1,2 Apart from younger age, the number of human leukocyte-antigen (HLA) mismatches, calcineurin inhibitor (CNI) minimization protocols and CNI-free mammalian target of rapamycin inhibitor (mTORi)-based immunosuppression regi-mens, and nonadherence to immunosuppressive therapy have been identified as major risk factors for developing de novo donor-specific antibodies (DSAs) and subsequent graft loss.^3-9 In addition, the large intrapatient variability (IPV) regarding CNIs is associated with increased risk of acute and chronic rejection, increased risk of interstitial fibrosis and tubular atrophy, decreased kidney function, and increased risk of graft loss.^10-14

Tacrolimus is the cornerstone immunosup-pressant used after kidney transplant and is part of the standard-of-care immunosuppressive regimen approved by the US Food and Drug Administration. Historically, Prograf (Astellas Pharma, Deerfield, IL, USA), a tacrolimus formulation given twice daily, was and is still the most common tacrolimus formulation. However, Advagraf (Astellas Pharma), a tacrolimus formulation given once daily, has been developed and commercialized with the aim of improving adherence and reducing IPV. Several studies have shown that conversion from Prograf to Advagraf improves adherence and reduces IPV.^15-18 However, comparisons between these formulations regarding the development of DSAs have not been explored. Hence, in this study, our aim was to compare the development of DSAs and the incidence of acute rejection at 2 years posttransplant in non-HLA-sensitized de novo kidney transplant recipients given either Prograf or Advagraf, combined with mycophenolic acid with or without steroids.

Materials and Methods

Patients
Between January 2009 and December 2013, 794 kidney transplant procedures were performed in our institution. Of these, 469 non-HLA-sensitized patients received an isolated first ABO-compatible kidney transplant. Of this total, 250 patients received an immunosuppressive regimen based on tacrolimus with either Prograf (n = 168) or Advagraf (n = 82), plus mycophenolic acid with or without steroids. We excluded the 169 patients who did not receive tacro-limus after kidney transplant. Another 50 patients who received a different once-a-day tacrolimus formulation (ie, Envarsus, Chiesi Farmaceutici, Parma, Italy) were also excluded, as the difference in the pharmacokinetic profiles between Envarsus and Advagraf could affect IPV.

The tacrolimus twice-daily formulation is part of our standard immunosuppression protocol. The once-daily formulation was used when patients were included in clinical trials. When enrollment in clinical trials was possible, all consecutive patients were included in the clinical trial without any selection. Until December 2012, all patients were given induction therapy by basiliximab; however, those at high risk of delayed graft function received polyclonal antibodies. In all patients, mycophenolic acid was started just before transplant at a dose of 2 g/day and continued until day 15. A steroid pulse (10 mg/kg) was given before surgery. Methyl-prednisolone was given at 250 mg on day 1 and then decreased to 1 mg/kg/day for 1 week, with doses then tapered to 20 mg on day 15 and 10 mg on day 30.

The Toulouse University Hospital Institution Review Board approved the study. All patients were followed for 2 years posttransplant or until graft loss or death or until the last-follow-up under the initial form of tacrolimus (Prograf or Advagraf). The median follow-up period was 24 months (range, 0.3-24 mo). All patients were screened for anti-HLA antibodies before transplant, at 6, 12, and 24 months posttransplant, and each time the patient presented with impaired kidney function.

No protocol kidney biopsies were performed. However, each time kidney function was impaired, a kidney biopsy, C4d staining, and SV40 staining were performed. Kidney biopsies were analyzed according to the Banff 2013 criteria. Cellular acute rejection epi-sodes were treated using steroid pulses. Antibody-mediated rejection episodes were treated with plasmapheresis (6 sessions) and rituximab (2 infusions at 1-week intervals at 375 mg/m² each).

Immunologic analyses
Luminex assays (Luminex, Austin, TX, USA) determined the specificity of class I HLAs in A/B/C and class II HLAs in DR/DQ/DP immunoglobulin G antibodies in serum samples of recipients (centrifuged at 10,000g for 10 min) using Labscreen single-antigen HLA class I and class II detection tests (One Lambda, Inc., Canoga Park, CA), according to the manufacturer’s instructions. The presence and specificity of the antibodies were then detected using Labscan 100 (One Lambda, Inc.), and the mean fluorescence (baseline value) of each sample in each bead was evaluated. The baseline value was calculated as follows: (raw sample mean fluorescence intensity [MFI] − raw negative serum control MFI) – (negative-bead raw MFI with sample − negative-bead raw MFI with negative serum control). A baseline value of > 500 was considered positive.

Calculation of tacrolimus variability among patients
Variability of tacrolimus was estimated using the coefficient of variability (CV). The CV was calculated as follows: CV (%) = (standard deviation/mean tac-rolimus trough level concentration) × 100. Variability of dose-corrected concentration (CV C0/D) was calculated using the tacrolimus trough level divided by dose of tacrolimus ingested the day before trough level measurement. Tacrolimus variability was calculated using trough level obtained at months 1, 3, 6, 12, 18, and 24. Because some patients were converted from 1 formulation to another during follow-up, we calculated CV and CV C0/D after excluding tacrolimus trough levels obtained after the switch.

Statistical analyses
Reported values are shown as means and standard deviation or medians with ranges. Proportions were compared using the Fisher exact test. Quantitative variables were compared using the Mann-Whitney nonparametric test or t test. Occurrence of DSA is presented with the use of Kaplan-Meier curves. P < .05 was considered statistically significant.

Results

Patient characteristics
The patient characteristics at transplant are presented in Table 1. No significant differences were observed between the 2 treatment groups.

Immunosuppressive therapy
At transplant, a significantly higher number of patients were given induction therapy with twice-daily tacrolimus (Table 2). In both groups, most patients received basiliximab. All patients were given tacrolimus (twice-daily or once-daily) plus myco-phenolic acid. At transplant, all but 2 patients from the tacrolimus once-daily group were also given steroids.

During follow-up, 28 patients were converted from twice-daily tacrolimus to once-daily tacrolimus, and 6 patients were switched from once-daily tacrolimus to twice-daily tacrolimus. Patients were switched from the twice-daily to the once-daily formulation at their request to improve quality of life. Six patients were converted from once-daily to twice daily dose because high doses of the once-daily formulation were required to achieve the target trough level. Hence, physicians preferred using the twice-daily formulation. The median time from transplant to conversion from twice-daily to once-daily tacrolimus was 174 days (range, 8-540 d). The median time from transplant to the conversion from once-daily to twice-daily tacrolimus was 108 days (range, 14-245 d).

In the tacrolimus twice-daily group, 12 patients were withdrawn from tacrolimus; of these, 2 patients were converted to belatacept (at months 6 and 9), 6 patients were converted to mTORi agents (at months 2, 3, 6, 9, 9, and 15), 1 patient had HLA full match, 1 patient developed kidney cancer, 1 patient had BK virus-associated nephropathy, and 1 patient was converted to once-daily LCP-Tacro MeltDose (Envarsus) tacrolimus.

In the tacrolimus once-daily group, 6 patients were withdrawn from tacrolimus; of these, 2 were converted to belatacept (at months 6 and 9) and 4 patients were converted to mTORi agents (at months 6, 9, 15, and 18) because of increasing creatinine levels.

Tacrolimus trough levels were similar during follow-up for both groups, except for at 6 months posttransplant, when the tacrolimus trough level was significantly lower in patients receiving once-daily tacrolimus.

Over the past several years, the immunosup-pressive strategy in our center has changed. Patients were often converted from mycophenolic acid to an mTORi to prevent viral infections and skin cancer. From 3 months posttransplant until the last follow-up, the proportion of patients given mycophenolic acid was significantly lower in the once-daily tacrolimus group. This is related to the fact that a proportion of patients were converted from myco-phenolic acid to mTORi. Consequently, the pro-portion of patients given mTORi was significantly higher in the once-daily tacrolimus group than in the twice-daily group. The proportion of patients given steroids was also significantly lower in the once-daily tacrolimus group.

Intrapatient variability
The CV results were not significantly different between the 2 groups, ie, 39.4 ± 15.3% in the twice-daily tacrolimus group versus 40.9 ± 12% in the once-daily tacrolimus group (P = .43). The CV C0/D result was also similar in both groups: 33.6 ± 18.5% in the twice-daily group versus 32.3 ± 17% in the once-daily group (P = .6). The proportion of patients with CV < 30% in the once-daily tacrolimus group (13.9%) was similar to that shown in the twice-daily group (22.8%; P = .1). After we excluded data obtained after conversion from 1 formulation to another, CV (39.8 ± 14.4% in the twice-daily tacrolimus group and 41.4 ± 11.7% in the once-daily tacrolimus group; P = .4) and CV C0/D (33.6 ± 17% in the twice-daily tacrolimus group and 32.8 ± 17% in the once-daily tacrolimus group; P = .73) were also shown to be similar between groups. The proportion of patients who had CV < 30% was 18.9% in the twice-daily tacrolimus group and 11.8% in the once-daily tacrolimus group (P = .24). Finally, the development of DSAs did not increase in patients who had CV ≥ 30% (3.27%) versus in those who had CV of < 30% (2.22%).

Incidence of donor-specific antibodies
Under tacrolimus therapy, 9 patients (5.35%) who received the twice-daily formulation and 2 patients (2.4%) who received the once-daily formulation developed anti-class I anti-HLA antibodies (P = .51). Eight other patients (4.76%) in the twice-daily group and 2 patients (2.4%) in the once-daily group developed anti-class II anti-HLA antibodies (P = .5). Thirteen patients (7.74%) in the twice-daily tacro-limus group and 2 patients (2.4%) in the once-daily tacrolimus group developed anti-class I and/or class II anti-HLA antibodies (P = .15). During follow-up and under tacrolimus therapy, 6 patients (3.6%) in the twice-daily group and 1 patient (1.2%) in the once-daily group developed anti-HLA DSAs (P = .43; Figure 1). Hence, the 2-year incidence of DSAs was 2.8%. Survival rates without development of a DSA at 2 years posttransplant are illustrated in Figure 2.

We note that none of the patients who were converted from 1 tacrolimus formulation to another developed a DSA before or after the switch. Similarly, all patients who were withdrawn from tacrolimus also developed no DSAs. During the study period, 1 graft loss, due to renal-artery stenosis and severe interstitial fibrosis/tubular atrophy, occurred at 8 months posttransplant in the twice-daily tacrolimus group; another graft loss caused by a vein thrombosis occurred at 7 months posttransplant in the tacro-limus group. In both cases, no DSAs were detected at graft loss. Three patients died during the study: 2 patients who had received twice-daily and 1 patient who had received once-daily tacrolimus. Deaths occurred at 12, 12, and 21 months posttransplant. The causes of deaths were lung cancer, suicide, and hepatocellular carcinoma. None of these patients had DSAs.

Incidence of acute rejection
Overall, during the 24-month follow-up, the inci-dence of acute rejection was 11.2% (28/250). For patients converted from 1 formulation to another (6 from once-daily to twice-daily tacrolimus and 28 from twice-daily to once-daily tacrolimus), the incidence of acute rejection in those who received twice-daily tacrolimus was 11.6% (17 of 146 patients) and was 10.6% (11 of 104 patients) in patients who received once-daily tacrolimus (P = .84). The incidence of T-cell-mediated acute rejection was similar in both groups: 11 of 104 patients (10.6%) who received once-daily tacrolimus and 14 of 146 patients (9.6%) who received twice-daily tacrolimus (P = .83).

The incidence of acute antibody-mediated rejection was similar in both groups, with 5 of 104 patients (4.8%) in the once-daily tacrolimus group and 4 of 146 patients (2.7%) in the twice-daily tacrolimus group (P = .5). Simultaneous T-cell-mediated rejection and antibody-mediated rejection occurred in 4 and 1 patients receiving once-daily and twice-daily tacro-limus, respectively. The time from transplant until an acute rejection was similar in both groups (252 ± 221 d in the twice-daily group and 168 ± 218 d in the once-daily group; P = .32). Three T-cell-mediated acute-rejection episodes occurred at > 3 months after conversion from twice-daily to once-daily tacrolimus. Comparisons between patients who experienced or not an acute rejection are presented in Table 3.

Kidney function and infection complications
No significant differences in kidney function were observed between the 2 treatment groups (Figure 3). The incidences of cytomegalovirus infection that required therapy, of BK viremia, or of bacterial or fungal complications that required hospitalization by day 30 or at months 3, 6, 12, and 24 were similar between the 2 groups (data not shown).

Discussion

Anti-HLA DSA-mediated rejection has become the main cause of graft loss after kidney transplant.¹ Some immunosuppressive regimens, such as CNI-free mTORi-based regimens,⁶ and minimization protocols⁵ are presently avoided to reduce the risk of developing a de novo DSA. Poor compliance with immunosuppressive therapy is also associated with acute rejection episodes and is considered a risk factor for development of de novo DSAs.^2,8,9 In addition, IPV has been associated with an increased risk of acute or chronic rejection.^10-14

The use of once-daily tacrolimus compared with a twice-daily formulation has been found to improve compliance and IPV.^17,18 Hence, it is expected that its use could decrease the risk of developing de novo DSAs. The aim of our retrospective study was to assess the incidence of developing de novo DSAs during the first 2 years posttransplant in patients receiving tacrolimus once or twice daily. Our findings were 3-fold: (1) the 2-year incidence of developing a de novo DSA in low-risk immunologic patients given a tacrolimus-based therapy was low, ie, 2.8%; (2) the incidence of DSAs and acute rejection episodes did not differ between the 2 formulations; and (3) IPV was similar for both formulations and was not associated with acute rejection episodes or development of de novo DSAs.

To compare the incidence of DSAs with the 2 tacrolimus formulations, we included all patients with a low immunologic risk, ie, those patients who did not have anti-HLA antibodies at pretransplant, had received a graft in our institution since the Luminex SA assay became available, and had initially received the criterion standard immunosuppressive regimen (ie, tacrolimus, mycophenolic acid, and low-dose steroids). Overall, the incidence of de novo DSAs was quite low (2.8%) and was lower than that shown in previously published reports. Indeed, it has been reported that patients given different combinations of immunosuppressive regimens had incidences of de novo DSAs ranging from 7% to 27%.^6,19-21 In a prospective study, a CNI mTORi-based immunosup-pression regimen was associated with risk of developing a DSA in de novo kidney transplant recipients.⁶ In the BENEFIT trial, the 7-year incidence of DSAs was 11.6% in patients given cyclosporine and ~2% in patients given belatacept.²² Our results are similar to those presented in a recent prospective study that reported a 1-year incidence of de novo DSAs of 3.14% in low-risk patients given tacrolimus once daily plus mycophenolic acid with or without steroids.⁵ However, in that study, all cases of de novo DSAs occurred in patients where low tacrolimus exposure was targeted (< 3 ng/mL).⁵ Hence, the combination of tacrolimus and mycophenolic acid (with or without steroids) is safe and is associated with few de novo DSAs in low immunologic risk patients.

To the best of our knowledge, no comparisons have been made regarding DSA incidence in patients given tacrolimus twice daily versus once daily to improve compliance. In the present study, we found the incidence of DSAs to be similar with both formulations. The lack of difference can be related to factors other than the tacrolimus formulation, including a higher proportion of patients having been given induction therapy with twice-daily tacrolimus, different proportions of patients on mycophenolic acid and mTORi beyond 3 months posttransplant, and the presence of an education program in our institution, which probably decreased the proportion of noncompliant patients. However, the overall incidence of DSAs was too low to allow a multivariate analysis to be conducted.

Results have shown, including in the OSAKA study, which included a large number of patients randomized to receive tacrolimus twice daily or once daily, a similar rate of acute rejection with both formulations and similar tolerance profiles.23 This was also observed in our study.

Although the effects of IPV of tacrolimus blood levels on acute rejection rate, histologic lesions, graft loss, and the development of DSAs have been reported, results are still controversial. Rodrigo and associates found that a CV > 30% was an inde-pendent predictive factor for developing a de novo DSA.¹⁴ Vanhove and colleagues reported that high IPV did not affect kidney function during a 2-year follow-up period.¹³ However, IPV was associated with accelerated progression of chronic histologic lesions. A tacrolimus once-daily formulation was found to improve IPV compared with a twice-daily formulation.^15-17 In our study, we did not observe any difference in IPV according to tacrolimus formu-lation. A CV of > 30% was not associated with an increased risk of developing a de novo DSA. This could be related to the low incidence of DSAs observed in our study. Because no protocol kidney biopsies were performed in our study, we were unable to assess the effects of IPV on histologic lesions.

Because of its retrospective design, our study has some limitations. The most important is the lack of evaluating patient compliance and quality of life. However, based on previous studies, it is reasonable to speculate that compliance and especially quality of life can be improved in patients given once-daily tacrolimus.²⁴ Second, the follow-up may not have been sufficiently long enough to assess improved compliance leading to the development of less DSAs. Indeed, it has been shown that compliance to treatment declines over time. A longer follow-up may be required to find differences in the incidence of DSAs. Finally, no systematic kidney biopsies were performed to evaluate the effects of IPV on kidney lesions.

In summary, the use of tacrolimus-based im-munosuppression along with mycophenolic acid is safe and is associated with a low risk of developing a de novo DSA. At 2 years, there was no difference between once-daily and twice-daily formulations of tacrolimus. A longer follow-up period is required to assess whether the expected improvements in compliance and quality of life can prevent the occurrence of DSAs.

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