Annals of Hematology

, Volume 83, Issue 12, pp 769–774

Delayed hepatitis B virus reactivation after cessation of preemptive lamivudine in lymphoma patients treated with rituximab plus CHOP

Authors

  • Ming-Shen Dai
    • Division of Hematology/Oncology, Department of Medicine, Tri-Service General HospitalNational Defense Medical Center
    • Division of Hematology/Oncology, Department of Medicine, Tri-Service General HospitalNational Defense Medical Center
  • Woei-Yau Kao
    • Division of Hematology/Oncology, Department of Medicine, Tri-Service General HospitalNational Defense Medical Center
  • Rong-Yaun Shyu
    • Division of Gastroenterology, Department of Medicine, Tri-Service General HospitalNational Defense Medical Center
  • Tan-Mei Liu
    • Division of Rheumatology and Immunology, Department of Medicine, Tri-Service General HospitalNational Defense Medical Center
Original Article

DOI: 10.1007/s00277-004-0899-y

Cite this article as:
Dai, M., Chao, T., Kao, W. et al. Ann Hematol (2004) 83: 769. doi:10.1007/s00277-004-0899-y

Abstract

Preemptive lamivudine in lymphoma patients undergoing intensive chemotherapy can effectively prevent chemotherapy-related HBV reactivation. Nevertheless, the safety profile after withdrawal of lamivudine and the impact of rituximab-containing chemotherapy on HBV reactivation has not been defined. To illustrate the necessity of prolonged surveillance after cessation of preemptive lamivudine in lymphoma patients treated with rituximab and chemotherapy, four patients with B-cell NHL carrying HBV received rituximab plus CHOP. Preemptive lamivudine therapy was administered 1 week before chemotherapy until 4 weeks after completion of chemotherapy. Serial serum alanine aminotransferase (ALT), total bilirubin, and HBV-DNA levels were prospectively monitored in three patients. The fourth patient was closely monitored for ALT. The HBV DNA was checked after development of clinical overt hepatitis. The peripheral blood CD20+ B-lymphocyte counts were analyzed periodically in two patients. All of the three patients studied prospectively had virological relapses with surgence of HBV DNA 6–8 months after completion of rituximab-plus-CHOP (R+CHOP) therapy. Two of the three patients had biochemical relapses and one of them developed severe hepatitis. Sequencing for HBV polymerase gene in these patients failed to show evident emergence of lamivudine-resistant mutations. The fourth patient developed a hepatitis flare-up 6 months after completion of chemotherapy. The CD20+ lymphocytes were totally depleted when HBV DNA started to increase. Delayed HBV reactivation can occur in lymphoma patients receiving R+CHOP after withdrawal of preemptive lamivudine. More protracted lamivudine therapy may be an alternative to close monitoring following chemotherapy, and further studies are needed to define optimal duration of lamivudine therapy.

Keywords

HBV reactivationLamivudineLymphomaPreemptive treatmentRituximab

Introduction

Chemotherapy-related reactivation of hepatitis B virus (HBV) has been increasingly described and its incidence reported to be as high as 49% in lymphoma patients carrying HBV surface antigen (HBsAg) [13]. The mechanism for this complication is believed to be increased viral replication and antigen expression in hepatocytes during cytotoxic chemotherapy, followed by cytolysis of HBV-infected hepatocytes upon immune recovery [2]. Although HBV reactivation may result in hepatic failure and death, the preemptive use of lamivudine has successfully reduced the fatal complication of HBV reactivation after chemotherapy [4]. Nevertheless, the long-term outcome for HBV-seropositive patients after withdrawal of preemptive lamivudine in chemotherapy-treated lymphoma patients was not clear.

Combination with rituximab and standard chemotherapy in the treatment of B-cell non-Hodgkin’s lymphoma (NHL) has been shown to be more effective than chemotherapy alone [5]. It may become the standard regimen of next-generation therapy for B-cell NHL. Rituximab can induce profound and durable B-cell depletion with impaired secondary humoral immunity and thereby increase vulnerability to viral infection according to isolated reports [6]. Therefore, the relationship between HBV reactivation and the incorporation of rituximab in the standard regimen for lymphoma treatment needs to be appraised closely.

Materials and methods

Subjects

The Human Subjects Institutional Review Board and Ethics Committee of the Tri-Service General Hospital of Taiwan granted approval for the execution of the study. Serum was prospectively and periodically collected from three consecutive HBsAg+ patients with diffuse large B-cell NHL receiving six cycles of rituximab (375 mg/m2/cycle) and CHOP (cyclophosphamide, doxorubicin, vincristine, and prednisone) chemotherapy (R+CHOP chemotherapy). All of them were seropositive for HBV surface antigen (HBsAg) and received lamivudine (Zeffix tablets; GlaxoSmithKline) 100 mg daily starting 1 week before chemotherapy and continuing for 4 weeks after the last course of chemotherapy. Serial serum alanine aminotransferase (ALT), total bilirubin, prothrombin time, and HBV-DNA levels were measured before and after chemotherapy at 4-week intervals. The fourth patient was monitored for ALT only. His serum was collected for HBV-DNA measurement after development of a hepatitis flare-up.

Amplification and sequencing of the core promoter and precore region of HBV DNA

The polymerase chain reaction (PCR) products of the core and precore region of HBV DNA in the sera collected before chemotherapy and after lamivudine withdrawal were sequenced directly. The segment of HBV-DNA spanning 216 nucleotides (from 1626 to 1984) was amplified by PCR with the use of a pair of synthetic oligonucleotide primers: nt 1606–1625 (5′-GCATGGAGACCACCGTGAAC-3′) and nt 1985–2003 (5′-GATCTCCTCGACACCGCCT-3′). Particular attention was directed to the nucleotide sequence of the 87 nucleotides in the PR (from nt 1814–1900) and core promoter (CP) region (upstream of nt 1814) of HBV DNA. Before chemotherapy and after discontinuation of lamivudine, basic core promoter (T(1762)/A(1764)) and precore (A(1896)) HBV variants were determined. Another segment HBV-DNA spanning 194 nucleotides (from 482 to 605) was amplified by PCR with the use of a pair of synthetic oligonucleotide primers: nt 463–481 (5′-GTGGGCCTCAGTCCGTTTC-3′) and nt 606–628 (5′-GGCTACTCCCTTAACTTCATGGG-3′). Particular attention was directed to the nucleotide 550–558 to see if there was any emergence of a lamivudine-selective mutant strain (the so-called tyrosine-methionine-aspartate-aspartate, YMDD mutation).

Quantitative HBV-DNA assay and serological markers

For assessing the HBV viral load, sera were collected monthly from the beginning of chemotherapy and for at least 12 months after the end of the chemotherapy, by means of the HBV-DNA real-time PCR quantitative assay (Ampicor monitor, Roche Diagnostic LightCycler Systems) with a detection sensitivity of 2×102 copies/ml. Hepatitis flare-up was defined as a threefold or greater increase in serum ALT level that exceeded 100 IU/l. The hepatitis or hepatitis flare-up was attributed to reactivation of HBV when there was a more than tenfold increase of serum HBV DNA compared with the baseline level. HBsAg, HBV envelope antigen (HBeAg), anti-HBV envelope antibody (HBeAb), and anti-HBV core antibody (HBcAb) were assayed with a commercial microparticle enzyme immunoassay (AxSym; Abbott Laboratories).

Analysis of lymphocytes subset

Whole blood (100 μl) was collected in EDTA. Twenty microliters was stained with 5 μl of anti-CD45 fluoroscein isothiocyanate (FITC; Becton Dickinson, USA). Twenty microliters was stained with IgG2a-phycoerythrin PE (Diaclone) or IgG1-FITC (Diaclone) antibodies (Ab) as isotype controls (Becton Dickinson, USA). Expression of activation markers on B cells was determined by two-color staining with CD19-PE-Cy5 Ab (Immunotech, Marseille, France) associated with CD3RA-FITC Ab (Diaclone, Besançon, France) and CD8RO-PE Ab (Immunotech) with CD4-FITC Ab (Diaclone). The stained cells were incubated for 20 min after lysis of red cells (FACS lysing solution; Becton Dickinson, USA) and then analyzed using a FACScalibur flow cytometer (Becton Dickinson, Mountain View, CA, USA). Analysis was performed using SimulSET software (Becton Dickinson). The percentage of cells expressing on their cell surface was determined. This was compared both to the lymphocytes stained with the isotype control and a normal sample. Markers were drawn so that less than 0.1% of the isotype control–stained cells were positive.

Results

The clinical characteristics of these four patients are described in Table 1. All of them received six cycles of R+CHOP and received preemptive lamivudine for 6–7 months. Coexisting hepatitis C virus (HCV) or hepatitis D virus (HDV) was excluded before chemotherapy. Sequencing analysis of the precore region of HBV DNA found a point mutation G1896A in all of these four patients with HBeAg/anti-HBeAb+ in serum. Rising levels of HBV DNA were observed in the three patients prospectively studied after withdrawal of lamivudine and gradually exceeded their baseline level (Figs. 1, 2, and 3). Subsequent overt hepatitis flare-up developed in two patients 7 and 5 months after cessation of lamivudine, respectively, and severe icteric hepatitis (peak bilirubin level 36 mg/dl) developed in one of them. Hepatitis A virus, HCV, HDV, cytomegalovirus, or EBV acute infection were excluded during HBV reactivation with hepatitis flare-up. The two patients with HBV reactivation and hepatitis responded to reinstitution of lamivudine well with complete recovery of liver function profiles. Repeated sequencing analysis of HBV-DNA polymerase gene yielded no evidence of lamivudine-selective resistant mutations. Subset analysis of peripheral blood lymphocytes revealed the CD20+ B cells to be depleted during the period of chemotherapy and virological relapse, but recovered until clinical hepatitis flare-up in the first patient (Fig. 1). Nevertheless, they remained depleted during hepatitis flare-up in the second patient (Fig. 2). All of these patients currently remain free of disease.
Table 1

Clinical characteristics of 4 HBsAg+ patients with diffuse large B-cell lymphoma treated with preemptive lamivudine. HBV Hepatitis B virus, ALT alanine aminotransferase (7–33 IU/l), CR complete remission, UA unavailable

Patient no.

Age/sex

Ann Arbor stage

Baseline HBV DNA (copies/ml)

Peak HBV DNA after lamivudine withdrawal (copies/ml)

Baseline ALT (IU/l)

Peak ALT (IU/l)

Hepatitis flare-up/virological relapse after last dose of rituximab (months)

Outcome and status of NHL

1

42/M

III B

2.3×102

8.5×108

45

4,400

8

Alive (CR)

2

33/M

II B

4.8×102

6.7×105

28

235

6

Alive (CR)

3

41/F

III B

2.5×103

5.6×105

47

34

4

Alive (CR)

4

21/M

II B

UA

UA

13

272

6

Alive (CR)

Fig. 1

HBV viral load, ALT level, lymphocyte count, and CD20+ lymphocytes in peripheral blood during and after chemotherapy in patient no. 1. ALC absolute lymphocyte count, HBeAg hepatitis B virus envelope antigen, anti-HBeAb hepatitis B virus envelope antibody

Fig. 2

HBV viral load, ALT level, and serial CD20+ lymphocyte count in peripheral blood during and after chemotherapy in patient no. 2. ALC absolute lymphocyte count, HBeAg hepatitis B virus envelope antigen, anti-HBeAb hepatitis B virus envelope antibody

Fig. 3

HBV viral load and ALT level in patient no. 3

In the retrospectively studied patient (patient no. 4), rising serum transaminase to 272 IU/l 4 months after discontinuation of preemptive lamivudine was noted. It was probably after a virological flare-up and subsequent overt hepatitis (Fig. 4). Measurement of serum HBV DNA after the peak of ALT, i.e., after the immune clearance, was 3×102 copies/ml without reinstitution of lamivudine.
Fig. 4

ALT levels in patient no. 4

Discussion

For the past decade, lamivudine has been the treatment of choice for chronic HBV infection because it is well tolerated and effective in suppressing HBV replication. Early rather than deferred preemptive lamivudine is recommended for HBsAg+ lymphoma patients undergoing intense chemotherapy, to reduce postchemotherapy HBV-related liver morbidity and mortality [4, 7]. Nevertheless, no independent risk factor for HBV reactivation in chemotherapy-treated cancer patients has been recognized [8]. Therefore, it is generally accepted that lamivudine should be preemptively administered in all lymphoma patients carrying HBsAg prior to cytotoxic chemotherapy. However, the optimal duration of lamivudine administration has not yet been defined. An additional 1–2 months administration of lamivudine after chemotherapy is recommended according to a study by Rossi et al. [9]. Nevertheless, prolonged use of lamivudine cannot guarantee its clinical benefit, due to the emergence of a lamivudine-resistant HBV strain. On the contrary, too short a period of antiviral prophylaxis may not be effective. In this study, all of our patients received 1 additional month of lamivudine after completion of chemotherapy. They all developed either a virological or biochemical reactivation of HBV. The small number of patients is the limitation of our study. However, our study is different from previous studies because this is the first study to prospectively examine HBV DNA levels by means of an assay with high sensitivity in patients undergoing concomitant rituximab and CHOP chemotherapy. Meanwhile, all of our patients have received preemptive lamivudine therapy.

The relationship between delayed fulminant HBV reactivation and the incorporation of rituximab into conventional chemotherapy has been discussed in isolated reports but remains unclear [11, 12]. Viral infection has occasionally been reported in association with the use of rituximab, but combined chemotherapy may also contribute to this complication [13, 14]. In this study, one patient demonstrated a unique course of disease in which HBV-related severe hepatitis flare-up developed 4 months after active viral replication. The lag in HBV reactivation could be attributed to previous viral suppressive therapy and impaired immune responsiveness related to rituximab treatment. Although the immune attack against HBV-infected hepatocytes was mainly mediated by cellular immunity, i.e., CD8+ cytotoxic T lymphocytes (CTLs), B lymphocytes may also act as antigen-presenting cells, priming CTL-specific responses in HBV infection [15]. Rituximab treatment may play a role in HBV evading immune recognition and clearance and in its persistence [6]. In our study, CD20+ B cells recovered upon hepatitis flare-up in patient no. 1, nevertheless, they remained depleted during hepatitis flare-up in patient no. 2. The use of rituximab may provide a platform to study the role of B lymphocytes in the pathogenesis of hepatitis B reactivation in the future.

In a review of previously reported studies (Table 2), HBV-related hepatitis flare-up after chemotherapy including rituximab occurred during chemotherapy in two patients and within 5 months after chemotherapy in another two patients (0–5 months, mean 1.7 months) [1619]. In comparison with previously reported data, our patients had longer time lags (4–8 months, mean 6 months) to the development of HBV rebound viremia and overt hepatitis after R+CHOP therapy. This is probably because we used an HBV DNA quantitative assay with higher sensitivity. With the use of a more sensitive assay, one might be able to reintroduce lamivudine earlier to reduce the complications caused by hepatitis flare-up.
Table 2

Reported cases of hepatitis B reactivation after chemotherapy and rituximab treatment. CHOP Cyclophosphamide, doxorubicin, vincristine, and prednisone, COP cyclophosphamide, vincristine, and prednisone, C/T chemotherapy

Number

Disease HBV serology before treatment

Previous therapy

Overt hepatitis after last dose of rituximab

Therapy for hepatitis flare-up

Outcome

Reference

1

Low grade lymphoma

CHOP×5

Followed 5th course

No

Deatha

[16]

Unknown

Rituximab (weekly) ×4

Rituximab

2

B-prolymphocytic leukaemia

Previously fludarabine and cyclophosphamide

2 months

No

Deatha

[17]

HBsAg (+), HBeAg (−), Anti-HBe (+)

Rituximab (weekly) ×4

3

Follicular lymphoma

R+CHOP ×5

During C/T (5th–6th course)

Lamivudine

Recovery

[18]

HBsAg (+), HBeAg (−), Anti-HBe (+)

4

Follicular lymphoma

COP ×6

5 months

Lamivudine

Deatha

[19]

HBsAg (+), HBeAg (−), Anti-HBe (+)

Rituximab (weekly) ×4

aDeath related to fulminant hepatitis with hepatic failure

Conclusion

Preemptive lamivudine administration in HBsAg+ lymphoma patients undergoing cytotoxic chemotherapy is generally applied, as well as rituximab treatment in combination with chemotherapy. Physicians should be aware that late HBV reactivation can occur in these patients, and close surveillance of HBV serology after cessation of preemptive lamivudine is crucial.

Acknowledgments

This study was supported in part by a grant (TSGH-C92-08) from the Tri-Service General Hospital, Taipei, Taiwan.

Copyright information

© Springer-Verlag 2004