The reduction in renal transplantation rejection rates achieved over recent decades has not translated into a commensurate improvement in long-term allograft survival. Therefore, increasing attention is being focused on the cause of late graft loss (1); the leading causes are chronic allograft nephropathy (2, 3) and death with a functioning allograft, often from progressive cardiovascular disease and various cancers (4). Although the causes and progression of chronic allograft nephropathy are multifactorial, the nephrotoxic effects of calcineurin inhibitor (CNI) drugs have emerged as important contributors to this process. Accordingly, immunosuppressive regimens that would permit early elimination or the complete avoidance of CNIs would be attractive, provided that adequate immunosuppression and acceptable rates of acute rejection were preserved. Initial reports of CNI-free or sparing mammalian target of rapamycin (mTOR) inhibitor-based immunosuppressive regimens (5–7) suggested better preservation of renal function and improved histological chronicity scores (8, 9), when compared with CNI-based protocols. However, results of larger multicenter trials have been disappointing and plagued by excessive patient dropout and switching to a CNI drug in the CNI-free groups due to problems with delayed wound healing, formation of lymphoceles, higher rates of biopsy-proven acute rejection (BPARs), and increased incidences of possible sirolimus (SRL)-related side effects during the early period after transplantation (10–13).
Therefore, we hypothesized that early CNI withdrawal may be the best option by delivering CNIs during the early period of immunologic graft injury and wound healing and then converting patients to a less nephrotoxic mTOR-inhibitor before significant renal damage occurs.
We carried out a 12-month, prospective, open-label, multicenter randomized clinical trial in two parallel groups of adult renal transplant recipients in compliance with the provisions of the Declaration of Helsinki and good clinical practice (GCP) guidelines (Controlled-trials.com, ISRCTN no. 74429508). The institutional review board at each site had approved the protocol, and patients had given written informed consent before enrollment. A permuted block randomization scheme was used to assign trial participants to one of the treatment groups at each of the individual study sites. Allocation concealment was secured by a centralized distribution of sequentially numbered, opaque, sealed envelopes, and a confirmatory randomization fax to the clinical research organization (CRO) (Fig. 1).
Inclusion and Exclusion Criteria
Patients between the ages of 18 and 65 years who were scheduled to receive a single organ renal transplant from a living donor or a deceased donor were eligible.
Exclusion criteria at baseline (day 0) included a current or historic panel-reactive antibody titer of more than 30%; a positive cross-match; a gastrointestinal disorder that might interfere with the ability to absorb oral medication; a history of cancer, except successfully treated; the receipt of a new investigational drug within the previous 3 months and a body mass index greater than 32 kg/m2. Patients were also required to have a white blood cell count more than or equal to 4000 mm−3 (4.0×109 L−1), platelet count more than or equal to 100,000 mm−3 (100×109 L−1), fasting triglycerides less than or equal to 4.6 mmol/L (400 mg/dL), and fasting cholesterol less than or equal to 7.8 mmol/L (300 mg/dL).
Conditions that may compromise patients' safety by an early conversion to an antiproliferative combination with SRL and mycophenolate mofetil (MMF) were excluded for all patients before randomization by protocol. These predefined exclusion criteria included a persistent delayed graft function (defined as continuous need for dialysis at the time of randomization), a steroid-resistant or persistent/unresolved rejection, an active wound healing problem, and surgical site infection and a symptomatic lymphoceles.
Intravenous antithymocyte globulin (ATG-F, provided by Fresenius Biotech, Germany) was administered as a single dose of 4 mg/kg in the first 25 patients. As we experienced a higher than expected rate of BPAR of 41.2%, we decided to amend the protocol and increased the ATG induction to 9 mg/kg in the following 173 patients. All patients received 500 mg methylprednisolone intraoperatively and afterward, a maintenance dose of corticosteroids according to the local practice of the participating center. The minimum maintenance corticosteroid doses were 20 mg of prednisone (or the equivalent) for the first 2 weeks after transplantation, 15 mg from week 3 to week 8, 10 mg from week 9 until the end of month 4, and a minimum of 2 mg thereafter. Cyclosporine A (CsA) was started within 24 hr after transplantation with an initial oral dose of 3 to 5 mg/kg twice daily and was adjusted to achieve a target trough level of 200 to 250 ng/mL for the first 2 to 3 weeks. Oral MMF was initiated at a dose of 3 g/d.
After randomization between day 10 and 24, CsA trough levels in the control group were lowered to 150 to 200 ng/mL and to 100 to 150 ng/mL by month 4. MMF was given at a daily dose of 2 g, and maintained as tolerated.
In patients randomized to the CNI-free protocol, SRL was initiated with a loading dose of up to 0.1 mg/kg followed by 2 to 4 mg/d once daily, aiming for an initial target trough level of 8 to 12 ng/mL. At this time, CsA was reduced by 50% and eliminated 3 days later. After 3 months, SRL was tapered to achieve target levels of 5 to 10 ng/mL. In the CNI-free SRL group, MMF was intentionally decreased to 1.5 g/d to account for CNI-related induction of mycophenolic acid (MPA) metabolism in the CsA arm.
Recipients with a high-risk constellation (D+/R−) received cytomegalovirus (CMV) prophylaxis according to local center practice. Low, intermediate, and high-risk patients after prophylaxis were monitored for CMV viremia when presenting for routine clinic visits (weekly for the first 6 weeks, alternate weeks until month 3 and monthly, thereafter). In case of CMV viremia above the detection limit (200 copies/mL); preemptive treatment was initiated with intravenous ganciclovir or oral valganciclovir. Pneumocystis carinii pneumonia prophylaxis was not uniformly administered but was recommended for the first year after a pneumocystis carinii (PCP) outbreak in one study center (see discussion).
Efficacy and Safety
The primary endpoint was the estimated glomerular filtration rate (eGFR) 12 months after transplantation, calculated from serum creatinine measures by using the Nankivell formula (eGFR=6.7/SCr+0.25×weight−0.5×urea−100/height2+35 [or 25 if female], adjusted for body surface) and the modification of diet in renal disease formula (GFR [mL/min/1.73 m2]=186×SCr−1,154×age−0.203×[0.742 if female]×[1.21 if black]). Secondary efficacy endpoints included BPAR, time to the first episode of BPAR, and recipient and allograft survival at 12 months. As a combined secondary endpoint, we analyzed the frequency of treatment failure during the first 12 months. Treatment failure was defined as the occurrence of any of the following: BPAR, discontinuation of any study medication for more than 42 cumulative days, allograft loss, or death. Incidence and severity of biopsy BPARs were classified according to Banff ‘97 criteria.
Sample size calculations were based on the results of Flechner et al. (14) and Oberbauer et al. (15), who reported differences in mean serum creatinine values of 1.78±0.76 mg/dL and 167±68 μmol/L for a CsA-based and 1.32±0.33 mg/dL and 128±66 μmol/L for a SRL-based immunosuppressive regimen, respectively. Sample size calculation based on the t test procedure resulted in a sample size of 50 and 55 patients per treatment arm (α=0.05 and power=0.85). To allow for a 20% drop out rate, the sample size was increased to 70 patients per treatment group.
The primary analysis was based on the intent-to-treat (ITT) population where all patients who started the randomized treatment were included. A second analysis was restricted to patients who completed the trial after 12 months on the randomized treatment. Although the same size estimation and initial analysis plan was based on t test and ANOVA procedures, a data review before the final analysis demonstrated that the normality assumption had to be rejected by inspection of diagnostic plots and use of the Shapiro-Wilk test on the 10% level for serum creatinine and eGFR. Therefore, a nonparametric approach was considered more adequate and overall group differences for primary endpoints and their changes in time were tested by the Wilcoxon rank sum test. Missing values for serum creatinine, urea, and weight were imputed by the “last observation carried forward” method. Patients who died were excluded from the analysis and in case of graft failure, serum creatinine was set to the screening value, and eGFR was set to 10 mL/min per 1.73 m2. A linear mixed model repeated measures analysis (SAS PROC MIXED) with unstructured covariance was used for longitudinal data. All P values are two sided, and P less than 0.05 was considered statistically significant. All statistical analyses were performed using SAS version 9.1.3 for windows, SAS Institute Inc., Cary, NC.
From February 2005 to April 2007, a total of 198 patients were included in six centers. Fifty-three patients were excluded for failing inclusion or meeting exclusion criteria; four patients had not received a transplant; 141 patients were randomized and 140 were converted to the assigned treatment (Fig. 1). The median (Q1-Q3) time of conversion within the time window from 10 to 24 days after transplantation was 18 (16–21) days for the SRL-conversion group and 18 (15–21) days for the CsA group (P=0.2769).
Baseline demographic, clinical, and donor-recipient characteristics showed no significant difference between the treatment groups (Table 1).
Target trough levels for immunosuppression were generally met (Fig. 2a,b). Mean SRL levels achieved were largely within the target range; however, they were more commonly at the lower end of this target range. The actual administered doses of MMF (SRL vs. CsA group, ITT population) at the time of conversion were 2058±649 mg/d vs. 2331±601 mg/d (P<0.05), 1376±296 mg/d vs. 1707±498 mg/d (P<0.05) at month 3, 1349±325 mg/d vs. 1622±505 mg/d (P<0.05) at month 6, and 1238±352 mg/d vs. 1521±545 mg/d (P<0.05) at month 12. Although MMF was given as per protocol at lower doses in the SRL group, MPA levels in this group were significantly higher than in the CsA group, as determined by MPA level measurements performed in all patients of one trial center (Fig. 2c). There was no difference in the administered prednisolone dose between the study groups at any time point. At 1, 6, and 12 months after transplantation, the administered doses for prednisolone were 15.25±6.91, 5.66±4.08, 3.95±2.62 mg/d in the SRL group and 15.69±6.66, 7.54±8.34, 5.13±6.93 mg/d in the CsA group, respectively.
The primary ITT analysis at 12 months showed significantly better renal function in patients converted to a CNI-free, SRL-based immunosuppression. Differences were significant for serum-creatinine (Δ=0.23 mg/dL), eGFR calculated by Nankivell (Δ=10.0 mL/min/1.73 m2) and the modification of diet in renal disease formulas (Δ=10.9 mL/min/1.73 m2). Analysis of the patients who continued their assigned treatment for up to 12 months showed similar differences for the median values of serum creatinine and eGFR (Table 2). The observed positive effects for patients in the SRL conversion group were consistent, irrespective of the donor type, whether patients received low or high dose ATG-F, or went through a phase of delayed graft function after transplantation (data not shown). By subtracting the baseline value from the target value at 12 months the median increase of eGFR was 11.8 (Q1-Q3: 1.0-19.8) in the SRL group and 3.9 (Q1-Q3: −3.3-13.0) in the control group (P=0.0266). At the time of conversion, patients in the SRL group had numerically but insignificantly higher eGFR median values in the SRL group (50.9 mL/min/1.73 m2) and in the control group (45.5 mL/min/1.73 m2; P=0.14).
Seven days after conversion, differences reached significance for eGFR, with a ΔGFRNankivell of 8.27 mL/min per 1.73 m2 (P=0.0049) in favor of the converted patients. Correcting for baseline GFR, ΔGFRNankivell was expressed as a difference of GFR (7 days after conversion) and GFR (at the time of conversion), was significantly better in the SRL group (7.0 mL/min/1.73 m2 [2.4; 11.7]) compared with the CsA group (4.7 mL/min/1.73 m2 [0.0; 8.3]) (median [interquartile range], P=0.0484). Although in both groups renal function improved up to 3 months after transplantation, after this time a negative trend in the renal function was observed in the CsA ITT population. (Fig. 3a).
Longitudinal data analyses by a piecewise linear mixed model showed a significant difference in the eGFR slopes for the period between 3 and 12 months after transplantation. In this time interval, after reaching optimal renal function in both groups, the slope (eGFR, Nankivell) in the ITT population for SRL was positive (+0.43 mL/min/1.73 m2 per month) and negative for the CsA group (−0.35 mL/min/1.73 m2 per month) (P=0.0011 for the group×time interaction). Similar results were obtained with a slope calculated from day 7. This is consistent with the median ΔGFR analysis expressed as a difference (GFR[M12]−GFR[M3]), which is positive in the SRL group (1.1 mL/min/1.73 m2 [−3.4; 8.9]) and negative in the CsA group (−1.0 mL/min/1.73 m2 [−8.1; 3.1]; P=0.0239) (values denote medians with interquartile range and P values of the Wilcoxon ranks sum test).
New onset proteinuria after transplantation was detected in 5 (7.3%) of the SRL-treated patients and in 1 (1.4%) CsA-treated patient (P=0.113).
During the recruitment of the first 25 patients, from which 17 patients were subsequently randomized in one of the two treatment arms, we experienced a higher than expected rate of BPAR of 41.2%. After amending the protocol, early rejection rates before randomization decreased significantly and overall 12 months BPAR rates decreased to 25.2% in the remaining 123 patients within the trial.
The BPAR rate of patients on CsA, MMF, and corticosteroids before randomization was 15% (Banff grade 4). The incidence of clinically suspected and treated Banff grade 4 rejections in the first 12 months after randomization was 17.4% for the SRL group and 15.5% for the CsA group. Borderline lesions were treated in 6% of patients before randomization and in 0% in the SRL and 2.8% in the CsA group after randomization, respectively. All rejection episodes in the ITT population responded to steroid bolus treatment. Incidence of BPAR within 12 months and the freedom from rejection are detailed in Figure 3(b, c).
Overall Survival and Allograft Survival
Overall 1-year survival rates for patients were 99% in both groups. Allograft survival was 99% in the SRL group and 97% in the CsA group. In the CsA group, 1 graft had to be removed because of the diagnosis of a non-Hodgkin lymphoma in the transplanted kidney. In total, two patients died, one in each arm of the study; one patient in the SRL group developed a fatal pulmonary embolus as an outpatient 82 days after transplantation, another patient in the CsA group developed a fulminant Pneumocystis jirovecii pneumonia 140 days after transplantation.
Treatment Failure and Discontinuations of Trial Medication
Treatment failure was more frequent in SRL treated than in CsA-treated patients (51% vs. 32%, P=0.0392). Death and allograft loss had a negligible effect on the rate of treatment failure. BPAR contributed in 23.2% of treatment failures in the SRL group and in 19.7% in the CsA group (P=0.6828). Treatment failure due to BPARs was not necessarily associated with discontinuation of the assigned study treatment. Sixty-three percentage of these patients remained on the randomized treatment after BPAR. Differences in the rate of treatment failure were mainly due to differences in the discontinuation of study treatment; 26% in the SRL group and 10% in the CsA group (P=0.0151). These discontinuations were mainly related to adverse events (AEs), which may have been attributable to one of the primary immunosuppressive drugs (22% SRL group vs. 6% CsA group, P=0.0064). The most frequent adverse events leading to discontinuation from study treatment were pneumonia (SRL, 4 patients; CsA, none) and wound healing disorder (SRL, 3 patients; CsA, 1 patient). Overall, treatment failure occurred rather early after transplantation, usually in the first 5 months after initiation of therapy (Fig. 3d).
Serious AEs were reported by 54% of patients in the SRL group, compared with 66% in the CsA group (Table 3). The severity (mild/moderate/severe) of AEs was similar in both groups: SRL, 46.6%, 40.2%, 13.1% and CsA, 51.5%, 35.3%, and 13.2% of the events. A relationship between the immunosuppressive drug and AEs was inferred in 39% of the events with SRL, 35.7% with CsA, and 31.1% with MMF. The requirement for any kind of treatment was similar in both groups (76.8% of the patients in the SRL group versus 76.1% in the CsA group, P=1.00). No significant difference was found for the rate of rehospitalization for AEs (SRL 49.3% vs. CsA 62.0%, P=0.1733) and the need for surgical interventions (SRL 27.5% vs. CsA 32.4%, P=0.5827).
Skin disorders, mainly acneiform skin problems and aphtous ulcers, were more frequent in the SRL group (Table 3). SRL-treated patients showed the typical temporary increase in triglycerides and cholesterol levels reaching a peak value between month 3 and 6; at 12 months these differences had resolved.
In general, infections tended to be more frequent in CsA-treated patients. Although there was no significant difference in the CMV risk profile (Table 1), CMV infection rates were significantly lower in SRL-treated patients. BK virus infections were detected in two patients in the SRL ITT group. One BK virus infection developed 1 month after a rejection episode, mandating the conversion to tacrolimus and a steroid bolus treatment. In this patient, BK virus infection could be controlled by the reduction of immunosuppression. Another case of BK virus infection resolved with antiviral therapy (Cidofovir).
Cancer developed in four patients in the CsA group (6%). These included a renal cell cancer, a colon cancer, a squamous cell cancer of the nasal cavity, and a non-Hodgkin lymphoma of the transplanted kidney. All cancers, including the squamous cell cancer, underwent surgical resection; the graft had to be removed in the patient with the non-Hodgkin lymphoma. No cancers were detected in the SRL group (P=0.1198).
Concerns over possible delays in functional recovery, increased acute rejection rates, coupled with the potential for impaired wound healing, have led many clinicians to introduce mTOR inhibitors only after the early posttransplant period to pre-empt or avoid the adverse effects of other immunosuppressive agents (often CNI-related nephrotoxicity) (11). However, late conversion with acceptable AR rates achieved modest to little renal function improvement in patients with progressive allograft dysfunction (7). Recognizing these considerations, we have designed an early conversion protocol from a CsA-based therapy to a CNI-free SRL-based therapy in patients younger than 65 years of age with a low-to-moderate immunologic risk. Acknowledging known potential side effects of an early mTOR-inhibitor based therapy, we intentionally excluded patients ' conditions, which may selectively compromise safety in patients randomized to SRL and MMF. As exclusion criteria were applied before randomization and to both study groups, we do not assume that these protocol-defined exclusion criteria would bias the principle results of this trial.
The primary endpoint of this study was renal function at 12 months after transplantation. Renal function both measured and estimated, was significantly better in the SRL group, with a ΔeGFR of approximately 10 mL/min as compared with patients continuing on CsA. This result is very similar to differences reported when CsA was eliminated at 3 months (16). The improvement was observed soon after conversion and is therefore likely to have a direct consequence of eliminating the effects of CsA on renal blood flow and glomerular filtration. As numerical differences in baseline eGFR values may have some potential to confound these data, we also analyzed longitudinal measures of renal function (ΔGFR and the GFR slopes) for various time intervals (Δ12-3 months or Δ12 months - time of randomization) in an attempt to adjust for these baseline differences. The improvement in renal function was significantly better in the SRL conversion group irrespective of the time interval analyzed. Whether the better slope of renal function observed after the initial recovery is truly accompanied with less chronic CNI-induced kidney damage needs to be explored in future studies that incorporate routine protocol kidney biopsy specimens (17).
However, our study also yielded some unexpected results: while the induction with ATG-F and an initial phase of CNI (CsA) coverage were chosen to provide effective protection of immunologic graft injury within the first 2 to 3 weeks after transplantation the initial protocol did not fulfill this expectation but resulted in an unexpected high rate of BPAR in the first 17 study patients. Lymphocyte subpopulation analysis in some of these initial patients showed an early preferential clonally expansion of activated alloreactive lymphocyte subpopulations, suggesting an ineffective induction treatment; and in contrast, a mechanism referred to as the “lymphopenic homeostatic proliferation of alloreactive T cells” may even have triggered these rejections (18). In fact, after amending our protocol by increasing the dose of ATG-F induction from 4 to 9 mg/kg, which was shown to be effective in other protocols (19), we were able to reduce BPAR to overall 25% in the remaining 123 study patients.
Furthermore, an important question to be addressed is the potentially increased risk of acute rejection after conversion. Within the statistical power of this study, we could not detect any difference in the rejection rates between both treatment groups after the conversion process. A similar incidence of BPAR (17%) was recently reported after conversion to a SRL/MMF regimen at 6 months (CONCEPT trial, ). In the CONCEPT trial, increased BPAR in the SRL/MMF group versus the CsA/MMF group were temporally associated with steroid withdrawal at month 8, suggesting reduced efficacy with SRL at blood levels less than 10 ng/mL in the first year after transplantation. Accordingly, we have also observed that some of the BPARs in the SRL group were clearly associated with low (potentially subtherapeutic) or borderline target levels, especially in the first 3 months. Although, rejection episodes after conversion were mild and all responded to steroid pulse therapy, these events illustrate the challenge of a SRL-based immunosuppression maneuvering in a narrow therapeutic window (20).
The adverse event profile reported here is consistent with those previously reported. The most frequent causes for drug discontinuation in the SRL group were suspected pneumonitis and recurrence of wound healing problems. Interestingly, the total rate of pulmonary infections was not significantly different between SRL- and CsA-treated patients. Although suspected more often, only one patient was confirmed to have SRL-related pneumonitis. Other suspected cases turned out to be PCP infections occurred during a PCP outbreak in one study center (21). SRL discontinuations were often preventive in nature especially when additional surgical interventions were necessary.
The inconsistency of drug discontinuations and objective safety parameters (number and severity of AEs, rate of rehospitalization or reintervention), raises the question whether some of the discontinuations in the SRL group could have been avoided with better patient selection and/or more refined management of SRL-(un)related AEs.
Also, the contribution of MMF as a second antiproliferative agent in the occurrence of certain AEs such as mouth ulcers and diarrhea is unclear. It is reasonable to believe that crosswise adjustment of SRL and MMF with MPA measurements may further improve tolerability of this combination.
Most notably CMV viremia/infection was significantly reduced in patients on SRL. This anti-CMV effect of SRL has been seen in other studies (10). However, it is not clear whether effects on CMV are specific or simply reflect a lower state of immunosuppression. There is some experimental evidence to believe that mTOR inhibitors may directly interfere with CMV virus replication, signaling (22, 23), and may positively regulate memory T-cell response against viruses (24).
The encouraging results of this study should be kept in context. The results presented here pertain to low risk subjects (mainly Caucasian recipients younger than 65 years of age, no DCD donors, relatively short ischemic times) and therefore cannot be generalized to higher risk subjects without careful study. Additionally, as some of the patients in the SRL group developed proteinuria, the 1-year results of this study cannot be simply extrapolated. This surrogate for renal injury needs to undergo critical long-term analysis. In summary, we report improved renal transplant function in patients with a new early conversion strategy from a CsA-based to a SRL-based regimen. A concern for patient compliance is the high rate of dropouts due to adverse events. This will limit the benefits of this early CNI-free strategy to a selected group of patients. Nevertheless, one-year ITT data suggest that this strategy may be a useful approach to improve renal function, while avoiding potential negative effects of SRL in the first 2 to 3 weeks after transplantation.
The authors thank Michael Eder and Franziska Brandenburg for their outstanding help in organizing this trial and Karl Fehnle for assistance in manuscript preparation. The authors thank Charbel Sandroussi and Ian McGilvray for proofreading the manuscript.
Additional investigators of the SMART-Study group: Helmut Arbogast and Markus Rentsch, Department of Surgery, Munich University Hospital, Campus Grosshadern, Munich, Germany; Ulf Schönermarck, Department of Internal Medicine, Grosshadern, University of Munich, Germany; Mirian Opgenoorth, Department of Medicine, Division of Nephrology, University of Erlangen, Erlangen, Germany; Carsten Böger, Department of Internal Medicine II, Nephrology and Transplantation, University Medical Center, Regensburg, Germany; Dirk Burmeister, Department of Urology, Krankenhaus Güstrow, Germany; Stefan Farkas, Department of Surgery, University of Regensburg, Germany; Heiner Wolters, Department of Urology, University of Rostock, Rostock, Germany.
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