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ORIGINAL ARTICLE
A prospective randomized trial comparing tacrolimus
and steroids with tacrolimus monotherapy in liver
transplantation: the impact on recurrence of hepatitis C
Carlos Margarit,
1
Itxarone Bilbao,
1
Lluis Castells,
2
In
˜igo Lopez,
1
Leonor Pou,
3
Elena Allende
4
and Alfredo Escartin
1
1 Liver Transplantation Unit, Department of General Surgery, Hospital Vall Hebro
´n, Universidad Auto
´noma Barcelona, Barcelona, Spain
2 Hepatology Unit, Department of Internal Medicine, Hospital Vall Hebro
´n, Universidad Auto
´noma Barcelona, Barcelona, Spain
3 Department of Biochemistry, Hospital Vall Hebro
´n, Universidad Auto
´noma Barcelona, Barcelona, Spain
4 Department of Pathology, Hospital Vall Hebro
´n, Universidad Auto
´noma Barcelona, Barcelona, Spain
Introduction
Steroids, which cause important side-effects, are still used
for induction as well as for maintenance immunosuppres-
sion in the majority of transplant centres [1]. With the
introduction of cyclosporin, steroid dosage could be
successfully reduced or even withdrawn in some individu-
als, particularly children who were likely to suffer severe
side-effects such as growth retardation [2,3]. Few investi-
gators have been willing to risk induction therapy free of
steroids [4–6] because of their extremely low cost, famili-
arity with their management and their effectiveness in
Keywords
hepatitis C virus patients, immunosuppression,
liver transplantation, steroid-free, tacrolimus.
Correspondence
Carlos Margarit MD, PhD, Liver
Transplantation Unit, Hospital General Vall
Hebro
´n, 08035 Barcelona, Spain. Tel.: 34
932746113; fax: 34 932746112; e-mail:
cmargarit@vhebron.net
Received: 3 November 2004
Revision requested: 25 March 2005
Accepted: 31 August 2005
doi:10.1111/j.1432-2277.2005.00217.x
Summary
The aim of this prospective randomized trial was to study the efficacy and
safety of tacrolimus monotherapy (TACRO) and compare it with our standard
treatment of tacrolimus plus steroids (TACRO + ST) after liver transplant
(LT). Furthermore, the impact of steroid-free immunosuppression on outcome
of hepatitis C virus (HCV) was analysed. Between 1998 and 2000, 60 patients
(mean age: 57 years) were included in the study and randomized to receive
TACRO (n¼28) or TACRO + ST (n¼32). Indication for LT was postnec-
rotic cirrhosis in all cases (58.3% were HCV-positive). Mean follow-up was
44 months. Survival, incidence of rejection, infection and side-effects were
compared between the two groups. In patients with HCV infection, incidence
and severity of acute hepatitis C, long-term outcome of recurrent hepatitis C
and survival were studied in an intention-to-treat analysis or in the real group
analysis (real-TACRO versus real-TACRO + ST). Patient survival at 1, 3 and
5 years, tacrolimus pharmacokinetics, incidence of rejection infections and
side-effects were similar. In patients with HCV, the incidence and severity of
graft hepatitis C tended to be lower in TACRO (47%) compared with TAC-
RO + ST (67%) (P¼NS), and also in real-TACRO (42%) compared with
real-TACRO + ST (61%) (P¼NS). A poor outcome considered as evolution
to cirrhosis at 3 years was observed in one (9%) living patient in real-TACRO
and nine (45%) in real-TACRO + ST (P¼0.04). Patient survival at 1, 3 and
5 years was 92%, 92% and 73% for real-TACRO and 78%, 61% and 51% for
real TACRO + ST (P¼0.07). Steroid-free immunosuppression appears to be
safe and efficacious. The main advantage of this regimen could be in HCV
patients, as recurrence of hepatitis in the graft was less severe in the group of
patients in whom steroids could be avoided completely.
Transplant International ISSN 0934-0874
1336 Transplant International 18 (2005) 1336–1345 ª2005 European Society for Organ Transplantation
control of rejection. However, early cessation of steroid
therapy at 3 months post-liver transplantation (LT) [7,8],
or as early as 15 days post-LT [9], has demonstrated a
reduction in and better control of side-effects such as
arterial hypertension, hypercholesterolaemia, diabetes and
other complications. The next logical step was to try to
avoid steroids from the beginning. Several pilot studies,
and few randomized trials, have explored this possibility
with mixed results [4–14]. Other studies explored a ster-
oid-free regimen including a calcineurin inhibitor with
mycophenolate mofetil (MMF) [6], rapamicine [10], rab-
bit antithymocyte globulin plus MMF [2] or MMF and
daclizumab [14]. The incidence of rejection with these
associations was lower, between 8.5% and 28%, than with
calcineurin inhibitor monotherapy. Therefore, the ques-
tion remains as to whether a short course of steroids in
the peritransplant period is safe or an unnecessary risk.
Hepatitis C viral (HCV) cirrhosis is currently the main
indication for LT; unfortunately, recurrence of HCV
infection in the graft post-LT is universal. Ten to twenty
per cent of patients develop severe cholestatic hepatitis
with a high proportion of early graft failure soon after
LT, whereas around 40% of patients present cirrhosis at
5 years post-transplant. Consequently, long-term patient
survival in hepatitis C patients is lower than other indica-
tions [15–17]. Among other factors, the severity of graft
hepatitis and poor outcome after LT have been related to
steroid administration for prophylaxis and treatment of
rejection.
Objectives
The main purpose of the present prospective randomized
trial was to assess the efficacy and safety of tacrolimus
monotherapy without steroids in LT, and compare the
results with our standard treatment of tacrolimus plus
steroids. Efficacy was assessed by (i) patient and graft sur-
vival at 1, 3 and 5 years, (ii) incidence and cause of early
and follow-up mortality and (iii) incidence and severity
of rejection during the first 3 months post-transplant.
Safety was assessed by the rate of infectious complications
and side-effects such as renal insufficiency, arterial hyper-
tension, diabetes, dyslipaemia, neurotoxicity and other
complications during the first 3 months post-transplant.
The second important objective was to study the impact
of steroid-free immunosuppression on the outcome of
HCV recurrence post-LT.
Patients and methods
Over a 2-year period between October 1998 and Septem-
ber 2000, a total of 82 adult patients underwent LT in
our Unit. Institutional review board approval for the
study was obtained and all patients provided written
informed consent before enrolment into each of the
groups with 1:1 randomization. Of the potential patients
available over the recruitment period, 22 liver transplant
recipients were excluded. The reasons for exclusion were
combined liver–kidney transplant (n¼5), re-transplants
(n¼6), renal failure pretransplant (n¼4), fulminant
liver failure (n¼3) and pre-LT oral consumption of
steroids (n¼1). Three patients were excluded after rand-
omization because of perioperative death (n¼2) and
positive cross-match (n¼1). Finally, 60 patients were
randomized to receive tacrolimus alone (TACRO) or tacro-
limus plus steroids (TACRO + ST), just before the surgical
procedure.
Patients
Patient demographic characteristics and indications for
LT are presented in Table 1. Mean age of patients was
57 years; 50% were over 60 years of age, and there was a
male predominance. All patients suffered from end-stage
liver cirrhosis or hepatocellular carcinoma (HCC) in cir-
rhosis. The main aetiology of cirrhosis was HCV infection
(58%), with or without HCC.
Donors
The mean age of the patients was 43 years and mean cold
ischaemia time was 8 h.
Operation
Recipient hepatectomy with inferior vena cava preserva-
tion was performed in all cases. Choledocho-choledochos-
tomy without T-tube was performed in 63.5% of cases. A
500 mg steroid bolus was administered to all patients
during the anhepatic phase before liver reperfusion.
Immunosuppression and drug measurements
Tacrolimus treatment was started at the end of the opera-
tion and administered through a nasogastric tube at
0.05 mg/kg body weight every 12 h in both groups. Tacro-
limus dosage was set to achieve a trough level between 10
and 15 ng/ml over the first few weeks and between 8 and
12 ng/ml thereafter. Tacrolimus trough levels were meas-
ured daily during the hospital stay and at each outpatient
visit. Pharmacokinetic studies were performed on days
3 and 9 post-LT. Whole blood samples were taken at 1, 2, 4
and 6 h postdose and predose for calculation of C
min
,C
max
and area under the curve (AUC
0)12
) of tacrolimus. In
patients randomized to TACRO + ST, a steroid taper was
instituted starting at 100 mg b.i.d. of methylprednisolone
Margarit et al.Tacrolimus monotherapy in liver transplantation
Transplant International 18 (2005) 1336–1345 ª2005 European Society for Organ Transplantation 1337
post-LT day 1 and decreasing to 20 mg/day by day 6.
Patients were weaned off prednisone, if possible, within
3 months post-LT.
Diagnosis and treatment of rejection
Acute rejection episodes were diagnosed by alteration in
liver function tests and confirmed by liver biopsy. The
Banff 97 criteria [18] were used for histopathological
diagnosis. Ultrasound examination was used to rule out
technical complications. Acute rejection was treated by
increasing tacrolimus doses if levels were under 15 ng/ml,
together with steroid therapy. A 3-day 500 mg i.v. bolus
of methylprednisolone was administered followed by a
steroid taper from 200 to 20 mg/day over 6 days in cases
of severe rejection. When liver graft dysfunction persisted
despite steroid pulses, administration of 1–2 g/day of
MMF was started. Orthoclone monoclonal antibodies
against T lymphocytes was not used in any case. Severe
acute rejection was defined when there was an incomplete
response to steroid treatment and other immunosuppres-
sive drugs, such as MMF or rapamicine, were necessary
to control rejection. Refractory rejection was recorded
when no response was obtained, and re-transplantation
or death were derived from rejection.
Definition of side-effects
Renal insufficiency was classified as mild (creatinine
>1.5 mg/dl), moderate (creatinine >3 mg/dl) and severe
(need for dialysis or extrarenal depuration) [19]. Arterial
hypertension was recorded if treatment with drugs was
required to control blood pressure values. Diabetes was
defined as the need for insulin for more than 1 week,
when the patient was not receiving total parenteral nutri-
tion or i.v. glucose infusion. Hypercholesterolaemia and
hypertriglyceridaemia were defined as values over 300 and
280 mg/dl, respectively, in two consecutive analyses separ-
ated by at least 1 week. Only bacterial, viral and fungal
infections that required active treatment were considered
as infections.
Hepatitis C viral infection
Viral load and genotype were evaluated pretransplanta-
tion. HCV RNA levels were measured at 1 week, 2 weeks,
1 month, 3 months, 6 months, 9 months and 1 year
post-transplantation. Complete data were available in 31
of 35 HCV-positive patients: 17 in the TACRO and 14 in
the TACRO + ST groups. Results are expressed as log
10
HCV RNA copies/ml serum [20]. Diagnosis of recurrent
hepatitis C in the graft was based on elevation of liver
enzymes together with histologic confirmation from liver
biopsy tissue. Mild hepatitis was recorded for patients
with transaminase concentrations <400 IU, total bilirubin
(TB) <2 mg/dl and with no general symptoms such as
malaise, fever and tenderness of the right upper abdomen.
Moderate hepatitis was recorded when transaminases were
>400, TB >2 but <10 mg/dl and no symptoms. Severe
hepatitis was considered when TB >10 mg/dl and with
symptoms. The clinical progress of HCV-positive patients
at 3 years, or at the end of follow up, was monitored
using the measurement of liver enzymes, liver biopsy and
clinical symptoms. Liver function was considered normal
Table 1. Characteristics of recipients, donors, surgery and post-trans-
plant complications.
Characteristic
TACRO
(n¼28)
TACRO + ST
(n¼32) P-value
Recipient
Mean age 57 ± 7 56 ± 8 NS
Patients over 60 years 15 (54) 14 (44) NS
Male/female 18/10 25/7 NS
Diagnosis
HCV 20 (71) 15 (47) 0.05
ETOH 5 11
HBV 3 2
Cryptogenic 0 2
Haemochromatosis 0 2
HCC over cirrhosis 13 (46) 11 (34)
Child–Pugh A/B/C (%) 18–28–54 12–37–51 NS
Donor
Mean age (years) 42 ± 18 45 ± 19 NS
Graft steatosis 3 (11) 11 (35) 0.037
Surgery
Mean RBC units 6.3 ± 5 5.7 ± 4 NS
Cold ischaemia time(min) 498 ± 118 481 ± 120 NS
Post-transplant
Mod/severe ischaemic injury 7 (25) 7 (22) NS
Mean post-op stay (days) 26 ± 14 36 ± 17 NS
Re-transplantation (%) 0 5 (15) NS
Mortality 7 9
Early (<3 months)
PDF 1 2
Sepsis-MOF 2 1
Late (>3 months)
HCV recurrence 3 3
HCC recurrence 1 0
De novo tumour 0 2
HAT-retx 0 1
Patient survival (%)
1, 3 and 5 years 85, 81, 66 84, 78, 73 NS
Graft survival (%)
1, 3 and 5 years 85, 81, 66 75, 60, 60 NS
Percentage values are given in parentheses.
HCV, hepatitis C virus; ETOH, cirrhosis due to alcohol; HBV, hepatitis
B virus; HCC, hepatocellular carcinoma; RBC, red blood cells; PDF, pri-
mary dysfunction; MOF, multiorgan failure; HAT, hepatic artery
thrombosis.
Tacrolimus monotherapy in liver transplantation Margarit et al.
1338 Transplant International 18 (2005) 1336–1345 ª2005 European Society for Organ Transplantation
when transaminases and total bilirubin were lower than
twofold normal values. Liver biopsies were performed
when clinically indicated (alteration of liver function tests
or at time of death) and protocol liver biopsies were per-
formed to almost all HCV patients at 3 years. Histology
grading according to the Ishak K classification system was
used [21]. Chronic hepatitis grading score (scale of 0–18)
representing necro-inflammatory activity was the sum of
piecemeal necrosis score (0–4), confluent necrosis score
(0–6), focal lytic necrosis, apoptosis and focal inflamma-
tion score (0–4) and portal inflammation score (0–4).
Chronic hepatitis fibrosis score (0–6) was based on the
extent of fibrosis and the development of cirrhosis. At
the close of the study, ‘good outcome’ was considered if
the patient had normal liver function or chronic active
hepatitis shown on liver biopsy, and ‘poor outcome’ if
the patient had compensated or decompensated cirrhosis
or death related to the HCV recurrent cirrhosis. Risk fac-
tors for poor outcome of HCV recurrence were analysed.
Data analysis
The SPSS software program was used throughout. Stu-
dent’s t-test or the Mann–Whitney U-test were used for
quantitative data and Pearson’s chi-square or Fisher’s
exact test for categorical data. Differences were consid-
ered statistically significant when the P< 0.05. Data are
shown as the mean ± SD, or as percentages. The
Kaplan–Meier method was used for survival analysis.
Demographic data of recipient and donor and surgery-
related factors were compared to assess comparability
of the two treatment groups (TACRO and TAC-
RO + ST). End points between groups were compared
to evaluate efficacy and safety. To evaluate the impact
of a steroid-free protocol in hepatitis C-positive
patients, this subgroup of patients was analysed inde-
pendently and special attention paid to incidence and
severity of rejections, recurrence of hepatitis C in the
graft and toxicities directly related to immunosuppres-
sion. In HCV-positive patients, both treatment groups
were compared as intention-to-treat (TACRO versus
TACRO + ST) and as real groups: patients who
received steroids either for protocol or for rejection
(real-TACRO + ST) and those who received no steroids
at all during follow up (real-TACRO). A Cox regression
analysis of risk factors for poor outcome of HCV
recurrence was performed, including recipient, donor,
surgery, immunosuppression and postoperative factors.
Results
Randomization assigned 28 patients to the TACRO group
and 32 patients to the TACRO + ST group. Demographic
characteristics, clinical indications, donor characteristics
and surgical variables are presented in Table 1. No differ-
ences were found between treatment groups except for a
higher incidence of graft steatosis in TACRO + ST and of
HCV-positive patients in the TACRO group. Primary
graft dysfunction secondary to ischaemic–reperfusion
injury that could affect tacrolimus metabolism and phar-
macokinetic parameters was similar in both groups. The
majority of technical complications and re-interventions
were of biliary origin.
Patient and graft outcome
Actuarial survival at 1, 3 and 5 years was 85%, 81% and
66% in the TACRO group and 84%, 78% and 73% in the
TACRO + ST group. Seven deaths occurred in the TAC-
RO group: three early (<3 months) and four late deaths
(three of them because of hepatitis C recurrence after
1 year). There were nine deaths in the TACRO + ST
group: three early and six late deaths (three of them
because of hepatitis C recurrence, two in the first year
post-LT and one after 1 year). Mean follow-up was
44 months (range: 3–60) and all patients have been fol-
lowed up for more than 4 years. Five re-transplants in
four patients were performed during follow up, all of
which were in the TACRO + ST group, and were the
result of primary non-function (in two patients), hepatic
artery thrombosis (two re-transplants in the same patient)
and HCV recurrence (in one patient).
Incidence of rejection
Incidence of acute rejection and severe acute rejection
was similar in TACRO versus TACRO + ST (39% vs.
32% and 14% vs. 9%, P¼NS; see Table 2). Rejection
with low tacrolimus levels was found in one patient of
the TACRO group and two patients of the TACRO + ST
group. Seven patients presented severe acute rejection and
received a mean accumulative MMF dose of 13 g/patient
in the TACRO group versus 20 g/patient in the TAC-
RO + ST group. Steroid withdrawal in these seven
patients was possible in the sixth month for the TACRO
group and in the fourth month for the TACRO + ST
group.
Incidence of infections, toxicity and other side-effects
During the first 3 months post-LT, 26 episodes of infec-
tions occurred in the TACRO group (62% bacterial, 23%
viral and 15% fungal) and 30 in the TACRO + ST group
(40% bacterial, 37% viral and 23% fungal); (P¼NS). All
cytomegalovirus (CMV) infections appeared in patients of
the TACRO + ST group: positive CMV antigenaemia was
Margarit et al.Tacrolimus monotherapy in liver transplantation
Transplant International 18 (2005) 1336–1345 ª2005 European Society for Organ Transplantation 1339
found in three patients and only one patient developed
CMV disease with pneumonitis and sepsis. All episodes of
fungal infection were the result of Candida.
No statistically significant differences were found
regarding side-effects and toxicity, although trends
towards a higher incidence of renal insufficiency were
observed in the TACRO group and arterial hypertension
and de novo diabetes mellitus in the TACRO + ST group.
With regard to neurological complications, three patients
in each group had tremors.
Conversion to MMF (n¼3) because of renal insuffi-
ciency, rapamicine (n¼1) due to refractory ascites plus
renal insufficiency, or neoral (n¼1) due to intestinal
problems, was required in two patients in TACRO group
and three patients in the TACRO-ST group.
Pharmacokinetic measurements
Pharmacokinetic studies were available for 21 patients in
the TACRO and 24 in the TACRO + ST study groups
(Fig. 1). A trend was observed towards higher trough lev-
els of tacrolimus with lower doses in the monotherapy
group, probably because of steroid induction on cyto-
chrome P450 system. However, tacrolimus doses, trough
levels and AUC were similar in both groups. Comparison
of tacrolimus pharmacokinetic profile between patients
with and without rejection showed no significant differ-
ences.
Table 2. Rejections, infections and
toxicity comparing TACRO versus
TACRO + ST over the first 3 months
post-transplant.
Event TACRO (n¼28) TACRO + ST (n¼32) P-value
Rejections
Patients with acute rejection 11 (39.3) 10 (32.3) NS
Number of episodes 12 11
Methyl-prednisolone (g)/episode 1.5 1.5 NS
Steroid-sensitive rejection 8 (28.57) 8 (25) NS
Rejection requiring MMF treatment 4 (14.28) 3 (9.37) NS
Refractory rejection and chronic rejection 0 0
Patients without steroids 3, 6, 12 months (%) 64, 78, 93 19, 44, 86
Conversion to MMF, neoral 2 (7) 3 (9.3)
Episodes of Infections (3 months) 28 34 NS
Bacterial/viral/fungal (%) 62/23/15 40/37/23
Side-effects and toxicity
Pre-LT renal failure 3 (11) 4 (12.5) NS
Renal insufficiency 20 (71.4) 17 (53) NS
Mild/moderate/severe (dialysis) 14/0/6 10/2/5
Pre-LT arterial hypertension 3 (10.7) 6 (18.8) NS
De novo arterial hypertension 1 (3.5) 3 (9.6) NS
Pre-LT diabetes 6 (21.4) 5 (15.6) NS
De novo diabetes mellitus 2 (7.1) 6 (18.7) NS
Dyslipaemia 5 (17.8) 4 (14.2) NS
Neurologic complications 9 (32) 10 (31.3) NS
Diarrhoea 4 (14.2) 5 (15.6) NS
Percentage values are given in parentheses.
Doses (mg/kg/day)
Tacrolimus Tacrolimus + steroids
Trough levels (ng/ml)
P = ns 3rd day 9th day
Cmin Cmax AUC (0–12 h) Cmin Cmax AUC (0–12 h)
Tacrolimus (range)
Tacro + steroids
(range)
13.1
(3.9–31)
22
(8–54.4)
205.3
(80–479)
12
(5.2–20)
22.5
(11–51.2)
183
(107–367)
12.3
(8.8–21)
33.5
(14–58)
217.2
(130–353)
13.2
(4.4–24)
31.5
(8.2–66)
229.6
(62–416)
0.20
0.15
0.10
0.05
25
30
20
15
10
5
1 3 5 7 9 11 13 15 17 19 25 31 60 90 180
Days
1 3 5 7 9 11 13 15 17 19 25 31 60 90 180
Days
Figure 1 Comparison between mean doses and trough levels in
TACRO and TACRO + ST during the first 6 months post-transplant.
C
min
(ng/ml), C
max
(ng/ml) and AUC
0)12 h
(ng h/ml) at third and ninth
days post-transplant.
Tacrolimus monotherapy in liver transplantation Margarit et al.
1340 Transplant International 18 (2005) 1336–1345 ª2005 European Society for Organ Transplantation
Hepatitis C virus recurrence
Thirty-five patients were HCV-positive: 20 were random-
ized to the TACRO and 15 to the TACRO + ST groups
(see Table 3). Three patients in the TACRO group and
one patient in the TACRO + ST group died within
3 months post-transplant from unrelated causes and were
excluded from the analysis because the main end point
was to evaluate the evolution of HCV graft re-infection.
Mean age was 59 ± 6 years and distribution of HCC over
cirrhosis were similar in both groups. Incidence of acute
rejection, CMV infection and side-effects was similar in
both groups. Postoperative mortality and patient and
graft survival showed no statistically significant differ-
ences. All re-transplantations were performed in the TAC-
RO + ST group.
Complete viral load and genotype were available in 31
of the 35 HCV-positive patients: 17 in the TACRO group
and 14 in the TACRO + ST group. All patients were
HCV genotype 1b, except one patient who was type 1a,
and one who was type 1a-b. Quantification of HCV RNA
pre-LT and throughout the first year was similar in both
groups when analysed on an intention-to-treat basis.
Clinical hepatitis C recurrence in the graft showed a
trend towards a higher incidence in TACRO + ST with
10 patients (71%) compared with nine patients (53%) in
the TACRO group (P¼NS). Graft status at 3 years, as
assessed by liver function, clinical symptoms and histolo-
gical findings, showed no statistically significant differ-
ences. Protocol liver biopsies were performed in all but
six cases (three in each group), the reason being the
excellent clinical status and normal liver enzymes during
follow up. Hence, with respect to histological findings,
there were 14 patients in the TACRO group and 11
patients in the TACRO + ST group. A good outcome was
observed in 76% of patients in the TACRO group versus
57% in the TACRO + ST group (P¼NS) (Table 3).
A comparison was then made between the 12 patients
who received no steroids at all (real-TACRO) and the 23
patients who received steroids (real-TACRO-ST) either
from the beginning (randomization assignment, n¼15)
or to treat rejection (n¼8). (Table 4). Viral load was
lower for real-TACRO and statistically different from the
period of 2 weeks to 3 months post-transplant. Recur-
rence of hepatitis C virus in the graft showed a trend
towards a lower incidence in patients with no steroids at
all: five patients (45%) in real-TACRO, all of whom were
classified as mild hepatitis, compared with 14 (70%) in
real-TACRO-ST classified as mild (n¼7), moderate
(n¼5) and severe (n¼2). These differences were not
statistically significant (P¼0.06). Graft status at 3 years
or at the end of the follow-up period indicated more
cases of poor outcome: nine patients (45%) in the real-
TACRO + ST group developed cirrhosis or HCV-related
death compared with one (9%) in the real-TACRO group
(P¼0.046). The six patients who died as a result of
HCV recurrence belonged to the real-TACRO + ST
group. With respect to histological findings, necro-
inflammatory activity did not differ. The only statistically
significant difference (P¼0.005) was observed in fibrosis
score: 1.78 ± 1 (n¼9) vs. 3.73 ± 1.94 (n¼15). Patient
survival at 1, 3 and 5 years was 92%, 92% and 73%
for real-TACRO versus 78%, 61% and 51% for real-
Table 3. Demographic characteristics, incidence of rejection, infec-
tions, side-effects and mortality in hepatitis C virus patients.
TACRO TACRO + ST P-value
Demographic characteristics (n)20 15
Early deaths <3 months
excluded (n)
17 14
Viral load pre-LT HCV-RNA 5 ± 1.2 4.7 ± 1.3 NS
1 week 4.6 ± 2 5.4 ± 1.6
2 weeks 4.6 ± 2 5.4 ± 2.3
1 month 5.2 ± 2 5.5 ± 2
3 months 5.4 ± 2 6 ± 2
6 months 5.7 ± 2.2 5.8 ± 1.8
9 months 5.8 ± 1.7 5.5 ± 2
12 months 5.6 ± 1.2 5.9 ± 2
Postoperative outcome
Acute rejection 8 (42) 7 (46) NS
Severe AR requiring MMF 3 0
Recurrence of HCV in graft (n)17 14
Acute graft hepatitis C 9 (53) 10 (71) NS
Mild (AST <400, TB <2) 7 (41) 5 (36)
Moderate (AST >400, TB>2) 2 (12) 3 (21)
Severe (TB >10 + symptoms) 0 2 (14)
No hepatitis 8 (47) 4 (29)
IFN-ribavirin treatment 1 3
Graft status at 3 years (n)17 14
Mean follow-up in months 43 ± 12 39 ± 15 NS
(a) Normal liver function test 6 (35) 5 (36)
(b) Chronic active hepatitis 7 (41) 3 (21)
(c) Compensated cirrhosis 1 (6) 2 (15) NS
(d) Decompensated cirrhosis 0 1 (7)
(e) HCV-related death 3 (18) 3 (21)
a + b (good outcome) 13 (76) 8 (57) NS
c + d + e (poor outcome) 4 (24) 6 (43)
Histology findings (n)14 11
Piecemeal necrosis score 1.77 ± 0.93 2 ± 1.18 NS
Confluent necrosis score 2.46 ± 0.88 2.55 ± 1.21 NS
Focal lytic necrosis/inflam. 2.31 ± 0.63 2.27 ± 0.90 NS
Portal inflammatory score 1.85 ± 0.69 1.91 ± 0.94 NS
Fibrosis score 2.77 ± 2 3.27 ± 1.8 NS
Survival
Patient survival (%)
1, 3, 5 years 89, 83, 61 81, 61, 61 NS
Re-transplantation
Technical 0 2
HCV recurrence 0 1
Percentage values are given in parentheses.
Margarit et al.Tacrolimus monotherapy in liver transplantation
Transplant International 18 (2005) 1336–1345 ª2005 European Society for Organ Transplantation 1341
TACRO + ST, almost reaching statistical significance
(P¼0.07).
Risk factors for poor outcome of HCV recurrence
Recipient age over 60 years, graft steatosis, pre-LT viral
load higher than 5 (log
10
HCV RNA copies/ml) and treat-
ment with steroids (either at baseline or subsequently)
were found to be statistically significant for poor outcome
of HCV recurrence by univariate analysis (Table 5).
Discussion
In our experience, immunosuppression regimen with
tacrolimus in monotherapy after LT is feasible and safe.
Patient survival, acute rejection rates and immune graft
loss were similar to our standard regimen of tacrolimus
and steroids. Sixty per cent of patients in the study group
never received steroids in the post-LT period whereas
40% were treated with steroids for rejection. Steroid with-
drawal was started at 3 months post-LT in the TAC-
RO + ST group, and at 10 months of follow-up 90% of
patients in both groups were free of steroids. The inci-
dence of rejection in our study was lower than in previ-
ous studies with monotherapy with either neoral or
tacrolimus [4,5], probably because of the high proportion
of older patients, all of whom had postnecrotic cirrhosis
mainly of alcohol or HCV origin. It is well known that
senior and alcoholic patients have lower immunoreactivity
and a lower incidence of acute rejection. This protocol
may not be applicable to younger patients with
autoimmune or cholestatic diseases who have higher
Table 4. Graft hepatitis analysed as ‘real’ groups receiving and not
receiving steroids.
Real-TACRO
(n¼12)
Real-TACRO +
ST (n¼23) P-value
Viral load RNA-VHC
Pre-LT 4.7 ± 1.5 4.9 ± 1.5 NS
1 week 4.1 ± 2.3 4.1 ± 1.6 NS
2 weeks 3.6 ± 2 5.6 ± 1.8 0.02
1 month 4.2 ± 2.2 5.8 ± 1.8 0.05
3 months 4.6 ± 2 6.1 ± 1.8 0.05
6 months 4.8 ± 2.8 6.1 ± 1.6 NS
9 months 5.2 ± 2.1 5.8 ± 1.8 NS
12 months 5.3 ± 1.6 5.9 ± 1.7 NS
Recurrence of HCV (n)11 20
(Early deaths excluded) (n)1 3
Graft hepatitis C 5 (45) 14 (70) NS
Mild/mod/severe 5/0/0 7/5/2 0.06
IFN–ribavirin treat. 1 3
Follow-up at 3 years (n)11 20
Follow-up (months) 42.9 ± 10.5 37.9 ± 12 NS
Progress of HCV
(a) Normal liver
function test
4 (36) 7 (35) NS
(b) CAH 6 (55) 4 (20)
(c) Compensated cirrh. 1 (9) 2 (10)
(d) Decomp. cirrhosis 0 1 (5)
(e) HCV-related death 0 6 (30)
a + b (good outcome) 10 (91) 11 (55) 0.046
c + d + e (poor outcome) 1 (9) 9 (45)
Histology findings (n)9 15
Piecemeal 1.67 ± 1.12 2 ± 1 NS
Confluent necrosis 2.33 ± 1 2.60 ± 1 NS
Focal lytic necrosis 2.44 ± 0.73 2.20 ± 0.77 NS
Portal inflammatory 1.67 ± 0.71 2 ± 0.85 NS
Fibrosis score 1.78 ± 1 3.73 ± 1.94 0.005
Patient survival (%)
1, 3, 5 years 92, 92, 73 78, 61, 51 0.07
Percentage values are given in parentheses.
Table 5. Risk factors for poor outcome for HCV recurrence in graft
(univariate analyses).
Factors
Good outcome
(n¼21)
Poor outcome
(n¼10) P-value
Donor
Mean age (years) 40 ± 24 48.5 ± 16 NS
>60 years 6 (29) 4 (40) NS
Graft steatosis 2 (9.5) 6 (60) 0.004
Recipient
Mean age (years) 58 ± 5 62 ± 6 NS
Age >60 years 10 (48) 9 (90) 0.049
HCC 10 (48) 5 (50) NS
HBV 3 (14) 0 NS
Pre-LT viral load 4.6 ± 1.2 6 ± 0.4 0.06
Viral load >5 7 (47) 8 (100) 0.022
Surgery
Ischaemic time >10 h 3 (14) 0 NS
Postoperation
Mod/sev ischaemic injury 3 (15.8) 4 (36) NS
Acute rejection 7 (33) 5 (50) NS
Severe acute rejection 2 (10) 1 (10) NS
Intent-to-treat groups
TACRO 13 (62) 4 (40) NS
TACRO + ST 8 (38) 6 (60)
Real groups
Real TACRO 10 (47) 1 (10) 0.055
Real TACRO + ST 11 (53) 9 (90)
Intent-to-treat
groups – according to rejection
TACRO no rejection 10 (48) 1 (10) NS
TACRO + rejection 3 (14) 3 (30)
TACRO + ST no rejection 4 (19) 4 (40)
TACRO + ST + rejection 4 (19) 2 (20)
Percentage values are given in parentheses.
Good outcome: patients with normal liver function test, no clinical
evidence of HCV recurrence on the graft and no clinical signs of cir-
rhosis plus patients with chronic active hepatitis on the graft but no
clinical signs of cirrhosis. Poor outcome: Patients with cirrhosis on the
graft and clinical signs of compensated or decompensated cirrhosis or
patients who had died or had been retransplanted because of HCV
recurrence on the graft.
Tacrolimus monotherapy in liver transplantation Margarit et al.
1342 Transplant International 18 (2005) 1336–1345 ª2005 European Society for Organ Transplantation
inmunoreactivity and, consequently, suffer more frequent
and severe rejection episodes. In terms of safety, we could
not demonstrate a significant benefit of avoiding steroids,
probably because of the low number of patients included
in the study. However, a trend was observed towards less
de novo arterial hypertension and diabetes in the mono-
therapy group, whereas renal insufficiency was higher in
the TACRO group.
Concerning pharmacokinetics studies, in the TAC-
RO + ST group, higher doses of tacrolimus were required
to reach the same blood levels achieved with tacrolimus
alone. This finding confirms the increase in tacrolimus
metabolism due to cytochrome P450 3A4 iso-enzyme
(CYP3A4) induction by steroids, and shown in the report
of van Duijnhoven et al. [22].
HCV subgroup of patients
Recurrent hepatitis C viral infection is universal post-LT.
Almost half the patients present acute graft hepatitis early
after LT and 90% develop chronic active hepatitis with
evolution to cirrhosis in 30% of cases at 5 years post-LT
[23]. Evolution to liver failure and death is very rapid
once these patients present decompensated cirrhosis.
Results obtained with retransplantation for recurrent
HCV infections are poor. Recent data from UNOS and
the Spanish Liver Transplant Registry have shown lower
patient and graft survival in HCV patients [16,17,24].
Moreover, reports suggested that graft outcome of LT for
HCV may have deteriorated in recent years. Hence, the
subgroup of hepatitis C patients was evaluated independ-
ently to assess the potential benefit of a steroid-free im-
munosuppression regimen.
In our experience, there was a tendency for the subgroup
of HCV patients to have more acute rejections diagnosed
than HCV-negative patients. Difficulty in liver biopsy inter-
pretation between acute rejection and HCV hepatitis, or
the coexistence of both, has sometimes led to patients with
recurrent hepatitis C being treated for rejection with steroid
boluses. TACRO and TACRO + ST groups had similar
demographic characteristics and acute rejection rates. Ana-
lysis of HCV recurrence in the graft showed a lower,
although not significant, incidence of graft hepatitis in the
TACRO monotherapy group as well as a higher proportion
of patients with good outcome. However, these differences
did not reach statistical significance, probably because of
the scant number of patients studied.
Although further grouping down patients into real-
TACRO and real-TACRO + ST leaves only few patients
in each group, we were interested in knowing the evolu-
tion of graft hepatitis in the patients who never received
steroids and compared this group with the remaining
patients who received steroids either for protocol or to
treat acute rejection episodes. Significantly lower levels of
hepatitis C viraemia were found during the early weeks
and months in patients without steroids, thereby confirm-
ing the findings of Garcia Retortillo et al. [25] and Boker
et al. [26]. This may have been the reason for the lower
incidence of acute graft hepatitis and its milder course in
all 45% of steroid-free patients who presented it. We
observed that a cut-off point in viral load pre-LT of
>log
10
5 was predictive of a poor outcome. At 12 months,
viral loads in our study were similar in both groups,
probably because at that time point most of the patients
in both groups were free of steroids and received the
same amount of immunosuppression. In contrast to our
findings, Papatheodoridis et al. [15] reported higher levels
of HCV RNA at 3 months in patients with single initial
therapy compared with those receiving double therapy.
However, a correlation was found between viral load at
12 months, duration of steroid treatment and extent of
fibrosis. All these reports indicate a correlation between
viral load and the amount of immunosuppression.
After a mean follow up of 44 months, evolution of
HCV graft infection was significantly better in the ster-
oid-free group. Only one patient presented a poor out-
come (compensated cirrhosis), whereas the rest had good
outcome (normal liver function or mild chronic active
hepatitis). These findings were confirmed by a signifi-
cantly lower fibrosis score in the liver biopsy study. Nev-
ertheless, the possible effects of steroids on HCV
recurrence and fibrosis remain controversial. A recent
study by Fasole et al. [27] defended the beneficial effect
of steroids on avoiding fibrosis in graft HCV recurrence.
The hypothesis of Berenguer [28] is that the immune sys-
tem becomes reconstituted following steroid withdrawal
and dose reduction in calcineurin inhibitors and
immune-mediated liver damage may then occur, together
with progression to severe forms of chronic hepatitis.
There are many factors which influence the outcome of
graft hepatitis C re-infection. Liver graft quality is extre-
mely important: suboptimal donor liver either due to
steatosis, old donor age, fibrosis, etc. results in important
ischaemic–reperfusion injury, activation of inflammatory
processes, greater susceptibility to acute rejection and
probably more severe HCV graft re-infection. Recipient
age over 60 years appeared to be significant in our study
as well as in other studies [16,29–33]. In a previous study
[34] concomitant CMV hepatitis was found to aggravate
the evolution of hepatitis C; no case of CMV hepatitis
was found in the present study. The link between anti-
rejection therapy and worsening of hepatitis C seems very
clear. Many authors and ourselves have demonstrated an
association between more aggressive recurrence of hepati-
tis and evolution to fibrosis and cirrhosis and episodes of
treated rejection [31,35,36].
Margarit et al.Tacrolimus monotherapy in liver transplantation
Transplant International 18 (2005) 1336–1345 ª2005 European Society for Organ Transplantation 1343
Finally, patient survival was much better in real-TACRO,
although the low number of patients in each group did not
allow to reach statistical significance. Altogether, we can
affirm that outcome of HCV graft infection was better in
patients who could be maintained without steroids after
LT. All HCV-related deaths occurred in the group receiving
steroids from the beginning, or during follow-up. This
study is one of the few prospective and randomized trial of
immunosuppression regimen based on tacrolimus in
monotherapy since the first post-transplant day, and
although it was not originally designated to look at hepati-
tis C this is the first prospective randomized study of
monotherapy regimen and recurrent hepatitis C infections.
Another strong point of the trial is the long-term follow-up
presented in patients without steroids. Other authors have
reported differences in recurrent hepatitis but not long-
term evolution of the graft. The few number of patients
enrolled and the lack of periodically protocol biopsies in
HCV patients are the weak points of the study. However,
we should be able to learn about these findings and in fact,
this study has prompt us to change our standard immuno-
suppressive regimen in HCV positive patients, which is
based at the moment on tacrolimus plus MMF.
In summary, (i) a steroid-free immunosuppression pro-
tocol based on tacrolimus monotherapy is safe in a cohort
of senior patients transplanted mainly for alcoholic or viral
cirrhosis. Patient and graft survival and acute rejection and
steroid-resistant rejection rates were similar to those of our
standard tacrolimus short-term steroid protocol. (ii) No
significant benefit was obtained in terms of side-effects of
this steroid-free protocol although a trend was observed
towards less hypertension and diabetes but more nephro-
toxicity. (iii) The principal benefit could be in patients
transplanted for HCV cirrhosis, as a trend towards lower
incidence and milder course of acute recurrent HCV hepa-
titis and better long-term outcome was found in the group
without steroids. (iv) This benefit was more evident in
patients receiving no steroids at all after LT. Therefore, the
key of future protocols for HCV patients would be a more
effective steroid-free immunosuppression to achieve acute
rejection rates of <10%; consequently, more than 90% of
HCV LT patients will be steroid-free.
Acknowledgements
The study was supported, in part, by a grant from Fuji-
sawa GM.
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