Accepted Manuscript
Safety of Complete and Sustained Prophylaxis Withdrawal in Patients Liver
Transplanted for HBV-Related Cirrhosis at Low Risk of HBV Recurrence
Ilaria Lenci, Giuseppe Tisone, Daniele Di Paolo, Fabio Marcuccilli, Laura
Tariciotti, Marco Ciotti, Valentina Svicher, Carlo Federico Perno, Mario
Angelico
PII: S0168-8278(11)00032-8
DOI: 10.1016/j.jhep.2010.12.036
Reference: JHEPAT 3700
To appear in: Journal of Hepatology
Received Date: 16 July 2010
Revised Date: 13 December 2010
Accepted Date: 14 December 2010
Please cite this article as: Lenci, I., Tisone, G., Di Paolo, D., Marcuccilli, F., Tariciotti, L., Ciotti, M., Svicher, V.,
Perno, C.F., Angelico, M., Safety of Complete and Sustained Prophylaxis Withdrawal in Patients Liver Transplanted
for HBV-Related Cirrhosis at Low Risk of HBV Recurrence, Journal of Hepatology (2011), doi: 10.1016/j.jhep.
2010.12.036
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1
RE-REVISED VERSION
SAFETY OF COMPLETE AND SUSTAINED PROPHYLAXIS WITHDRAWAL IN
PATIENTS LIVER TRANSPLANTED FOR HBV-RELATED CIRRHOSIS AT LOW RISK
OF HBV RECURRENCE
Ilaria Lenci1, Giuseppe Tisone2, Daniele Di Paolo1, Fabio Marcuccilli3, Laura Tariciotti2, Marco
Ciotti3, Valentina Svicher3, Carlo Federico Perno3 and Mario Angelico1
1Hepatology and 2Liver Transplant Unit, Tor Vergata University, Rome, Italy; 3Laboratory of
Molecular Virology, Tor Vergata University, Rome, Italy
Running Title: HBV prophylaxis withdrawal after liver transplantation
Address of correspondence:
Ilaria Lenci, MD
Chair of Gastroenterology
Dep. of Internal Medicine
Hepatology Unit, Policlinico Tor Vergata, Rome
Tor Vergata University
Via Montpellier, 1
00133 Roma
Phone and fax number: +39-06-72596803
2
Word Count:
Abstract: 249
Text: 4.832
Figure: 2
Table: 3
Abbreviations:
LT: Liver Transplantation
DNA: Deoxyribonucleic acid
ccc-DNA: covalently closed circular DNA
total HBV-DNA: including relaxed circular DNA, intermediate e forms and cccDNA
HBIg: Anti-hepatitis B immunoglobulins
HBV: Hepatitis B virus
HBsAg: Hepatitis B surface antigen
HBcAb: Hepatitis B core antibodies
HBV-DNA: Hepatitis B virus DNA
Anti-HBs: antibodies against hepatitis B surface antigen
HCV: Hepatitis C virus
HDV: Hepatitis D virus
ALT: alanine amine transferases
PCR: polymerase chain reaction
3
The Authors who have taken part in this study declared that they do not have anything to disclose
regarding funding or conflict of interest with respect to this manuscript
This study was awarded in 2009, during the EASL Conference placed in Copenhagen, as “Best
Clinical Abstract” and in 2010, during the EASL Conference placed in Vienna, with the prize “Best
Poster Award”.
4
ABSTRACT
Background and Aim: HBV reactivation after liver transplantation may be related to persistence
of covalently closed circular (ccc)DNA. We investigated the safety of HBV prophylaxis withdrawal
in selected HBV transplanted patients.
Methods: thirty patients transplanted 64-195 months earlier (23 males, median age 56 yrs),
HBsAg-positive, HBeAg and HBV-DNA negative at transplant (43% HCV/ HDV coinfected), with
undetectable intrahepatic total and ccc-DNA were enrolled. All underwent HBIg withdrawal and
continued lamivudine with monthly HBsAg and HBV-DNA monitoring and sequential liver
biopsies. Those with confirmed intrahepatic total and ccc-DNA undetectability 24 weeks after
stopping HBIg, underwent also lamivudine withdrawal and were followed-up without prophylaxis.
Results: Twenty-five patients did not exhibit signs of HBV recurrence after prophylaxis withdrawal
(median follow-up 28.7 months, range 22-42). Five patients became HBsAg-positive: one early
after HBIg withdrawal, the other 4 after HBIG and lamivudine withdrawal. None of these
experienced clinically relevant events. In the first patient HBIg were reinstituted with prompt
HBsAg negativization. Of the other 4, one remained HBsAg-positive with detectable HBV-DNA
and mild ALT elevation and was successfully treated with tenofovir. In the remaining 3, HBsAg
positivity was transient and followed by anti-HBs seroconversion, thus no antiviral treatment was
needed.
Conclusions: Patients with undetectable HBV viremia at transplant and no evidence of intrahepatic
total and cccDNA may safely undergo cautious weaning of prophylaxis, showing low rate of HBV
recurrence after a two year follow-up. Undetectability of intrahepatic ccc-DNA may help to identify
patients at low-risk of recurrence, yet studies with longer follow-up are needed.
5
INTRODUCTION
The management of patients liver transplanted for HBV-related cirrhosis has considerably evolved
over the past two decades. The introduction of intravenous hepatitis B immunoglobulins (HBIg)
therapy dramatically reduced HBV recurrence rates and increased patient’s survival [1]. This
strategy, however, had several limitations, such as very high costs, low effectiveness in patients
with high levels of HBV replication before LT and potential selection of HBsAg escape mutants.
When oral antiviral agents became available, most transplant centres switched from HBIg
monotherapy to a combined approach with HBIg plus nucleoside analogues (NUCs), which allowed
a further decrease in rates of HBV reinfection after LT [2,3]. The protection granted by the
indefinite use of HBIg and NUCs allowed these patients to obtain survival rates comparable, or
even better, than those of patients transplanted for non HBV-related disease, with a risk of HBV
recurrence lower than 10% [4-6]. Although the current schedules of HBV prophylaxis are very
effective, several approaches have been attempted in the last few years to reduce or discontinue the
requirement of i.v. HBIg [5-7], including the use of intramuscular HBIg, antiviral monotherapy
[8,9] and HBsAg vaccination [10-12]. Nevertheless, several open issues still remain in relation to
the efficacy of these strategies, which require further clinical evaluation. A major confounding
factor present in most of the available studies is the wide variability of patients transplanted for
HBV, due to viral heterogeneity and/or differing levels of viral replication before and at time of
transplant. This heterogeneity suggests that different prophylactic strategies could be necessary in
relation to the pre-LT HBV replication status and that optimal treatment should be tailored on the
individual patient characteristics.
On the other hand, the persistence of low levels of HBV-DNA has been reported even after 10 years
after-LT despite a successful anti-HBs prophylaxis [13]. Current concepts indicate that HBV-DNA
eradication is an elusive goal, as the virus may persist in the liver, and possibly in other extrahepatic
reservoirs, as intrahepatic covalently closed circular DNA (ccc-DNA), even in the presence of a
HBsAg-negative status. Accordingly, the presence of intrahepatic total and ccc-DNA was detected
in most cases in a US cohort of patients after LT, suggesting that these tools are not helpful in
predicting viral recurrence after transplant [14]. In these studies, however, the majority of patients
were HBeAg-positive and showed active HBV replication at time of transplant, two factors known
to independently predict HBV recurrence [15].
Recently, we studied a cohort of patients liver transplanted more than 5 years earlier, all HBeAg-
negative and with undetectable HBV-DNA at transplant, in whom intrahepatic total and ccc-DNA
were found to be undetectable in the vast majority of cases [16]. Although in this study the
6
possibility of HBV persistence in extrahepatic sites, such as lymph nodes or peripheral blood
mononuclear cells, was not excluded, we considered patients with undetectable intrahepatic total
and ccc-DNA long-term after LT to be at low risk of HBV recurrence. This prompted us to verify
whether indefinite continuation of HBV prophylaxis was really necessary in this selected cohort of
patients, or whether a cautious withdrawal could be attempted.
In the present study we report the results of the efficacy and safety of a gradual weaning protocol of
HBIg and NUCs prophylaxis in the same group of patients, all with undetectable intrahepatic total
and ccc-DNA and the clinical outcome in the subsequent two-year follow-up.
PATIENTS AND METHODS
Study population
Thirty patients (23 males, median age 56 years), recruited from an initial cohort of 44 subjects liver
transplanted for HBV-related cirrhosis previously studied for the presence of intrahepatic total and
ccc-DNA [16], were enrolled in this study. Baseline clinical features are summarized in Table 1 and
are reported separately for patients transplanted before and after 2001, due to the different HBV-
DNA assays being used. All patients were HBsAg-positive and HBeAg-negative in serum before
LT, 6 (20%) were coinfected with hepatitis C virus (HCV) and 7 (23%) with hepatitis D virus
(HDV). Median follow-up after transplant was 139 months (range, 64-195). Nine (30%) patients
were treated with 100 mg/day of lamivudine (LAM) (Zeffix, Glaxo Wellcome, UK) for an average
of 16±22 (range 4-92) months before LT because of detectable serum HBV-DNA at time of listing.
In 2 patients (6.6%), 10 mg /day of adefovir-dipivoxil (ADV) (Hepsera, Gilead Science, CA) were
added to LAM due to development of YMDD lamivudine-resistant mutants before LT.
All patients showed undetectable serum HBV-DNA at the time of transplant. This was analyzed in
19 patients (transplanted before 2001) using the hybridization assay (HBV Digene Hybrid.Capture
II, Digene Corp., USA, lower limit of detection: 105 cp/ml) and in 11 (transplanted after 2001)
using polymerase chain reaction (PCR) (Cobas Amplicor HBV Monitor, Roche Diagnostic, lower
limit of detection: <400 cp/ml). All patients received anti-HBV prophylaxis with HBIg after
transplant: in 28 in combination with LAM and in 2 combined with LAM and ADV. In all cases,
HBIg (Hepatect CP, Biotest Pharma GmbH, Germany) were given intravenously, at the dose of
10.000 IU during the anhepatic phase, followed by a daily dose of 10.000 IU for the first 4 days
after transplant. Subsequently, HBIg were administered at the dose of 5.000 IU i.v. per month until
month 6, and then at the dose of 2.000 IU given “on demand”, whenever the anti-HBs titre dropped
7
below 70UI/L (17). Serum monitoring for HBV-DNA and HBsAg after LT was performed at 6-12
months intervals (6 month after 2001).
Most patients received cyclosporine A as maintenance immunosuppression, with or without
mycophenolate-mofetil, and a minority received tacrolimus or sirolimus.
Study protocol
The study protocol was approved by the Ethics Committee of our Institution and the research was
conducted according to the principles of the revised Helsinki Declaration. All patients gave written
informed consent prior to enrolment. Patients were eligible if they fulfilled the following criteria: 1)
liver transplantation due to HBV-related cirrhosis performed at least 3 years earlier; 2) adherence to
anti-HBV prophylaxis with HBIg and NUCs since LT; 3) evidence of spontaneous or drug-induced
undetectable serum HBV-DNA at time of LT; 4) sustained normal biochemistry and sustained
undetectable serum HBsAg and HBV-DNA after LT; 5) undetectable total and ccc-DNA in liver
tissue before entering the study.
As shown in the Figure 1, patients who tested negative for both total and ccc-DNA in the baseline
liver biopsy (LB1) underwent HBIg withdrawal, while being maintained on LAM (+ ADV), with
monthly monitoring of serum HBsAg, anti-HBs titre and HBV-DNA. After 6 months, a second
liver biopsy (LB2) was obtained. Patients in whom intrahepatic total and ccc-DNA undetectability
was confirmed also underwent LAM (+ ADV) withdrawal and were monthly monitored for serum
HBsAg and HBV-DNA for the next 6 months, when a third liver biopsy (LB3) was obtained.
Patients who were confirmed to be negative for all HBV virological assays, both in serum and liver
tissue, continued a monthly follow-up without receiving anti-HBV prophylaxis for the next 12
months, when a fourth liver biopsy (LB4) was obtained. Patients again testing negative at this stage
continued to be followed without prophylaxis, with regular virological checks every 3 months.
Compliance to study protocol was assessed by verifying clinical and pharmacological records at
each monthly visit.
Virological assays in serum and liver tissue
Serum HBV markers (HBsAg, HBeAg, HBcAb) were detected using chemiluminescence (Modular
E170, Roche Diagnostics, IN, USA) and serum HBV-DNA by polymerase chain reaction (PCR)
(Taqman 48, Roche Diagnostics, lower limit of detection <20 IU/ml).
Percutaneous liver biopsies were performed using a Menghini modified needle (Surecut, 16G, TSK
Laboratories, Japan). Liver specimens were divided in two parts, one was transferred into vials
containing fresh RPMI 1640 culture medium (Invitrogen, CA, USA) to preserve cell viability and
8
detection of total and ccc-DNA and the other was formalin-embedded and used for conventional
histological analysis. Histological analysis was performed using the Ishak score.
Intrahepatic total and ccc-DNA were assessed as previously reported [16]. The lowest detectable
levels of total and ccc-DNA in liver tissue were 20 cp/mg and 45 cp/mg (0.07 cp/cell), respectively.
A positive and negative HBsAg specimens obtained from liver biopsy were included in each PCR
run and used as controls. Total and ccc-DNA was as follows: total HBV-DNA variability was <3.2
% in inter-assay experiments and <4.3 %, in intra-assay experiments, respectively. For ccc-DNA,
the inter- and intra-assays variability was <3.8 and <2.9%, respectively.
All virological analyses were performed at the Laboratory of Molecular Virology of the University
of Tor Vergata.
Statistics
Data were collected and analyzed with NCSS software package (Kaysville, Ut, USA) using
parametric and non parametric tests, as appropriate. Predictors of HBV recurrence (defined as any
HBsAg positivitizion during the study), were analyzed by univariate analysis The rate of HBV
recurrence was assessed according to Kaplan-Meier.
RESULTS
Safety and (clinical and virological) outcome of the weaning protocol
Of the 30 patients with undetectable total and ccc-DNA at baseline, 25 (83.3%) did not exhibit
serological signs of HBV recurrence after a median follow-up of 28.7 months (range 22-42). Five
patients (16.6%) became serum HBsAg-positive (Table 2): one shortly after HBIg withdrawal,
while still on LAM, and 4 after NUCs withdrawal. Notably, the anti-HBs titre was greater than 70
IU/ml in all patients (range 70-150) at time of HBIg withdrawal, while it dropped to 18 IU/ml
(range 10-26) at time of NUC withdrawal. In the first patient with HBsAg recurrence HBIg were
reinstituted with prompt HBsAg negativization. Of the other 4, only one remained HBsAg-positive
and developed detectable HBV-DNA levels (up to 5.6x102 IU/ml) with mild ALT elevation, and
was given tenofovir as rescue therapy. In this case sequencing analysis of the entire HBV genome
(ABI 3100 genetic analyzer, Applied Biosystems, Foster City, CA, USA) showed the same LAM
resistant virus (L180M/L mutation) detected before transplant (genotype D, subtype ayw2). The
other 3 patients had only transient, short-term HBsAg serum positivity and always remained with
undetectable serum HBV-DNA, which did not allow HBV genome sequencing. In addition, in all of
them HBsAg clearance was followed by anti-HBs sero-conversion, with development of anti-HBs
9
titres ranging from 25 to 100 IU/L after a median follow-up of 7 months (range 4-9). No adverse or
clinically relevant events occurred during the study.
Virological Findings in Liver Tissue
Twenty-three patients without HBsAg recurrence had undetectable total or ccc-DNA in all protocol
biopsies. Among the 5 patients who had serological HBV recurrence, the single patient who became
HBsAg positive after HBIg withdrawal was found to be positive in LB2 for total HBV DNA, but
not for cccDNA. Of the remaining 4 with HBV recurrence, only one was found to be positive for
total and ccc-DNA in LB3 and LB4, respectively. This was the only patient who also showed active
viral replication as evidenced by detectable serum HBV-DNA: in LB3, total intrahepatic HBV-
DNA was detected at the first round of PCR and ccc-DNA levels were 7x102 copies/mg of liver
tissue. The other 3 patients with HBV recurrence were positive only for total HBV DNA in LB4,
but had no evidence of ccc-DNA either in LB2, LB3 or LB4. Notably, the latter 3 patients were
coinfected with HCV (n=1) or HDV (n=2). In addition, two further patients without HBV
recurrence (i.e., who were never HBsAg or even HBV-DNA positive) were found positive for total
HBV DNA, but not for cccDNA in LB4.
Predictors of HBV recurrence
None of the demographic pre-LT or post-LT features differed significantly between patients with or
without HBV recurrence, as shown in Table 3. In particular, there were no differences in relation to
time after LT and type of maintenance immunosuppression. No patients in either group had
received steroids beyond 3 months after LT. Thirteen patients were co-infected, including six with
HCV and seven with HDV. Of these coinfected patients, 1 HCV- and 2 HDV-coinfected (23%),
underwent HBV recurrence, compared to only 2 (11.8%) of 17 with HBV monoinfection. This
difference was not statistically significant.
DISCUSSION
The current strategy to prevent HBV-disease recurrence after LT is based on the combined use of
HBIg and nucleos(t)ide analogs, usually LAM. This prophylaxis is maintained indefinitely and has
proved in recent years to be associated with a very low rate of HBV disease recurrence [2-6]. A
recent meta-analysis [18] has shown that the combination of HBIg and lamivudine, compared with
HBIg alone, is associated with greater reduction of HBV recurrence, HBV-related deaths and
mortality.
10
Because of the high costs and lifelong commitment of this prophylactic approach, there are
continuous efforts to challenge this strategy, despite the fact that it has allowed the best LT
outcomes for HBV related disease compared to all other indications. Post-transplant withdrawal of
HBIg associated with continued LAM monotherapy has been attempted and initially showed
encouraging results, even though, after longer follow-up, many patients experienced HBV
recurrence [19]. Yet, little is known on the safety and efficacy of minimizing/withdrawing HBV
prophylaxis in relation to HBeAg status and the extent of HBV replication at time of transplant,
which are the only two independent predictors of HBV recurrence [15]. Moreover, to our
knowledge, no study has investigated the possibility of withdrawing anti-HBV prophylaxis in
patients thought to be at low risk of recurrence.
In the present study we show that a complete withdrawal of prophylaxis for HBV recurrence is
feasible and safe in a selected group of patients at low risk of recurrence, defined by undetectable
HBV-DNA in serum at time of LT, negative HBeAg and undetectable intrahepatic total and ccc-
DNA at least 3 years after LT.
Five patients (16.6%) underwent HBV recurrence (as defined by HBsAg serum positivization), but
none experienced virological and/or clinically relevant events under careful monthly monitoring for
up to almost 4 years (median 28 months). Indeed, HBsAg positivity was transient in 4 out of 5
patients and in 3 of them HBsAg clearance was followed by spontaneous mounting of an anti-HBs
titre. Interestingly, all these 3 patients were coinfected with HCV or HDV. In addition, only one
patient showed detectable HBV DNA in serum positivity and this was the only one who tested
positive for intrahepatic ccc-DNA.
Study implications
Although preliminary and based on a relatively small number of cases, our findings may have
significant implications in relation to the definition of HBV recurrence and the use of new
virological tools in this setting, and could be regarded as a proof-of concept in relation to their
clinical relevance.
1. Definition of HBV recurrence after LT
HBV recurrence after LT is conventionally defined as persistent HBsAg or, in those who become
seronegative after surgery, HBsAg seroreversion at any time after LT [24]. This definition derives
from the evidence that adequate perioperative HBIg administration is immediately effective in
causing the disappearance of HBsAg in blood and that a later premature HBIg interruption, or poor
compliance to long-term HBIg prophylaxis, is associated with high risk of HBsAg positivization.
11
We believe that this definition is nowadays obsolete and does not match the current concept that
HBV infection cannot be eradicated. Indeed, persistence of HBV infection may be underestimated
based only on the presence of serum HBsAg. This is well demonstrated in the present study, both
by the evidence that immediate reinstitution of HBIg promptly reverted one patient with HBV
recurrence to a negative HBsAg status and that recurrence may occur even when patients have been
HBsAg negative for a long time. Moreover, false negative HBsAg results may also be due to the
presence of HBsAg escape mutants. On the other hand, this and other studies demonstrate that
many tools, more precise or informative than serum HBsAg, can be usefully employed before and
after LT to better stratify patients at different risk of HBV recurrence, analogous to the decision
processes recommended in non transplant patients [25,26]. We believe that a better definition of
recurrent/persistent HBV infection should nowadays be based on the presence, at any time after LT,
of one or more of the following: HBsAg positivity, HBV-DNA detectability in serum, ccc-DNA
detectability in liver tissue; and that the definition of recurrent HBV-related disease would, in
addition, require ALT elevation and biopsy-proven liver damage.
2. Validity and usefulness of new virological methods
At the present stage of understanding of HBV molecular biology, a key point is whether the current
methodology for detecting intrahepatic total and ccc-DNA is ready for clinical use, thus justifying
the use of protocol liver biopsies for this purpose. In the present study, the sensitivity of total and
ccc-DNA assays were comparable to those reported by other investigators [14,17,23]. We found
that, although undetectability of ccc-DNA in the baseline liver biopsy was a strong argument to
predefine a low risk of recurrence, this did not prove to be an absolute measure, as indeed 5 patients
underwent HBV recurrence. Thus, our study shows that the usefulness of these virological tools in
clinical practice remains unproven, mainly due to insufficient sensitivity and lack of
standardization. In addition, the significance of detectable total HBV-DNA in the absence of ccc-
DNA, found in two patients without HBV recurrence in our series, is also uncertain and may reflect
poor specificity of the test, leading to false positive results. These patients will, however,
undoubtedly require careful monitoring for potential later viral recurrence. Given all the above
considerations, we believe that the clinical use of frequent liver biopsies to monitor intrahepatic
HBV replication is currently unjustified.
3. Clinical implications
In contrast to the remarkable achievements occurring over the last few years and the current
consensus in the management of HBV infection [25,26], little has changed in the transplant setting,
12
despite the availability of new potent drugs. The reluctance to adopt new strategies is likely
explained by the fear of cholestatic fibrosing hepatitis (FCH), a devastating form of recurrent HBV-
related disease [27] which, however, has nowadays become rare. In many centres throughout the
world no cases of recurrent FCH have indeed been observed over the last 10 years. In addition,
most FCH were observed when nucles(t)ide analogues were not available and virological methods
were unable to detect viral loads below 105 log copies/ml, suggesting that most patients had been
transplanted with significant viral replication. Current guidelines, however, recommend the pre-
treatment of HBV-infected LT candidates in order to transplant them with the lowest HBV viremia
as possible [26] and to protect HBV recipients with specific prophylaxis indefinitely.
Our data show that no clinically relevant events occurred in the 5 patients with recurrent HBV,
including the only patient with pre-LT LAM-resistance and one patient with HCV or two with HDV
coinfection. An important issue is whether our findings can be extrapolated to different clinical
settings. Notably, our transplant recipients were all HBeAg negative and with undetectable HBV-
DNA at transplant. In addition, they were long-term survivors under low grade of
immunosuppression and no evidence of transient low-levels of HBV-DNA after LT with the
available methods (although the PCR sensitivity changed over the time), nor with high grade of
liver disease. We believe that there is no evidence that these patients should be managed as other
categories of patients in whom long-term HBV prophylaxis is recommended, such as highly
immunosuppressed neoplastic patients receiving steroids or other potent immunosuppressants [28],
or cirrhotic patients in whom HBV reactivation may be life threatening [29] In contrast, more
caution should be certainly used in HBeAg positive patients transplanted with detectable or
unknown HBV replication, in transplant recipients with pre-LT LAM resistance, or evidence of
significant liver disease and eventually in HDV coinfected patients, in whom treatment options are
limited.
Another clinical implication of this study is that HBV recurrence, in the current era of potent
antivirals, could be easily manageable even in the post-LT setting, as in fact occurred in the only
patient in our series who also developed detectable serum HBV-DNA. On the other hand, frequent
monitoring of serum HBV-DNA and detection of resistance profiles through viral genome
sequencing [30] would allow prompt intervention with the most appropriate antiviral drug.
Finally, prophylaxis withdrawal may allow significant cost reduction. We estimated that in our
country HBIg and LAM withdrawal will result in the saving of approximately 16.000 euros per
patient per year, a figure remarkably higher than the yearly cost of TDF (3500 euros) or LAM (1200
euros) per patient. Thus, even if all 5 patients with recurrent HBV had been treated with TDF
(instead of 1 actually treated) there would have been an advantage of the withdrawal strategy.
13
In conclusion, cautious HBV prophylaxis withdrawal in long-term LT survivors at low risk of HBV
recurrence is feasible and safe in clinical practice, is associated with low rate of HBV recurrence
after two years of follow-up, and results in considerable cost reduction. Identification of patients at
low risk of recurrence through ccc-DNA assay in liver biopsies may be helpful, but the
methodology still needs to be improved. Clinically relevant events in patients with HBV recurrence
are unlikely to occur with the current available drugs, but a strict clinical monitoring is required.
Further studies with extended follow-up are needed to evaluate whether these findings may have an
impact on the current clinical practice.
.
14
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17
Figure: 1. Flow-chart of the study protocol displaying the timing of liver biopsies performed
during the study and withdrawal of HBIG and NUCs
LB1: baseline liver biopsy performed during the screening phase, before HBIg withdrawal
LB2: liver biopsy performed 6 months after HBIg withdrawal
LB3: liver biopsy performed 6 months after LAM withdrawal
LB4: liver biopsy performed 18 months after LAM withdrawal
2. Kaplan-Meier recurrence figure
18
Figure 1
LB1LB1
Screening phase
NUC(s) only
No prophylaxis
6 6 months months
monthly monitoring of serum HBsAg and HBV - DNA
HBIg withdrawal
NUC(s) withdrawal
6 6 monthsmonths 12 12 monthsmonths6 6 months months
LB2LB2 LB3LB3 LB4LB4
19
Figure 2
HBIgwithdrawal
0,000
0,250
0,500
0,750
1,000
0,0 11,3 22,5 33,8 45,0
Follow-up (months)
Rec
uren
ce-f
ree
surv
ival
(%)
Number of patient at risk
30 29 28 27 26 25 20 9
HBIgwithdrawal
0,000
0,250
0,500
0,750
1,000
0,0 11,3 22,5 33,8 45,0
Follow-up (months)
Rec
uren
ce-f
ree
surv
ival
(%)
Number of patient at risk
30 29 28 27 26 25 20 9
20
1. Baseline clinical features of the enrolled patients
LT before 2001 (N=19)
LT after 2001 (N=11)
Patients M/F 16/3 7/4
Age (median) 59 (41-71) 51 (35-65)
Follow-up after LT (median, months) 146 (94-142) 88 (65-102)
Serum HBV-DNA pre-LT <105 cp/ml * <400 cp/ml **
LAM pre-LT 4 (21%) 5 (45.4%)
ADV therapy pre-LT - 2 (18.1%)
Prophylactic therapy after LT HBIG+LAM HBIG+LAM (+ADV in 2 pts)
Immunosuppression after LT (CyA, FK, MMF, Sir) 15/2/3/2 9/5/4/1
Coinfected patients 3 HBV+HCV 5 HBV+HDV
3 HBV+HCV 2 HBV+HDV
Donor’s anti-HBc positive status 9 (47%) 3 (27.2%)
Presence of HCC at LT 1 1
Mean grading at LB1*** (pre-prophylaxis withdrawal) 4.6 ± 3.4 3 ± 2.6
Mean staging at LB1*** (pre-prophylaxis withdrawal) 2 ± 1.8 1.8 ± 1.2
*Hybridization Assay (lld: 105 cp/ml); ** PCR Cobas Amplicor (lld : <400 cp/ml) LAM : lamivudine ; ADV : adefovir-dipivoxil ; HBIG : hepatitis B immunoglobulins ; CyA : cyclosporine A ; FK : tacrolimus ; MMF : mycophenolate mofetil ; Sir : sirolimus ; HCC : hepatocellular carcinoma ; ***histological score according to Ishak.
22
2. Characteristics of patients with recurrent HBV (HBsAg positivization)
Patient 1 Patient 2 Patient 3 Patient 4 Patient 5
Age, sex 38, M 47, M 74, M 54, F 69, M
BMI 23 25 27 30 29
Etiology HBV HBV HBV+HDV HBV+HDV HBV+HCV
HBeAg/anti-HBc neg/pos neg/pos neg/pos neg/pos neg/pos
HBV-DNA at LT <105 cp/ml* <400 cp/ml** <105 cp/ml <400 cp/ml <105 cp/ml
NUCs pre-LT (months of therapy) none LAM+ADV
(14) LAM
(6) none LAM (9)
Follow-up post-LT (months) 108 60 156 72 132
Prophylaxis post-LT HBIg+LAM HBIg+ LAM+ADV HBIg+LAM HBIg+LAM HBIg+LAM
Immunosuppression tacrolimus sirolimus CyA CyA CyA
total/ccc-DNA at baseline (LB1) neg /neg neg /neg neg /neg neg /neg neg /neg
Months of HBIG withdrawal*** 1 10 8 9.7 12
Months of NUCs withdrawal*** n.a. 4 2 3.7 6
total/ccc-DNA (LB2) pos /neg neg /neg neg /neg neg /neg neg /neg
total/ccc-DNA (LB3) neg /neg pos /pos neg /neg neg /neg neg /neg
total/ccc-DNA (LB4) neg /neg pos /pos pos / neg pos / neg pos / neg
HBV-DNA § at time of HBV recurrence <64 cp/ml 5.6x104 cp/ml <64 cp/ml <64 cp/ml <64 cp/ml
ALT (<42UI) at time of HBV recurrence 32 58 28 30 34
Rescue Therapy HBIg restitution Tenofovir none none none
HBsAg/anti-HBs (IU/ml) at the end of follow-up§ neg/>70 pos/<10 neg/40 neg/85 neg/110
Clinical outcome no AE no AE no AE no AE no AE
* Hybridization Assay (lld: 105 cp/ml); ** PCR Cobas Amplicor (lld : <400 cp/ml) *** before HBV recurrence ; § TaqMan 48 (lld : <64 cp/ml); CyA: cyclosporine A; AE: adverse event; § 28.7 month after HBIg and LAM withdrawal
23
3. Main characteristics of patients without or with HBV recurrence analyzed by
univariate analysis
LT= liver Transplantation; * Fibrosis stage at baseline liver biopsy (LB1) according to Ishak
Variables Patients without HBV recurrence
(n= 25)
Patients with HBV recurrence
(n= 5) Statistics
Age (mean±SD) 54.3 ± 8.2 53 ± 15 p=0.7
Sex (M/F) 19/6 4/1 p=0.8
BMI (mean±SD) 25.2 ± 4.1 27.2 ± 2.2 p=0.3
Coinfection with HCV or HDV (n, %) 10 (40%) 3 (60%) p=0.4
Months of follow-up after LT (mean±SD) 131.6 ± 36.1 110.8 ± 44.5 p=0.2
LT before 2001* (n, %) 17 (68%) 2 (40%) p=0.2
Use of cyclosporine A (n, %) 14 (56%) 3 (60%) p=0.8
Use of tacrolimus (n, %) 6 (24%) 1 (20%) p=0.8
Use of mycophenolate mofetil (n, %) 9 (36%) 0 p=0.1
Use of sirolimus (n, %) 2 (8%) 1 (20%) p=0.4
Fibrosis stage at baseline >2 in LB1* 8 (32%) 1 (20%) p=0.5
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