Most immunosuppressive protocols in liver transplantation are currently based on calcineurin-inhibitors granting excellent graft acceptance yet inducing a number of undesired side effects that can dramatically impact patient mortality, morbidity, and quality of life.
Various studies have demonstrated that calcineurin inhibitor (CNI)-induced renal insufficiency can be partially reversed when CNI-dose is substantially reduced. To provide adequate immunosuppression and avoid increased risk of rejection, mycophenolate mofetil (MMF) can be introduced into immunosuppressive protocols (1, 2).
Karie-Guigues et al. (3) confirmed these findings recently in the TRY study. Orlando et al. (4) demonstrated impressive results of monotherapy with 1.5 g of MMF daily in a prospective trial including 42 liver transplant patients. Some authors, however, have reported considerably more and severe rejection episodes when patients were switched from CNI to MMF monotherapy (5).
Large retrospective trials have demonstrated the strong immunosuppressive virtues of MMF in combination with CNIs (2, 6, 7). Increased incidence of infectious episodes under MMF-therapy has been described; however, currently no convincing prospective data are available concerning this issue (2, 8, 9). Schlitt et al. (10) and Stewart et al. (11) were among the first to publish small studies on MMF monotherapy in liver transplant patients with renal insufficiency (5, 9, 12).
Various small single-center studies have been performed to further evaluate MMF monotherapy (12, 13); however, no larger prospectively randomized trials have been conducted up to this date.
From a total of 150 patients, seven patients withdrew their consent; the main reason was the necessity of increased frequency in outpatient clinic appointments which caused unbearable inconvenience to these patients. One patient was lost to follow-up.
Patient characteristics were largely similar in both study groups (Table 1), mean serum creatinine was elevated in the MMF study group compared with controls (P<0.05). No differences were detected for age, gender, and underlying disease, time of study enrollment, or comorbidities. Of all the 142 patients included in this study, 44 (30.8%) had experienced acute rejection (AR) before study entry: 19 patients in the MMF group (27.1%) and 25 patients in the CNI group (34.2%), this difference was not significant. All these rejection episodes were acute cellular rejection and could be treated successfully by steroid-pulse therapy and increase of CNI levels. The mean time between liver transplantation (LT) and study enrollment was 4.9±3.3 years (CNI group) and 5.7±3.9 years (MMF group).
Five-year survival after study entry was 90% for MMF monotherapy patients and 94% for CNI monotherapy patients. Seven deaths occurred in the MMF group (three malignancies, two cardiac diseases, one sepsis, and one liver failure) compared with four deaths in the CNI group (three tumors and one liver failure), no statistical significance (Fig. 1).
Five years after study entry immunosuppression of all patients was as depicted in Table 2.
Biopsy-proven acute cellular rejection occurred in two patients of the CNI group (2.9%) and eight patients (11.1%) in the MMF monotherapy group (P=0.055). Of the eight rejections in the MMF group, two were grade 1 and six were grade 2 (Banff classification). Mean tacrolimus trough levels were below detection limit, mycophenolic acid plasma levels were not measured. Median MMF dose was 2 g/d, median tacrolimus dose was zero. Seven of the eight rejection episodes occurred within the first 4 months after complete CNI cessation.
Of the two rejections in the CNI group, one was grade 0.5, one was grade 1. For these patients, mean tacrolimus trough level was 5.8 μg/L with a median dose of 5 mg daily.
Rejection was successfully treated by steroid-pulse therapy in all cases for 3 to 5 days (500 mg methylprednisolone daily); for all rejection patients in the MMF monotherapy group, tacrolimus was added with a target trough levels of 5 μg/L. For the patients with rejection in the CNI group, a starting dose of 500 mg/d of MMF was introduced additionally two times per day, increasing it to 2× 1000 mg daily.
Liver biopsy was performed on 122 (86%) patients included in the study during the 5-year follow-up period (mean time point of biopsy 2.5 years) and 20 (14%) refused liver biopsy. None of the patients displayed any characteristics of chronic rejection.
In the MMF, monotherapy group low-dose tacrolimus (target trough level of ≤5 μg/L) was added in 11 cases for patients presenting with moderately elevated transaminases (2× upper limit of normal [ULN]); no liver biopsy was performed in these cases.
Regarding the entire study population no significant difference concerning the course of renal function (glomerular filtration rate [GFR]) could be detected between the two groups (Fig. 2). Serum creatinine levels were significantly higher in the MMF study population at initiation of the study (Table 3); in the further course, the cumulative change of GFR and creatinine values did not differ between the two groups.
Nine patients experienced defined renal complication in the CNI monotherapy group during the course of the study, no patient in the MMF monotherapy group experienced renal complication (P=0.018). Under MMF monotherapy significantly more patients improved their renal function compared with CNI-treated individuals (Table 3). This was especially relevant for patients with impaired renal function (serum creatinine >106 μmol/L) before study entry. Of all patients with compromised renal function before study entry, 19 patients showed improvement of their renal function under MMF monotherapy whereas only one patient improved under CNI monotherapy. Renal function improvement started within 6 weeks after switching to MMF monotherapy and reached a stable level after approximately 4 months in the majority of patients. Impairment of renal function was seen in seven patients under MMF monotherapy and nine patients under CNI monotherapy.
Reasons for Treatment Failure or Change of Immunosuppressive Protocol
In the MMF group, 22 patients received CNI or mammalian target of rapamycin-inhibitors additionally to or instead of MMF monotherapy. Biopsy-proven acute cellular rejection was the reason for CNI addition in eight cases, for 11 patients CNI was added due to significant increase of transaminases (2× ULN) under the suspicion of immunological activation (no aspects of AR in biopsy). Pregnancy, recurrent primary biliary cirrhosis, and tumor occurrence were the reasons for MMF discontinuation in three cases.
In the CNI group, MMF was added after acute cellular rejection in two cases and in one case for increase of transaminases to doubled ULN. Deterioration of renal function (decrease of GFR ≥20%) prompted introduction of MMF and CNI dose reduction in nine patients. New onset diabetes or deterioration of preexisting diabetes caused MMF introduction and CNI tapering in five patients. For one patient, tumor occurrence led to CNI discontinuation.
In the MMF group, six de novo malignancies were detected: one breast cancer, one leukemia, one posttransplant lymphoproliferative disease, one gastric cancer, one prostate cancer, and one pancreas carcinoma. In the CNI group, nine de novo malignancies were recorded: three lung cancers, two laryngeal carcinomas, one colorectal carcinoma, one basal cell carcinoma, and two recurrent liver tumors. No significant difference in incidence or distribution of cancer types could be detected.
No significant difference in number and intensity of infectious episodes was recorded between both study groups. Overall complications were equally distributed between groups with 12.4% (CNI group) and 14.1% (MMF group), respectively. Bacterial complications dominated slightly over viral complications, symptomatic CMV-infection (diarrhea) was seen in two (CNI group) and three (MMF group) patients, respectively.
Eighty-one biopsies were available for all 142 patients at initiation of the study: 39 for the MMF group patients and 42 for the CNI group. No significant difference was detected in distribution of fibrosis.
Five years after study entry, biopsies were available for 39 of 72 patients in the MMF group (54.2%) and 42 patients in the CNI group (60%) without significant differences between groups. In a separate analysis of the subgroup of hepatitis-C positive patients (14 vs. 13 patients), we did not detect differences in progression or severity of fibrosis.
Overall 38 patients experienced deterioration of their preexisting cardiovascular disease: 21 in the MMF group, 17 in the CNI group. Of these cases, 17 of 21 in the MMF group and 14 of 17 in the CNI group were exacerbation of preexisting arterial hypertension; the other seven cases were severe cardiovascular complications such as myocardial infarction, stroke, or lung embolism. Although six patients suffered of new-onset hypertension in the CNI group, only one patient developed hypertension in the MMF group during the course of the study. Four patients (MMF group) compared with three (CNI group) patients saw improvement of their cardiovascular problems when under study monitoring. For 24 patients, no change concerning their preexisting cardiovascular disease status was recorded during the 5-year study period (13 MMF group/11 CNI group). None of these differences were statistically significant.
Gastrointestinal complications were recorded for 24 patients (MMF) vs. 23 (CNI) (P>0.05).
Nine of the 19 diabetic patients in the CNI group experienced deterioration of diabetes, in the MMF group preexisting diabetes worsened in five of 23 patients. Diabetic metabolism improved for three patients in the MMF group and none in the CNI group (P>0.05).
Most published reports concerning MMF monotherapy are of retrospective and nonrandomized nature (9, 12). In the majority of available studies, patients were converted to MMF therapy after CNI-related side effects occurred. Furthermore, histological data for patients under MMF (mono-) therapy are limited. The present study was conducted to reassess the available retrospective data in a prospectively randomized trial investigating safety and efficacy of MMF monotherapy. Potential reduction of CNI-induced side effects was a secondary end point of this analysis.
Although the actual dropout rate of patients was lower than expected, the immunosuppressive protocol had to be modified for a large proportion of patients during the 5-year course of the study. This, of course, complicates straightforward interpretation of the results.
After a 5-year follow-up, we detected an increase in the number of rejection episodes in the MMF monotherapy study group, though this difference was without statistical significance. Taking into account that some patients in our MMF monotherapy group were treated with additional low dose CNIs in the case of elevated liver enzyme values (2× normal) without biopsy-proven diagnosis of rejection, it becomes clear that MMF monotherapy does not provide equally potent immunosuppression as CNI treatment does. However, the majority of our MMF monotherapy patients who experienced AR or liver enzyme increase, interpreted as “immunological activation,” did so shortly after CNI cessation and shift to MMF monotherapy. Once patients had received MMF monotherapy for more than 6 months successfully, we saw only one rejection episode. This fact underlines that the risk for AR is the greatest in the early phase after complete CNI cessation. For this reason, many authors have continued low-dose CNI treatment in their patients treated with MMF 2 g/d for impaired renal function (1, 3, 14, 15). Not only the underlying disease but also the frequency of previous rejection episodes and the cumulative dose of CNI may have a significant impact on the risk of AR. Because of the heterogeneous patient population and limited numbers, we were unable to generate significant predictors of increased AR risk under or after immunosuppression transition.
Regarding one of the major side effects of CNI therapy, nephrotoxicity, we found rather a heterogeneous picture. Although patients with impaired renal function (creatinine clearance <50 mL/min) before study entry improved significantly under CNI cessation and MMF monotherapy, we did not detect a significant change in renal function compared with CNI-treated individuals when looking at the entire study collective. However, this finding is limited by the fact that at the beginning of our study mean creatinine values were elevated for patients in the MMF monotherapy group compared with controls. Furthermore, nine patients who were initially randomized to the CNI treatment group received MMF additionally in the further course due to dramatically deteriorating renal function under CNI monotherapy. We defined these individuals as suffering from “renal complication,” a secondary end point of our analysis, which did not occur in the MMF monotherapy group (P>0.05). Most of these patients stabilized or improved their GFR when CNI dose was reduced and MMF introduced. Improvement of renal function occurred faster and to a greater extent in patients who had a short latency between LT and initiation of MMF monotherapy; however, numbers are too small to draw significant conclusions.
We detected improvement of glucose metabolism in diabetic patients under MMF monotherapy; this difference, however, was short of statistical significance. No significant differences were found concerning the defined cardiovascular or gastrointestinal events.
It becomes clear that the inability of up to 40% of the patients to remain in their assigned study group represents a major limitation of this study. However, neither continuous CNI monotherapy treatment in the case of strongly deteriorating renal function nor withholding stronger immunosuppressants from MMF monotherapy patients in the case of apparent immunological activation would have been ethical.
In 2004, Wiesner et al. analyzed a large collective of patients who had received MMF after LT; although there are some limitations to the study such as only short-term MMF application, the potential assets of MMF to the 4-year survival are suggested to be based on the initial postoperative period up to the 6th month after LT.
Consequently, early introduction of MMF into immunosuppressive protocols provides the opportunity to reduce CNI dosage and thereby CNI-induced side effects. It is unlikely, though, that MMF monotherapy may be an option at this early stage after LT.
Safety results of MMF monotherapy treatment are heterogeneous: although some authors describe fairly low rejection rates (16), others report irreversible rejection episodes with consecutive deaths in their small study (17). A common drawback to most studies is their retrospective, single-center character lacking randomization protocols. Most authors chose a relatively “safe” setting converting patients rather late after LT.
In 2005, Reich et al. (18) published a small prospective randomized study including 27 patients with MMF monotherapy or MMF+low CNI treatment and detected higher rejection rates after cessation of cyclosporine A compared with tacrolimus. We could not confirm these findings in our collective.
Mammalian target of rapamycin inhibitors may become a valuable alternative to MMF treatment for patients with CNI-induced side effects. However, they have not yet been approved for clinical use in LT patients although some recent studies suggest good efficacy and tolerability (19–21).
MMF monotherapy is applicable for a majority of patients in long-term immunosuppression after liver transplantation. Approximately one third of all patients develop immunological activation or even acute cellular rejection after conversion from CNI to MMF monotherapy. This occurs early after conversion to MMF in most cases. CNI-related side effects can be reduced by conversion to MMF monotherapy in liver transplant recipients. Especially for patients with preexisting renal function impairment MMF monotherapy may provide a good alternative.
MATERIALS AND METHODS
A prospective randomized study on the safety and efficacy of long-term conversion of CNI to MMF after liver transplantation was performed. Inclusion criteria were liver transplantation at our center at least 6 months ago, age 18 to 70 years, and liver enzyme values within 2× normal range. Exclusion criteria were renal insufficiency (serum creatinine >177 μmol/L), anemia (Hb <6.2 mmol/L) or thrombocytopenia (<50.000/mL), chronic rejection, or secondary malignancy. The primary endpoint was failure of immunosuppression represented by biopsy-proven acute or chronic rejection. Additionally, for patients whose liver enzymes were only moderately elevated (2× ULN), we introduced the category “immunological activation.” In these cases, no liver biopsy was performed but reasons for liver enzyme elevation other than of immunological nature had been ruled out. For these individuals, immunosuppression was intensified by elevation of CNI dose and addition of MMF (CNI group) or addition of low-dose CNI (MMF group).
The study design included power calculation of 80% with 95% confidence interval and a significance level of P less than 0.05 to detect statistically significant differences of rejection between respective study groups. A study group size of at least 63 patients (Gpower 3.0.5) was determined. We calculated a 20% drop-out rate. Randomization was performed as a standardized simple computerized procedure with a 1:1 ratio for 150 patients after LT with stable organ function (SAS software; SAS Institute Inc., USA) no earlier than 6 months after LT. Patients were recruited exclusively from our center having received LT between 6 months and 7 years before study entry. Not every consecutive LT patient was included in this analysis. Main reasons for not including patients were denial of consent, travel distance to our center, and lack of compliance. All patients had given their informed consent. The study was conducted according to the Helsinki declaration criteria and approved by the local ethics committee.
Renal function development was a secondary end point of this study. Deterioration of renal function requiring a change in the immunosuppressive protocol was defined as renal complication. Deterioration or improvement of renal function was defined as a decrease/increase in GFR of more than or equal to 20% compared with GFR at study onset (proven by three consecutive laboratory results). GFR was calculated using the Modification of Diet in Renal Disease formula (22).
Furthermore, cardiovascular side effects, gastrointestinal events, and diabetes development were recorded. Improvement/impairment of diabetes mellitus were categorized as reduction/increase of insulin dose by more than 20% or reduction/increase of oral antidiabetics. New onset or exacerbation of preexisting arterial hypertension, myocardial infarction, stroke, and pulmonary embolism were classified as cardiovascular complications. Exacerbation/improvement of arterial hypertension was defined as increase/decrease in systolic blood pressure more than 15 mm Hg during three consecutive visits and necessity for change of preexisting medication. Gastrointestinal complications were defined as new onset of consistent abdominal pain, diarrhea, or emesis after the exclusion of other reasons not related to immunosuppression. Infectious complications were also recorded.
A total of 83.6% of all patients included were on tacrolimus monotherapy at the time of study entry; 16.4% received cyclosporine A monotherapy. Steroids were included in immunosuppressive regimens at 10 to 20 mg/d for patients with autoimmune hepatitis or primary sclerosing cholangitis in combination with tacrolimus (n=6). Initially after LT, all patients had received dual immunosuppression with CNI+steroids. Steroids were tapered and eliminated over a 3 months period for all patients except those with autoimmune hepatitis or PSC. All patients had received induction therapy with Basiliximab (20 mg, two times a day on day 0 and day 4).
In the MMF monotherapy group, CNIs were withdrawn over a period of 3 months in five consecutive steps whereas MMF was introduced over a period of 4 weeks (Table 4). During the first 4 months, clinical examination and blood laboratory investigations were performed every 2 weeks, thereafter every 4 weeks.
Clinical check-up included measurement of blood pressure, heart rate, temperature, specific interview for gastrointestinal symptoms (stomach ache, diarrhea, and gastrointestinal bleeding) and interrogation for episodes of infectious diseases. Laboratory evaluation included liver enzymes, renal function parameters, electrolytes, fasting glucose levels, HbA1c, blood cell count, protein, clotting analyses, and CNI trough levels.
Routine liver biopsies were performed for all patients at time points 1, 3, 5, 7, 10, and 13 years after LT and in cases of suspected rejection or hepatitis C virus reinfection. Biopsies were evaluated by an experienced pathologist according to the Banff classification (23). For statistical analysis SPSS version 17.0 (SPSS Inc., Chicago, IL) was used.
The Kolmogorov-Smirnov test was used to prove normality; absolute values were compared by Fisher's exact test and the chi-square test. Survival was calculated using Kaplan-Meier analysis, comparison was performed by log-rank test. Cox regression was applied to check for the influence of individual variables in multifactorial settings.
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