Calcineurin inhibitor (CNI) therapy is known to induce nephrotoxicity, promoting interstitial fibrosis/tubular atrophy, and arteriolar hyalinosis in a dose-dependent manner (1, 2). The progression of chronic pathologic lesions is ameliorated if CNI is withdrawn during the first 6 months posttransplant (3, 4), but data are conflicting as to whether eliminating or reducing CNI exposure in longer term kidney transplant recipients with established CNI-related nephrotoxicity can improve graft function (2, 5). Evidence concerning the impact of CNI elimination on kidney graft survival is mixed (6–8).
Introduction of the mammalian target of rapamycin (mTOR) inhibitor class of immunosuppressants has offered an alternative to long-term CNI-based therapy. Everolimus with reduced-exposure CNI regimens has been shown consistently to offer effective immunosuppression in de novo kidney transplant recipients (9–13). The antiproliferative effects of everolimus have been shown to improve interstitial fibrosis/tubular atrophy in animal models (14, 15) and to reduce the severity and incidence of cardiac allograft vasculopathy in heart transplant recipients (16). Preemptive conversion of kidney transplant patients from CNI-based to mTOR inhibitor-based immunosuppression within the first 6 months posttransplant (17–24) has generally proven effective in preserving renal function without loss of efficacy. Although there was a trend to more acute rejection episodes in the CONCEPT study (17), these mostly occurred after protocol-stipulated steroid withdrawal. The large CONVERT study converted kidney transplant recipients at a mean of 3 years posttransplant from CNI treatment to sirolimus (25). In that study, no upper limit for renal function was specified, and 90% of patients had baseline GFR more than 40 mL/min. To date, no prospective study has investigated the impact of late conversion from CNI to mTOR inhibitor therapy in kidney transplant patients preselected to have a GFR between 30 and 70 mL/min at the time of study entry. Limited data in an unselected population of kidney transplant patients have suggested that CNI elimination may be associated with superior renal function compared with CNI reduction after introduction of mTOR therapy (26), but the two strategies have not been compared.
The ASCERTAIN study was undertaken to determine whether introduction of everolimus with elimination or minimization of CNI would improve graft function in maintenance renal transplant patients with renal impairment at the time of conversion.
Of 398 randomized patients, 394 provided at least one postbaseline assessment and comprised the intent-to-treat (ITT) population (Fig. 1). Baseline and demographic characteristics were generally comparable between treatment groups (Table 1). The mean age was 49.1 years, the majority of patients were men (65.7%), and 38.3% had received a graft from a living donor. The mean time from transplantation to study entry was 5.6 years. Fifty-seven patients (14.4%) had baseline measured glomerular filtration rate (mGFR) less than 30 mL/min/1.73 m2, whereas 140 patients had baseline mGFR less than 40 mL/min/1.73 m2 (35.5%) (Table 1) The majority of patients in each group were receiving cyclosporine A (CsA) at study entry, with mycophenolic acid (MPA) therapy and corticosteroids administered to approximately 70% of patients (Table 1).
Mean everolimus concentration in the CNI elimination group and the CNI minimization group was 7.6±3.1 ng/mL (n=119) and 6.4±3.4 ng/mL (n=136), respectively, at month 1, 7.1±2.5 ng/mL (n=88) and 5.0±2.1 ng/mL (n=118) at month 12, and 7.7±3.9 ng/mL (n=75) and 5.3±2.0 ng/mL (n=102) at month 24 after randomization (all P<0.001). In the CNI minimization arm, mean CsA C0 (C2) was 143±187 ng/mL (n=83) (517±198 ng/mL, n=82) at baseline compared with 32±36 ng/mL (n=62) (161±96 ng/mL, n=65) at month 24, a reduction of 78% (69%). Tacrolimus C0 decreased by 66% from 6.1±3.6 ng/mL (n=50) at baseline to 2.1±1.8 ng/mL (n=44) at month 24. In the control group, CNI exposure also decreased during the study: mean CsA C2 decreased by 20% (from 513±201 ng/mL at baseline [n=78] to 412±249 ng/mL at month 24 [n=63]) while mean tacrolimus C0 decreased by 28% from 7.1±4.4 ng/mL (n=33) at baseline to 5.1±1.8 ng/mL (n=31) at month 24.
In total, 282 patients (71.6%) had mGFR values available at both baseline and month 24, with a consistent proportion in each treatment group (88 [69.3%] CNI withdrawal, 101 [70.1%] CNI minimization, 93 [75.6%] controls). In 250 of these patients, GFR was measured using the same method at both time points. Renal function was stable in all three treatment groups (Table 2). There were no statistical differences in mGFR between the CNI elimination or CNI minimization group versus controls at month 24 (Table 2). A sensitivity analysis using the last observation carried forward method for all ITT patients confirmed the findings from the modified ITT population (P=0.64 for CNI withdrawal group vs. controls; P=0.47 for CNI minimization vs. controls).
Among the patients with creatinine clearance (CrCl) less than or equal to 50 mL/min at baseline, the change (least squares mean) from baseline to month 24 was 4.1 mL/min/1.73 m2 in the CNI minimization group (P=0.28 vs. difference in controls), −1.1 mL/min/1.73 m2 in the CNI elimination group (P=0.98 vs. difference in controls) and −1.2 mL/min/1.73 m2 in controls. The change in mGFR from baseline to month 24 was also similar between groups in patients with baseline CrCl more than 50 mL/min (−2.3 mL/min/1.73 m2 in the CNI elimination group [P=0.45 vs. difference in controls], 6.5 mL/min/1.73 m2 in the CNI minimization group [P=0.53 vs. difference in controls], and 2.1 mL/min/1.73 m2 in controls). No marked differences in change in mGFR to month 24 were observed in the subpopulations with CrCl less than or equal to 50 mL/min or more than 50 mL/min when comparing those who continued or discontinued study medication, but the number of patients who discontinued and had GFR measured at month 24 was low.
Urine protein and protein:creatinine ratio were significantly higher in the CNI elimination group at month 12 versus the control arm, but not at month 24 (Table 2).
In a post hoc multivariate analysis, mGFR at month 24 was found to have a significant association with donor age (P=0.009), baseline mGFR (P<0.001) and time since transplantation (P=0.047). Post hoc analyses also showed that among patients with baseline CrCl more than 50 mL/min (Cockcroft-Gault) who remained on the randomized treatment regimen (n=99), the increase in mGFR from baseline to month 24 in the CNI elimination group was significantly greater than in control patients (difference in least squares mean 11.4 mL/min/1.73 m2, 95% confidence interval [CI] for difference 2.1 to 20.8 mL/min/1.73 m2, P=0.017). Among on-treatment patients with CrCl more than 55 mL/min (Nankivell, n=97), the CNI elimination patients also showed a higher mGFR increase than controls (difference in least squares mean 11.8 mL/min/1.73 m2, 95% CI for difference 2.5 to 21.1 mL/min/1.73 m2, P=0.014). Among patients less than 1 year posttransplant at study entry who remained on study treatment, the improvement in mGFR from baseline to month 24 was significantly, greater in the CNI elimination arm versus controls (difference in least squares mean 8.3 mL/min/1.73 m2 [95% CI −14.1 to 30.8 mL/min/1.73 m2, P=0.041]) but the number of patients was too small to draw meaningful conclusions (5 and 4 patients, respectively). No other post hoc analyses, including mGFR at baseline, identified predictors of change in mGFR.
At month 24, 93.9% of patients (24 of 394) survived with a functioning graft. There were no significant between-group differences for the composite efficacy endpoint (biopsy-proven acute rejection [BPAR], graft loss, death, or loss to follow-up) or for any efficacy endpoint (Table 3). The 24-month incidence of BPAR was 5.7% in the CNI elimination group (3 IA/IB, 1 IIA, 1 IIB, 1 antibody-mediated, and 1 missing), 5.6% in the CNI minimization group (4 IA, 2 IIA, 1 III, and 1 missing) and 2.5% (1 IA, 2 missing) in the control group (local biopsy readings). The episode graded IIB in the CNI elimination group occurred the day after everolimus was discontinued at month 7 after randomization. The grade III rejection episode that occurred in the CNI minimization group took place 9 months after everolimus was discontinued (the patient was receiving tacrolimus and steroids at the time of the rejection). Protocol biopsies showed no significant differences in change from baseline to month 24 between intervention group versus the control arm (data not shown).
Table 4 summarizes the most frequent events reported in each treatment group, regardless of relation to study drug. The incidence of adverse events categorized as drug related was higher in the CNI elimination (77.2%) and CNI minimization (70.8%) groups compared with controls (25.2%; P<0.001 vs. both other groups), the most frequent of which were mouth ulceration (26.8%, 9.7%, and 0.8%, respectively), anemia (20.5%, 13.9%, and 1.6%), proteinuria (as identified by the investigator: 14.2%, 9.7%, and 4.1%), peripheral edema (12.6%, 9.0%, and 0%), and hypercholesterolemia (10.2%, 14.6%, and 3.3%). Serious adverse events were least frequent in the control arm (Table 4). The most frequent serious adverse events were urinary tract infection (3.9%, 6.9%, and 4.9%, respectively), diarrhea (4.7%, 8.3%, and 1.6%), pyrexia (3.1%, 7.6%, and 0.8%), increased serum creatinine (5.5%, 2.8%, and 5.7%), and graft loss (2.4%, 6.3%, and 3.3%). The incidence of infections and serious infections (i.e., requiring hospitalization) was similar between groups (Table 4). High triglyceride level (8.5 mmol/L) and low neutrophil count (≤1×109) occurred more frequently in the CNI elimination group (Table 4). Forty-six malignant or benign neoplasms were reported in 27 patients (Table 4). All differences were nonsignificant except for a lower incidence of skin papilloma in the CNI elimination group versus controls, all of which were benign (P=0.028).
Adverse events were the primary reason for study drug discontinuation in 36 (28.3%) CNI elimination patients, 24 (16.7%) CNI minimization patients and 5 (4.1%) controls (P<0.001 vs. CNI elimination; P=0.020 vs. CNI minimization) (Table 4). The most frequent events leading to discontinuation were graft loss (two CNI elimination, three CNI minimization, and three controls), increased serum creatinine (four, one, and three, respectively), acne (four CNI elimination), aphthous stomatitis (three CNI elimination and one CNI minimization), diarrhea, peripheral edema and pyrexia (each one CNI elimination and three CNI minimization), and interstitial lung disease (three CNI elimination and one CNI minimization). Three deaths occurred in the CNI elimination group, all with no suspected relation to study drug, and there were three deaths in the CNI minimization group, one suspected to be related to study drug (see Table 3 for causes of death). No deaths occurred in the control group.
These findings confirm that in kidney transplant patients who are, on average, 5 years posttransplant, introduction of everolimus with elimination of CNI or a marked (>65%) reduction in CNI exposure has no overall benefit on renal function. The primary endpoint, mGFR at month 24, did not differ between the intervention group and control patients. The reduction in CNI exposure (>60% in the minimization arm and 20%–30% in the control arm) may have contributed to stabilization of renal function in these patients and thereby contributed to the lack of between-group differences (27). Post hoc analyses indicated that patients with better renal function at the time of conversion to everolimus may be the most suitable candidates for CNI discontinuation.
The study population consisted of patients with impaired renal function. Mean baseline mGFR was less than 50 mL/min/1.73 m2 in all groups (46.1–49.4 mL/min/1.73 m2) with approximately 30% of patients having mGFR less than 40 mL/min/1.73 m2. Two-year follow-up data from the CONVERT (25) and Spare-the-Nephron (19) trials have shown no benefit in terms of renal function after sirolimus introduction with CNI elimination in patients who had estimated GFR less than 40 mL/min (Nankivell). Similarly, we found CrCl to be predictive of response to everolimus initiation with CNI elimination. The cut-off point for experiencing a significantly greater improvement in mGFR after CNI elimination (50 mL/min [Cockcroft-Gault]) was slightly higher than that seen in the CONVERT study (40 mL/min, Nankivell). In our study, CrCl of 41 or 44 mL/min (Cockcroft-Gault) showed no significant association with improvement in mGFR (data not shown). No significant difference in evolution of mGFR was seen after CNI minimization, regardless of baseline CrCl. It has been suggested that early low-grade proteinuria may provide an alternative marker to GFR for subsequent risk of graft deterioration (28–31).
It should be borne in mind that the primary efficacy endpoint, mGFR at month 24, was evaluated only in those patients for whom mGFR was measured at month 24 that is, patients who died, lost their graft, discontinued, or were lost to follow-up were not included. The proportion of such patients, however, was only approximately 10% with no pronounced differences between treatment groups.
Values for CrCl and serum creatinine did not vary between groups. Urine protein:creatinine was significantly higher at month 24 in the CNI elimination arm versus the control arm, but the absolute value for protein excretion in the CNI elimination arm was acceptable.
Although the protocol stated that patients need only be 6 months posttransplant at study entry, unexpectedly almost one-half of the patients were more than 5 years posttransplant, by which time ischemic glomerulosclerosis is likely to have been prevalent (2). By year 5, 90% of grafts show evidence of CNI-associated lesions (2), and while early acute nephrotoxicity is generally reversible with CNI dose reductions, chronic lesions cannot be reversed once established (2). Experience from randomized trials in thoracic (32, 33) and liver transplantation (34) has shown that patients converted earlier posttransplant experienced a clinically relevant improvement in renal function after conversion to everolimus. In heart transplant patients, a significant improvement in GFR versus controls was observed in patients converted to everolimus or to a CNI minimization regimen during years 1 to 3 posttransplant (mean baseline mGFR 50 mL/min) (32, 33), a finding not seen here. Despite similar GFR values, it is conceivable that the transplanted kidneys in the current study population had more extensive histologic damage than in the heart transplant population due to ischemia-reperfusion injury and previous rejections, but this remains speculation.
There were no significant differences in efficacy outcomes between the three groups. Moderate BPAR (grade IIB or III) occurred in one patient in each of these groups, but not during everolimus treatment. All other episodes were graded mild (IA/IB or IIA). In the CNI elimination arm, mean everolimus trough level was 7 to 8 ng/mL, consistently below the target level of 8 to 12 ng/mL, possibly in response to adverse events, but this did not appear to adversely affect immunosuppressive efficacy compared with the CNI minimization group.
The profile of adverse events in the two everolimus groups was consistent with those reported previously for mTOR inhibitors (35). The incidence of adverse events categorized as drug related was markedly higher among everolimus-treated patients than controls. Although this may have been partly due to the open-label nature of the study, rates of serious adverse events, and serious infections were also higher in the two intervention groups, contributing to the relatively high rates of everolimus discontinuation. Everolimus was discontinued more frequently in the CNI elimination arm than the CNI minimization arm. The mean everolimus trough concentration was, as per protocol, higher in the CNI elimination arm which may have contributed to this difference, although the rate of drug-related adverse events and serious adverse events or infections was comparable. The fact that everolimus exposure was slightly below the protocol-specified target range may have reflected attempts to reduce adverse events in the CNI elimination group. Generally, the safety profile of the CNI elimination and CNI minimization regimens was similar to each other except for a higher frequency of mouth ulceration and rash in the CNI elimination arm.
In conclusion, conversion to an everolimus-based regimen with CNI elimination or minimization in a population of kidney transplant patients a minimum of 6 months and an average of 5.6 years posttransplant was achieved but showed no overall benefit in terms of renal function. On the other hand, CNI elimination did not result in a deterioration of renal function and patients who need to avoid CNI for non-renal reasons can be converted to everolimus in most instances. In terms of safety and tolerability, outcomes were inferior in the CNI elimination and minimization groups compared with controls. Selected patients with good renal function may, however, benefit from switch even at a late stage posttransplant.
MATERIALS AND METHODS
ASCERTAIN was a 24-month, open-label, multicenter study in which maintenance kidney transplant patients were randomized to one of three treatment arms: the control group, in which patients' current CNI-based immunosuppressive therapy was continued unchanged; the CNI elimination group, in which everolimus was initiated with discontinuation of CNI; or the CNI minimization group, in which everolimus was initiated with a predefined reduction in CNI exposure. Randomization, stratified by center, was performed using a validated, automated system. The study was undertaken during February 2005 to August 2009 at nephrology centers in 25 counties after approval by the institutional review board of each center, and was conducted in accordance with the Declaration of Helsinki and Good Clinical Practice. All patients provided written informed consent. The trial is registered at www.clintrials.gov (NCT00170846).
Patients were eligible if they had undergone a primary or secondary renal transplant at least 6 months previously from a living or deceased donor and had renal impairment, defined as calculated GFR 30–70 mL/min (Cockcroft-Gault). At study entry, all patients were required to be receiving CsA with a C2 level more than or equal to 400 ng/mL or tacrolimus with a C0 level more than or equal to 4 ng/mL with or without MPA or azathioprine, and with or without steroids. Key exclusion criteria were receipt of a multiorgan transplant, treated acute rejection within the previous 3 months, presence of de novo or recurrent glomerular nephritis or BK polyomavirus nephropathy, and protein:creatinine ratio more than or equal to 150 mg/mmol.
In the CNI elimination and CNI minimization groups, patients received everolimus 2 mg two times per day from day 1 (the day after randomization), with dose adjustments from week 1 onward to target an everolimus C0 of 8 to 12 ng/mL in the CNI elimination group and 3 to 8 ng/mL in the CNI minimization group. On day 1, the CNI dose was reduced by 20%. In the CNI elimination group, the CNI dose was discontinued when everolimus C0 level was more than or equal to 8 ng/mL. In the CNI minimization group, it was reduced to 70% to 90% below baseline values when everolimus C0 was more than or equal to 3 ng/mL. In the control arm, CNI therapy remained unchanged. Baseline doses of MPA, azathioprine, and corticosteroids, where administered, were continued unaltered in all three groups.
The primary endpoint was mGFR at month 24. Secondary endpoints at month 24 were graft and patient survival, number and severity of BPAR episodes, the change in serum creatinine, 1/creatinine, CrCl (Cockcroft-Gault , Nankivell , and Modification of Diet in Renal Disease formulae ) from baseline.
Study visits took place at baseline (day −7 to −1), weeks 1, 2, 3, 4, and 8, and months 3, 6, 9, 12, 15, 18, 21, and 24. Drug concentrations were measured centrally. GFR was measured by Cr-labeled ethylenediaminetetraacetic acid, Tc-diethylenetriamine pentaacetate, iohexol clearance, inulin clearance, or iothalamate clearance at baseline and at months 12 and 24. The same methodology was used for each individual patient at every time point. Protocol renal biopsy was performed at baseline and at month 24. Biochemical and hematological measurements were recorded at all study visits.
Sample size calculation showed that 128 patients in each treatment group were required for 80% power to detect a mean difference of 5 mL/ min/1.73 m2 in 24-month mGFR between treatment groups assuming a two-sided, significance level of 0.05, a standard deviation of 20 mL/min/1.73 m2 in each group and a drop-out rate of approximately 15%. The primary analysis used analysis of covariance (ANCOVA) with mGFR as the dependent variable and treatment or baseline mGFR as independent variables. A post hoc multivariate analysis of mGFR at month 24 was performed using an ANCOVA model that included the following covariables: recipient age, donor age, time posttransplant, history of diabetes, baseline mGFR, baseline urine protein:creatinine ratio, calcium channel blocker treatment at baseline, and lipid modifying treatment at baseline. Post hoc ANCOVA analyses were also undertaken to investigate the effect of baseline mGFR (<40, 40–50, 50–70, and >70 mL/min/1.73 m2). Further post hoc analyses were performed to assess the impact of baseline CrCl according to the Cockcroft-Gault formula (>50 mL/min or ≤50 mL/min) and the Nankivell formula (>55 mL/min or ≤55 mL/min), and time posttransplant (by tertiles and <1 year, 1–3 years, 3–5 years, >5 years) on the change in mGFR from baseline to month 24 for the two everolimus groups versus controls.
Kaplan-Meier estimates of graft and patient survival were compared between groups using the log-rank test. Between-group comparisons were made using Wilcoxon's rank sum test for continuous variables and Fisher's exact test or chi-square test for categorical variables, as appropriate.
Efficacy analyses were based on the ITT population comprising all randomized patients who provided at least one postbaseline assessment. The primary analysis was performed on the modified ITT population, which included all ITT patients for whom mGFR or CrCl values were available at month 24. The safety population consisted of all randomized, treated patients who provided at least one postbaseline safety assessment.
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The ASCERTAIN Investigators
Argentina: Lorenzo Toselli, San Martin; Australia: Josette Eris, Camperdown; Robert Fassett, Launceston; Randall Faull, Adelaide; David Goodman, Fitzroy; Brian Hutchison, Nedlands; John Kanellis, Clayton; Philip O'Connell, Westmead; Dwarakanathan Ranganathan, Herston; Graeme Russ, Woodville; Michael Suranyi, Liverpool; Rowan Walker, Parkville; Belgium: Eric Goffin, Brussels; Yves Vanrenterghem, Leuven; Canada: Edward Cole, Toronto; Anil Kapoor, Hamilton; Martin Karpinski, Winnipeg; France: Jacques Dantal, Nantes; Lionel Rostaing, Toulouse; Greece: Ioannis Boletis, Athens; Dimitrios Takoudas, Thessaloniki; India: Sudarshan Ballal, Bangalore; George T John, Vellore; Vijay Kher, New Delhi; Raj K Sharma, Lucknow; S Sundar, Bangalore; CM Thiagrajan, Chennai; Italy: Giovanni Cancarini, Brescia; Mario Carmellini, Siena; Piergiorgio Messa, Milan; Gianbenedetto Piredda, Cagliari; Vito Sparacino, Palermo; Sergio Stefoni, Bologna; Netherlands: R J Hene, Utrecht; Norway: Kristian Heldal, Skien; Aud Høyeggen, Oslo; Inger Laegreid, Trondheim; Karsten Midtvedt, Oslo; Einar Svarstad, Bergen; Spain: Manuel Arias, Santander; Frederic Oppenheimer, Barcelona; Daniel Seron, Barcelona; Taiwan: Sheng-Hsien Chu, Lin-Ko; Jong-Da Lian, Taichung; Po-Huang Lee, Taipei; Kuo-Hsiung Shu, Taichung; Wu-Chang Yang, Taipei; Thailand: Kearkiat Praditpornsilpa, Bangkok; Turkey: Faruk Gonenc, Ankara; Alihan Gurkan, Antalya; Huseyin Toz, Izmir.