Between April 1986 and June 2022, we performed a total of 2,204 LTs at our institution. For this study, we recorded the data of 324 LT recipients among a total of 1,697 who underwent LT between January 1997 (the first patient included in MMF-MT) and June 2022 and were subsequently converted from immunosuppression with combined CNI-MT or CNI + MMF to MMF-MT. In this retrospective single-center cohort study, we analyzed the impact of MMF-MT on toxicity or adverse effects (CKD, hypertension, diabetes mellitus, and recurrent biliary infection) in patients previously undergoing immunosuppression with CNIs and the incidence of rejection and adverse effects related to the use of MMF-MT.

The inclusion criteria for conversion from CNI-MT to MMF-MT were as follows: patients >18 years usually with more than 2 years of follow-up after LT, stable liver graft function and the absence of acute rejection in the last year before MMF-MT conversion. This study was closed on December 2023, and all patients were followed for at least 1.5 years after conversion to MMF-MT. Medical history of liver retransplantation, previous renal transplant, and hepatocellular carcinoma recurrence were considered exclusionary for this study. Informed consent for MMF-MT was obtained from all patients included in this study.

This research was performed in accordance with the ethical guidelines of the 1964 Helsinki Declaration and its later amendments and was approved by our Institutional Review Board (Research Registry no 24/025). The need for local clinical research ethics committee approval was waived because of the retrospective nature of the research.

The following patient data were retrospectively collected: age, sex, body mass index (BMI), Child‒Pugh and MELD scores, presence of arterial hypertension or diabetes, LT indications, pre-LT recipient laboratory values, type of donors and graft steatosis, type of CNI, rate and grade of post-LT acute rejection, median time elapsed from LT to initiation of MMF-MT and median time on MMF-MT, indications of conversion to MMF-MT, rate of post-MMF-MT acute rejection and therapy, adverse effects related to MMF-MT, causes of MMF-MT withdrawal, and the need for dialysis and kidney transplant during the follow-up of MMF-MT patients.

Determinations were taken of post-MMF-MT laboratory parameters (serum glucose, hematological, kidney and liver function values), doses of MMF and blood levels of MPA throughout the follow-up at different periods [pre-MMF-MT (combined CNI-MMF), 3, 6, and 12 months post-MMF-MT, and at the end of the study]. Comparisons between the variables (diabetes, hypertension, and hematological, kidney and liver function) of pre-MMF-MT patients and the final outpatient review of MMF-MT patients were performed. The sample was divided into two eras (first era: 1999–2011; second era: 2012–2023), that were compared regarding acute rejection, adverse effects, causes MMF-MT withdrawal and patient survival was performed between both groups of patients.

CKD was defined as a GFR <60 mL/min/1.73 m² or markers of kidney damage, or both, of at least 3 months in duration, and estimation of the GFR was performed according to the CKD-EPI equation [27]. Arterial hypertension was defined as a systolic blood pressure >140 mmHg and/or a diastolic blood pressure >90 mmHg on three consecutive measurements within three to 6 months [28]. A diagnosis of diabetes mellitus was established if fasting plasma glucose was ≥126 mg/dL or if 2 h plasma glucose levels were ≥200 mg/dL, according to the ADA criteria [29]. Anemia was defined as hemoglobin <8 g/dL; leukopenia was defined as a white blood cell count <2,500/mm³; and thrombocytopenia was defined as a platelet count <60,000/mm³. The diagnosis of acute rejection was performed by liver graft biopsy or empirically by alteration of liver function tests. Acute rejection was classified according to the Banff grades [30].

The initial immunosuppressive regimen comprised CNI (cyclosporine or tacrolimus) and steroids with or without MMF. Steroids were discontinued between 3 and 6 months post-transplantation. The dose of tacrolimus was adjusted to achieve target blood trough levels of 10–15 ng/mL for the first month, 7–9 ng/mL within the first year, 5 ng/mL between the 2nd and 4th years, and between 4 and 5 ng/mL thereafter. The dose of oral cyclosporine was adjusted to maintain blood trough levels between 200 and 300 ng/mL for the first month and between 150 and 200 thereafter.

In the presence of severe adverse effects associated with CNIs, such as renal dysfunction, diabetes, and hypertension, MMF was introduced to reduce CNI levels by half. Conversion from CNI to MMF-MT was performed on long-term follow-up recipients with stable liver function, starting at a dose of 500 mg of MMF twice daily, which was subsequently increased up to 1 g twice daily, followed by a gradual reduction in CNI until complete withdrawal. For patients on MMF-MT, MMF was administered at a dose capable of maintaining MPA levels between 2–4 ng/mL. Currently, the period from the introduction of MMF to CNI withdrawal is between 1 and 2 months, with posterior review in the outpatient clinic at 15, 30, and 90 days and routine follow-up every 6 months thereafter.

Patients on MMF-MT who showed liver dysfunction or biopsy-proven acute rejection grade I/II were initially treated with increasing doses of MMF (up to 1 g/12 h) to achieve MPA levels between 2–4 ng/mL or with 0.5–1 g of methylprednisolone intravenously for 3 days. Tacrolimus, cyclosporine or mTORi were reintroduced in cases of resistant acute rejection. In the presence of moderate-severe adverse effects, MMF was reduced or withdrawn and CNI was reintroduced. The dose of MMF was adjusted according to the protocol based on blood levels and liver function.

Qualitative variables are expressed as absolute numbers, and relative frequencies are expressed as percentages. Associations were analyzed via the chi-square test or Fisher’s exact test, when applicable. Most quantitative variables did not have a normal distribution according to the Kolmogorov–Smirnov test; therefore, all the quantitative variables are expressed as medians and percentiles and are expressed between 0 and 100. The relationships between quantitative variables were analyzed via the Mann–Whitney U test. A repeated measures model was used, taking each patient as its own comparator, in order to evaluate different key parameters pre and post MMF-MT. Survival analysis was performed via the Kaplan–Meier estimator and the log-rank test. A p value of <0.05 was considered to indicate statistical significance. Statistical analysis was performed via SPSS Statistics, version 25 (SPSS, Inc., Chicago, IL, United States).

From January 1997 to June 2022, a total of 1,697 patients underwent LT and were immunosuppressed with CNI. A group of 324 patients was initially treated with CNI standard immunosuppression (252 with tacrolimus-based and 72 with cyclosporine-based). The median recipient age was 55 (19–70) years, and the median MELD score was 15 (6–35). Alcoholic cirrhosis, hepatitis C virus (HCV) cirrhosis and HCC were the most frequent indications for LT.

Concerning pre-LT laboratory variables, the median serum creatinine value was 1.1 (0.5–1.9) mg/dL, and the median GFR was 70.2 (36–111) mL/min/1.73 m². Livers from donation after brain death (DBD) were used in 289 (89.2%) patients, and livers from donors with uncontrolled circulatory death (uDCD) were used in 16 (4.9%) patients. The remaining characteristics of the recipients and donors are detailed in Table 1.

Immediately after LT, tacrolimus-based immunosuppression was used in 252 (77.8%) patients vs. the 72 (22.2%) who received cyclosporine-based immunosuppression. One episode of acute rejection after LT occurred in 90 (27.8%) patients, and two or more episodes in 23 (7.1%), with rejection grades I-II appearing in 106 (32.7%) patients.

The median time from LT to the initiation of MMF-MT was 67 (5–338) months, whereas the median time from the initiation of the combined MMF-CNI or CNI-alone regimen to CNI withdrawal and switch to MMF-MT was 18 (0–170) months. The overall median follow-up time of patients on MMF-MT was 68 (1–231) months.

The indication for switching from CNI to MMF-MT was CKD in 215 (66.4%) patients (CKD on its own in 88 patients and associated with hypertension in 55 patients, diabetes and hypertension in 46 patients, and associated with diabetes in 26 patients), diabetes mellitus on its own in 61 (18.8%) patients, hypertension in 42 (13%) patients, and recurrent biliary infection in 6 (1.8%) patients.

Just before shifting from tacrolimus to MMF-MT, 228 (70.3%) patients were on tacrolimus immunosuppression (associated with MMF in 220 patients and monotherapy in 8 patients), and 96 patients were on cyclosporine immunosuppression (associated with MMF in 91 patients and monotherapy in 5 patients).

Twenty-four (7.4%) patients experienced acute rejection after conversion from CNI to MMT-MT; 14 (4.3%) patients were diagnosed by liver biopsy (grade I/II in 13), and 10 (3.1%) were empirically diagnosed by liver dysfunction. All patients responded completely to rejection therapy (steroids and reintroduction of tacrolimus [20 patients], cyclosporine [1 patient], or mTORi [3 patients]). Forty-eight (14.8%) patients developed adverse effects related to MMF-MT, with the most common diarrhea (5.6%), vomiting (1.9%), and leukopenia (5.9%).

MMF-MT withdrawal was performed in 42 (12.9%) patients, due to de novo tumors in 13 (4%) patients (substitution of MMF-MT by mTORi monotherapy), biopsy-proven rejection in 8 (2.5%), liver dysfunction in 6 (1.8%), liver retransplantation in 4 (1.2%), kidney retransplantation in 3 (0.9%) and adverse effects in 8 (2.5%). With respect to side effects, MMF-MT withdrawal was performed in 5 (1.5%) patients with persistent chronic diarrhea despite a change from MMF to mycophenolate sodium salt and in 3 (0.8%) patients with leukopenia (Table 2). The remaining patients with adverse effects improved with a reduction in MMF dosage.

The dosage of MMF was adjusted according to MPA plasma levels, resulting in great variability among patients. The overall daily dose of MMF and median MPA plasma levels in patients on combined CNI-MMF therapy (just before conversion to MMF-MT) and in MMF-MT patients after 3, 6, and 12 months and at the last outpatient review (median period of 78 months) are detailed in Table 3, where it can be observed that MPA median plasma levels were similar in the pre-MMF-MT period or combined CNI-MMF [2.6 (0.1–15 ng/dL)] and at the last outpatient review [2.7 (0.2–15) ng/dL; p = 0.527].

The frequency of diabetes and hypertension and laboratory values of hematological, renal and liver function during long-term follow-up are shown in Table 4. In the comparison of the median values of variables between the pre-MMF-MT period (combined MMF-CNI or CNI alone) and the last outpatient review on MMF-MT (median of 78 months), only the median GFR value was significantly greater in the last review on MMF-MT [56 (15–126) mL/min/1.73 m² vs. 61 (7–134) mL/min/1.73 m²; p = 0.001], whereas the frequency of diabetes and hypertension and laboratory values of hematological variables, serum creatinine and liver function (AST and ALT) did not show significant differences between the two periods (Figures 1, 2). The 6 patients who were switched to MMF-MT due to biliary infection did not experience any new episodes of recurrent biliary cholangitis. On the other hand, using a repeated measures model, taking each patient as its own comparator, we found a statistically significant increase in GFR, statistically significant decrease in creatinine and statistically lower value of ALT at the last outpatient review in comparison with the pre-MMF-MT period (combined MMF-CNI or CNI alone) (Table 4).

Concerning comparison between both eras, we observed a significantly higher incidence of hypertension (p = 0.019) and diabetes mellitus (p = 0.023) before LT in the first era, and a significantly higher incidence of HCC (p < 0.001) in the second era, showing significant differences (p < 0.001) between the eras regarding indications of conversion to MMF-MT (Table 5).

The overall actuarial patient survival rates at 1, 3, 5, and 10 years after the onset of MMF-MT were 95.7%, 86.5%, 75.3%, and 54.6%, respectively (Figure 3). The actuarial patient survival rate at 1, 3, 5, and 10 years after the onset of MMF-MT were 93.8%, 82.3%, 70.1%, and 51.9%, respectively, in era 1, whereas in the second era patient survival rate was 97.9%, 91.8%, 80.9%, and 64.3%, respectively. (p = 0.089) (Table 5).