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

ilaria.lenci@uniroma2.it

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