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The long-term clinical outcome of 1-year treatment of chronic hepatitis B with lamivudine—5 years observation
Hepatology Research 27 (2003) 13 /17 www.elsevier.com/locate/ihepcom
The long-term clinical outcome of 1-year treatment of chronic hepatitis B with lamivudine* 5 years observation /
Shogo Ohkoshi a,*, Norio Ogata b, Takafumi Ichida a a
Third Department of Internal Medicine, Niigata University School of Medicine, 1-754 Asahimachi Dori, Niigata 951-8122, Japan b Department of Clinical and Laboratory Medicine, Toyama Medical and Pharmaceutical University, Toyama City, Japan Received 2 July 2002; received in revised form 24 March 2003; accepted 1 April 2003
Abstract The biochemical and virological outcomes of 19 patients with chronic hepatitis B who had been treated with 100 mg per day of lamivudine (LMV) for 1 year from 1995 to 1996 were evaluated. Fourteen patients were followed for 4.5 /5 years since the end of the treatment without any further active antiviral treatment. During the treatment, DNA levels of hepatitis B virus (HBV) were under the detection limit of a hybridization assay in all the 19 patients. However, YMDD mutants appeared in 5 (26%) patients during the course of treatment and were accompanied in all five by the elevation of serum alanine aminotransferase (ALT). Mutated HBV DNA was not detected at 1 year after the end of treatment in any of the 5 patients. Of the patients who were followed for 4.5 /5 years, the rate of seroconversion to anti-HBe and negativity for HBV DNA fluctuated during the course. Four of 11 patients who initially had been positive for hepatitis B virus e antigen (HBeAg) became positive for anti-HBe and all of them remained positive for HBV DNA by a transcription-mediated amplification test at the end of the follow-up. Thus, a 1-year treatment with LMV for chronic hepatitis B resulted in the relapse of HBV viraemia in most of the patients who had been positive for HBeAg, although the clinical course ameliorated in some patients. In addition, HBV DNA remained positive and ALT values were elevated at the end of the follow-up in the three patients who had been treated with interferon, with or without LMV, during the follow-up. # 2003 Elsevier B.V. All rights reserved. Keywords: Chronic hepatitis B; Lamivudine; Interferon
1. Introduction Lamivudine (2?, 3?-dideoxy-3?-thiacytidine) (LMV), a deoxycytidine analogue, has been shown to be effective in inhibiting hepatitis B virus (HBV) replication both in vitro and in vivo [1 /3]. Suppression of viral replication and subsequent disappearance of HBV DNA, detected using standard assays, can be achieved in most patients. A significant improvement in necroinflammatory activity following LMV treatment has also been shown [4,5]. However, the earlier clinical trials with LMV treatment for 12/52 weeks showed a high rate of relapse when therapy was discontinued [4 /6]. A sustained loss of HBV DNA and seroconversion from hepatitis B virus e antigen (HBeAg) to anti-HBe positivity occurs in
approximately 16% of patients treated for 1 year [4,5]. This indicates that short-term treatment with LMV cannot eradicate HBV in most patients. Deterioration of the clinical course also can result from the emergence of the YMDD variant during treatment both in patients receiving liver transplantation [7,8] and in those with chronic hepatitis [9 /11]. This causes an acute flare of alanine aminotransferase (ALT) and even can result in decompensation of the liver disease [12 /14]. These virological problems make it difficult to elucidate a standard period for LMV treatment. Here, we describe clinical outcomes of patients who were treated with LMV for 1 year from 1995 to 1996, as a phase III trial at Niigata University Hospital in Japan.
2. Patients * Corresponding author. Tel.: /81-25-227-2207; fax: /81-25-2270776. E-mail address: [email protected] (S. Ohkoshi). 1386-6346/03/$ - see front matter # 2003 Elsevier B.V. All rights reserved. doi:10.1016/S1386-6346(03)00193-1
Twenty patients with biopsy proven chronic hepatitis B were treated with 100 mg of LMV for 1 year (52
14
S. Ohkoshi et al. / Hepatology Research 27 (2003) 13 /17
weeks) as the Phase III LMV clinical trial (GlaxoWellcome, Stevenage, UK) undertaken in Japan [15]. The trial was carried out from June 1995 to March 1997. Fibrosing scores were: F0 in one patient, F1 in six, F2 in six, F3 in five and F4 in one. Hepatitis B surface antigen (HBsAg) and HBeAg/anti-HBe were determined by enzyme immunoassays (Abbott Diagnostics, Chicago, IL). HBV DNA was measured by a hybridization assay before and after treatment and the branched DNA signal amplification assay (Chiron Corp., Emeryville, CA) was used during the follow-up. HBV DNA was also examined in the stored sera which were taken at the end of the follow-up by the transcription-mediated amplification (TMA) test. YMDD mutants were detected by a direct sequencing method during the treatment [15] and by a mini-sequencing method (Genome Science Laboratory, in Japan) during the follow-up [16]. Patients who had signs of decompensated liver disease were excluded from the study. All the patients were negative for hepatitis delta virus, hepatitis C virus and human immunodeficiency virus. No patient had been treated with antivirals or corticosteroid before the entry. One patient dropped out from the study voluntarily and 19 patients completed the protocol (Fig. 1). All the 19 cases were male and their mean age was 40.6 years. Fifteen of the patients were positive for HBeAg and four were positive for anti-HBe. All the patients were positive for HBV DNA, except for patient 12 ( B/2.5 pg/ml). He was excluded from the analysis for Fig. 2A and B because of the inappropriate indication of LMV in a retrospective analysis. The patients were monitored every 4 weeks during therapy using biochemical liver function tests and HBV markers. The patients were then followed-up monthly or bimonthly at the outpatient clinic of Niigata University Hospital (Fig. 1). One patient (patient 15) was lost to follow-up at 19th month after completion of the LMV treatment. One patient (patient 16) was diagnosed with hepatocellular carcinoma 5 months after the completion of the protocol. Three patients (patients 17 /19) were retreated with LMV or treated with interferon (IFN). Thus, 14 patients were followed for biochemical and virological markers at our university hospital without further antiviral treatment for at least 5 years after the completion of the treatment, except for two patients (patient one and two) who were re-treated with LMV 4.5 years after the first treatment when it was licensed in Japan. In these two patients, laboratory data including HBV DNA titers, for the final point were taken at 4.5 years after the end of LMV treatment. Seven of these 14 (50%) patients were treated with oral administration of ursodeoxycholic acid to reduce their ALT levels. Treatment outcomes at the end of the follow-up were divided into the following three categories depending on HBV DNA titers by the TMA test and ALT values (Fig. 1). ‘Complete response (CR)’ was assigned for those
Fig. 1. Patients, their follow-up status and changes of ALT values. Upper limits of normal ALT values are revealed by straight lines. The patients were divided into four categories based on HBV DNA titers by the TMA test and ALT values at the end of the follow-up, those are, (A) HBeAg-positive patients who remained positive for HBeAg at the end of follow-up, (B) HBeAg-positive patients who became negative for HBeAg and positive for anti-HBe, (C) Anti-HBe positive patients, (D) the patients who dropped out from the follow-up or received another active antiviral treatment. Note, the patient one and two who received LMV 6 months before the end of the follow-up, were not included in the Group D, but in the A, because of the long (4.5 years), antiviral-free follow-up periods. Black and hatched bars show the LMV and IFN treatments, respectively. Black dots indicate the appearance of YMDD mutants during the LMV treatment.
who obtained both normalization of ALT values and negativity of HBV DNA by TMA. ‘Partial response (PR)’ was for those with normalization of ALT but positivity for HBV DNA. ‘Non-response (NR)’ was for those who could obtain neither CR nor PR.
3. Results During the administration of LMV, all of the patients showed an initial response leading to an undetectable level of HBV DNA, using a hybridization assay with a detection limit of 2.5 pg/ml. However, a breakthrough of HBV DNA occurred in five patients, all of whom had
S. Ohkoshi et al. / Hepatology Research 27 (2003) 13 /17
Fig. 2. Changes in mean ALT values (A) and HBV DNA titers (B) during the clinical course from the 13 patients who fulfilled complete follow-up. Small vertical bars at each time point show the value of standard deviation. The data from the patient 12 (inappropriate indication for LMV treatment) were excluded from the calculation. Students’ t -test was used for the statistical comparison between the data before and after the LMV treatment, and before treatment and the latest time point.
an elevated ALT level at the end of therapy. Direct sequence analysis of the region of the DNA polymerase gene encoding the YMDD motif showed a mutation resulting in a coding change to YIDD (four patients) or YVDD (one patient) [15]. Normalization of ALT was observed in 12/19 (63%). Of the 15 HBeAg-positive patients, anti-HBe appeared in 9 (60%) and seroconversion to anti-HBe (HBeAg-negative and anti-HBe positive) occurred in 4 (27%) when therapy was completed. After the cessation of the treatment, 17 (90%) had an elevation in ALT values due to reactivation of HBV DNA during the period of 2/10 months after treatment
15
(Fig. 2A and B). The mean interval after the cessation of LMV treatment to the peak of ALT was 5.1 months. No patients showed signs of jaundice or hepatic failure. The maximum value of total bilirubin among the patients was 2.4 mg/dl. Two patients (patient 12 and 15 in Fig. 1) did not show rebound of ALT values. Patient 15 (the youngest, 18 years-old), whose HBV DNA and ALT were 2500 pg/ ml and 171 IU/l, respectively, achieved complete seroconversion to a high titer of anti-HBe, with normalization of ALT values and negativity for HBV DNA, during the follow-up period. ALT and HBV DNA values decreased in all the patients after the flare-up. YMDD mutants were no longer detected at 1 year after the end of treatment in any of the five patients. The mean values of ALT were elevated during the follow-up in the 14 patients, who were followed-up for a long term without antivirals (Fig. 2A). The mean titer of HBV DNA (Fig. 2A) and the rate of seroconversion to anti-HBe in the 11 HBeAg-positive patients (Fig. 3) fluctuated during the course. Four of the 11 (36%) patients who initially had been positive for HBeAg seroconverted to anti-HBe at the end of the follow-up and none of them were negative for HBV DNA by TMA (Fig. 1, Group B). All the three antiHBe-positive patients remained anti-HBe-positive throughout the course (Group C). Patient 17 (anti-HBe-positive) was re-treated with LMV at the time of ALT rebound and continued. He had normalized ALT values for 33 months until YMDD mutant appeared. He was later treated with natural IFN a, but HBV DNA titer and ALT value remained high (6.1 LGE/ml and 192 IU/l, respectively) at the end of the follow-up. Patient 18 (HBeAg-positive) was treated with three MU of IFN b every day for 4 weeks against the elevation of ALT value and HBV DNA titer due to YMDD mutant, but was not seroconverted to anti-HBe. He was later treated with six MU of recombinant IFN a2a at the time of ALT elevation. He was seroconverted to anti-HBe soon after this treatment. However, he remained positive for HBV DNA (6.2 LGE/ml) and had an elevated ALT value (57 IU/l) at the end of the followup. Patient 20 (HBeAg-positive) was re-treated with LMV at the time of ALT rebound and continued thereafter. Despite the three-courses of IFN treatment, he remained positive for HBeAg with HBV DNA titer (8.0 LGE/ml) and high ALT value (171 IU/ml) at the end of the follow-up. Both patients (one and two) who were re-treated with LMV 4.5 years after the end of the first treatment did not obtain YMDD mutants and became negative for HBV DNA by TMA.
S. Ohkoshi et al. / Hepatology Research 27 (2003) 13 /17
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Fig. 3. Changes in seroconversion rate of the 11 patients who initially were positive for HBeAg and completed the follow-up.
4. Discussion LMV is an effective anti-HBV agent [1 /3]. In this study, serum HBV DNA decreased rapidly and became negative in all 19 patients. In addition, seroconversion from HBeAg to anti-HBe occurred in four of 15 (27%) patients at the end of the treatment. This effect is similar to that of past reports of 52 weeks treatment [4,5]. However, as was reported in a previous report on this trial [15], the mutation of the YMDD motif in the reverse transcriptase domain of the HBV DNA polymerase occurred in five of 19 (26%) patients at the earliest after 28 weeks. The emergence of mutant virus was always accompanied by an elevation of ALT levels, with 233 IU/l at maximum. However, because of the mandatory cessation of LMV according to the trial’s protocol, rebounding flare-ups of the ALT and HBV DNA levels overcame liver dysfunction attributable to the mutant virus. In total, 17 of the 19 patients including these five experienced a flare of ALT accompanied by an upsurge of HBV DNA. A relapse of the disease due to reactivation of HBV DNA has been noted in previous clinical trials with 26 /52 weeks of LMV treatment [3 / 6]. An incidental clinical benefit caused by the withdrawal of LMV followed by the elevation of HBV DNA could be an establishment of sustained HBe seroconversion, which results from the reconstitution of the immune response against HBV. However, the seroconversion rate to anti-HBe at 1-year after the end of the treatment, that is, after the flare of ALT due to LMV withdrawal was lower than that at the end of the treatment (Fig. 3). Thus, successful seroconversion because of the rebounding ALT elevation after LMV withdrawal is unlikely. A therapeutic choice for the patients who had YMDD mutants and the elevation of ALT values is IFN [17].
However, the effect is still controversial in general [18]. Although patients 17 /19 were actively treated with IFN when the mutants appeared and ALT elevated, none of the 3 patients achieved PR or CR at the end of the follow-up. Thus, IFN did not seem to be an effective therapy for the patients in this study. This study showed that a 1-year treatment with LMV did not lead to clinical remission in most of the patients who had been positive for HBeAg. Of the 11 HBeAgpositive patients who completed the follow-up without any active anti-viral treatment, only 2 (18%) achieved PR and none of them obtained CR. Considering the clinical remission that may occur during the natural course of chronic HBV infection, this result does not reveal any beneficial effect of 1-year treatment of LMV for HBeAg-positive patients. Recent reports have shown a good clinical outcome following an extended LMV treatment of Asian patients for more than 2 years [19,20]. Two years of LMV therapy resulted in incremental HBeAg seroconversion from 17% at week 52 to 27% at week 104 [19]. Forty (40%) of the patients who received 3 years of treatment with LMV achieved HBeAg seroconversion [20]. Although YMDD mutations appeared in 38/57% of the patients, they continued to clear serum HBeAg and did not show a major deterioration of liver function. However, because liver dysfunction caused by the mutants can be very severe, similar to that after withdrawal of LMV [12 /14], it remains controversial how long patients should be treated and how they should be managed when the mutants emerge. Serfaty et al. reported that sequential treatment with LMV and IFN result in a high rate of seroconversion to anti-HBe and negativity of HBV DNA [21]. Considering that high HBV titers may be one of the predictive factors for the appearance of YMDD mutant [22], pretreatment of IFN before LMV
S. Ohkoshi et al. / Hepatology Research 27 (2003) 13 /17
administration to lower HBV DNA level can be another choice of treatment.
References [1] Doong SL, Tsai CH, Schinazi RF, Liotta DC, Cheng YC. Inhibition of the replication of hepatitis B virus in vitro by 2?, 3?-dideoxy-3?-thiacytidine and related analogues. Proc Natl Acad Sci USA 1991;88:8495 /9. [2] Chang C-N, Doong S-L, Zhou JH, et al. Deoxycitidine deaminase-resistant stereoisomer is the active form of (///)-2?, 3? dideoxy-3?-thiacytidine in the inhibition of hepatitis B virus replication. J Biol Chem 1992;267:13938 /42. [3] Dienstag JL, Perrillo RP, Schiff ER, Bartholomew M, Vicary C, Rubin M. A preliminary trial of lamivudine for chronic hepatitis B infection. N Engl J Med 1995;333:1657 /61. [4] Lai C, Chien R, Leung N, et al. A one year trial of lamivudine for chronic hepatitis B infection. Asia Hepatitis Lamivudine Study Group. N Engl J Med 1998;339:61 /8. [5] Dienstag J, Schiff ER, Wright TL, et al. Lamivudine as initial treatment for chronic hepatitis B in the United States. N Engl J Med 1999;341:1256 /63. [6] Nevens F, Main J, Honkoop P, et al. Lamivudine therapy for chronic hepatitis B: a six month randomized dose-ranging study. Gastroenterology 1997;113:1258 /63. [7] Ling R, Mutimer D, Ahmed M, et al. Selection of mutations in the hepatitis B virus polymerase during the therapy of transplant recipients with lamivudine. Hepatology 1996;24:711 /3. [8] Bartholomew MM, Jansen RW, Jeffers LJ, et al. Hepatitis-Bvirus resistance to lamivudine given for recurrent infection after orthotopic liver transplantation. Lancet 1997;349:20 /2. [9] Allen MI, Deslauriers M, Andrews CW, et al. Identification and characterization of mutations in hepatitis B virus resistant to lamivudine Lamivudine Clinical Investigation Group. Hepatology 1998;27:1670 /7. [10] Buti M, Jardi R, Cotrina M, Rodriguez-Frias F, Esteban R, Guardia J. Transient emergence of hepatitis B variants in a patient with chronic hepatitis B resistant to lamivudine. J Hepatol 1998;28:510 /3. [11] Chayama K, Suzuki Y, Kobayashi M, et al. Emergence and takeover of YMDD motif mutant hepatitis B virus during long-
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term lamivudine therapy and re-takeover by wild type after cessation of therapy. Hepatology 1998;27:1711 /6. Liaw Y-F, Chien R-N, Yeh C-T, Tsai S-L, Chu C-M. Acute exacerbation and hepatitis B virus clearance after emergence of YMDD motif mutation during lamivudine therapy. Hepatology 1990;30:567 /72. Bruno R, Sacchi P, Malfitano A, Filice G. YMDD-mutant HBV strain as a cause of liver failure in an HIV-infected patient. Gastroenterology 2001;121:1027 /8. Bessesen M, Ives D, Condreay L, et al. Chronic active hepatitis B exacerbations in human immunodeficiency virus-infected patients following development of resistance to or withdrawal of lamivudine. Clin Infect Dis 1999;28:1032 /5. Ogata N, Fujii K, Takigawa S, Nomoto M, Ichida T, Asakura H. Novel patterns of amino acid mutations in the hepatitis B virus polymerase in association with resistance to lamivudine therapy in Japanese patients with chronic hepatitis B. J Med Virol 1999;59:270 /6. Kobayashi S, Shimada K, Suzuki H, Tanikawa K, Sata M. Development of a new method for detecting a mutation in the gene encoding hepatitis B virus reverse transcriptase active site(YMDD) motif. Hepatol Res 2000;17:31 /42. Someya T, Suzuki Y, Arase Y, et al. Interferon therapy for flareup of hepatitis B virus infection after the emergence of lamivudine-induced YMDD motif mutant. J Gastroenterol 2001;36:133 /6. Liaw Y-F. Management of YMDD mutations during lamivudine therapy in patients with chronic hepatitis B. J Gastroenterol Hepatol 2002(Suppl. 3):S333 /7. Liaw Y-F, Leung NWY, Chang TT, et al. Effects of extended lamivudine therapy in Asian patients with chronic hepatitis B. Gastroenterology 2000;119:172 /80. Leung NWY, Lai CL, Chang TT, et al. Extended lamivudine treatment in patients with chronic hepatitis B enhances hepatitis B e antigen seroconversion rates: results after 3 years of therapy. Hepatology 2001;33:1527 /32. Serfaty L, Thabut D, Zoulim F, et al. Sequential treatment with lamivudine and interferon monotherapies in patients with chronic hepatitis B not responding to interferon alone. Hepatology 2001;34:573 /7. Yuen M-F, Sablon E, Hui C-K, Yuan H-J, Decraemer H, Lai CL. Factors associated with hepatitis B virus DNA breakthrough in patients receiving prolonged LMV therapy. Hepatology 2001;34:785 /91.
The long-term clinical outcome of 1-year treatment of chronic hepatitis B with lamivudine* 5 years observation /
Shogo Ohkoshi a,*, Norio Ogata b, Takafumi Ichida a a
Third Department of Internal Medicine, Niigata University School of Medicine, 1-754 Asahimachi Dori, Niigata 951-8122, Japan b Department of Clinical and Laboratory Medicine, Toyama Medical and Pharmaceutical University, Toyama City, Japan Received 2 July 2002; received in revised form 24 March 2003; accepted 1 April 2003
Abstract The biochemical and virological outcomes of 19 patients with chronic hepatitis B who had been treated with 100 mg per day of lamivudine (LMV) for 1 year from 1995 to 1996 were evaluated. Fourteen patients were followed for 4.5 /5 years since the end of the treatment without any further active antiviral treatment. During the treatment, DNA levels of hepatitis B virus (HBV) were under the detection limit of a hybridization assay in all the 19 patients. However, YMDD mutants appeared in 5 (26%) patients during the course of treatment and were accompanied in all five by the elevation of serum alanine aminotransferase (ALT). Mutated HBV DNA was not detected at 1 year after the end of treatment in any of the 5 patients. Of the patients who were followed for 4.5 /5 years, the rate of seroconversion to anti-HBe and negativity for HBV DNA fluctuated during the course. Four of 11 patients who initially had been positive for hepatitis B virus e antigen (HBeAg) became positive for anti-HBe and all of them remained positive for HBV DNA by a transcription-mediated amplification test at the end of the follow-up. Thus, a 1-year treatment with LMV for chronic hepatitis B resulted in the relapse of HBV viraemia in most of the patients who had been positive for HBeAg, although the clinical course ameliorated in some patients. In addition, HBV DNA remained positive and ALT values were elevated at the end of the follow-up in the three patients who had been treated with interferon, with or without LMV, during the follow-up. # 2003 Elsevier B.V. All rights reserved. Keywords: Chronic hepatitis B; Lamivudine; Interferon
1. Introduction Lamivudine (2?, 3?-dideoxy-3?-thiacytidine) (LMV), a deoxycytidine analogue, has been shown to be effective in inhibiting hepatitis B virus (HBV) replication both in vitro and in vivo [1 /3]. Suppression of viral replication and subsequent disappearance of HBV DNA, detected using standard assays, can be achieved in most patients. A significant improvement in necroinflammatory activity following LMV treatment has also been shown [4,5]. However, the earlier clinical trials with LMV treatment for 12/52 weeks showed a high rate of relapse when therapy was discontinued [4 /6]. A sustained loss of HBV DNA and seroconversion from hepatitis B virus e antigen (HBeAg) to anti-HBe positivity occurs in
approximately 16% of patients treated for 1 year [4,5]. This indicates that short-term treatment with LMV cannot eradicate HBV in most patients. Deterioration of the clinical course also can result from the emergence of the YMDD variant during treatment both in patients receiving liver transplantation [7,8] and in those with chronic hepatitis [9 /11]. This causes an acute flare of alanine aminotransferase (ALT) and even can result in decompensation of the liver disease [12 /14]. These virological problems make it difficult to elucidate a standard period for LMV treatment. Here, we describe clinical outcomes of patients who were treated with LMV for 1 year from 1995 to 1996, as a phase III trial at Niigata University Hospital in Japan.
2. Patients * Corresponding author. Tel.: /81-25-227-2207; fax: /81-25-2270776. E-mail address: [email protected] (S. Ohkoshi). 1386-6346/03/$ - see front matter # 2003 Elsevier B.V. All rights reserved. doi:10.1016/S1386-6346(03)00193-1
Twenty patients with biopsy proven chronic hepatitis B were treated with 100 mg of LMV for 1 year (52
14
S. Ohkoshi et al. / Hepatology Research 27 (2003) 13 /17
weeks) as the Phase III LMV clinical trial (GlaxoWellcome, Stevenage, UK) undertaken in Japan [15]. The trial was carried out from June 1995 to March 1997. Fibrosing scores were: F0 in one patient, F1 in six, F2 in six, F3 in five and F4 in one. Hepatitis B surface antigen (HBsAg) and HBeAg/anti-HBe were determined by enzyme immunoassays (Abbott Diagnostics, Chicago, IL). HBV DNA was measured by a hybridization assay before and after treatment and the branched DNA signal amplification assay (Chiron Corp., Emeryville, CA) was used during the follow-up. HBV DNA was also examined in the stored sera which were taken at the end of the follow-up by the transcription-mediated amplification (TMA) test. YMDD mutants were detected by a direct sequencing method during the treatment [15] and by a mini-sequencing method (Genome Science Laboratory, in Japan) during the follow-up [16]. Patients who had signs of decompensated liver disease were excluded from the study. All the patients were negative for hepatitis delta virus, hepatitis C virus and human immunodeficiency virus. No patient had been treated with antivirals or corticosteroid before the entry. One patient dropped out from the study voluntarily and 19 patients completed the protocol (Fig. 1). All the 19 cases were male and their mean age was 40.6 years. Fifteen of the patients were positive for HBeAg and four were positive for anti-HBe. All the patients were positive for HBV DNA, except for patient 12 ( B/2.5 pg/ml). He was excluded from the analysis for Fig. 2A and B because of the inappropriate indication of LMV in a retrospective analysis. The patients were monitored every 4 weeks during therapy using biochemical liver function tests and HBV markers. The patients were then followed-up monthly or bimonthly at the outpatient clinic of Niigata University Hospital (Fig. 1). One patient (patient 15) was lost to follow-up at 19th month after completion of the LMV treatment. One patient (patient 16) was diagnosed with hepatocellular carcinoma 5 months after the completion of the protocol. Three patients (patients 17 /19) were retreated with LMV or treated with interferon (IFN). Thus, 14 patients were followed for biochemical and virological markers at our university hospital without further antiviral treatment for at least 5 years after the completion of the treatment, except for two patients (patient one and two) who were re-treated with LMV 4.5 years after the first treatment when it was licensed in Japan. In these two patients, laboratory data including HBV DNA titers, for the final point were taken at 4.5 years after the end of LMV treatment. Seven of these 14 (50%) patients were treated with oral administration of ursodeoxycholic acid to reduce their ALT levels. Treatment outcomes at the end of the follow-up were divided into the following three categories depending on HBV DNA titers by the TMA test and ALT values (Fig. 1). ‘Complete response (CR)’ was assigned for those
Fig. 1. Patients, their follow-up status and changes of ALT values. Upper limits of normal ALT values are revealed by straight lines. The patients were divided into four categories based on HBV DNA titers by the TMA test and ALT values at the end of the follow-up, those are, (A) HBeAg-positive patients who remained positive for HBeAg at the end of follow-up, (B) HBeAg-positive patients who became negative for HBeAg and positive for anti-HBe, (C) Anti-HBe positive patients, (D) the patients who dropped out from the follow-up or received another active antiviral treatment. Note, the patient one and two who received LMV 6 months before the end of the follow-up, were not included in the Group D, but in the A, because of the long (4.5 years), antiviral-free follow-up periods. Black and hatched bars show the LMV and IFN treatments, respectively. Black dots indicate the appearance of YMDD mutants during the LMV treatment.
who obtained both normalization of ALT values and negativity of HBV DNA by TMA. ‘Partial response (PR)’ was for those with normalization of ALT but positivity for HBV DNA. ‘Non-response (NR)’ was for those who could obtain neither CR nor PR.
3. Results During the administration of LMV, all of the patients showed an initial response leading to an undetectable level of HBV DNA, using a hybridization assay with a detection limit of 2.5 pg/ml. However, a breakthrough of HBV DNA occurred in five patients, all of whom had
S. Ohkoshi et al. / Hepatology Research 27 (2003) 13 /17
Fig. 2. Changes in mean ALT values (A) and HBV DNA titers (B) during the clinical course from the 13 patients who fulfilled complete follow-up. Small vertical bars at each time point show the value of standard deviation. The data from the patient 12 (inappropriate indication for LMV treatment) were excluded from the calculation. Students’ t -test was used for the statistical comparison between the data before and after the LMV treatment, and before treatment and the latest time point.
an elevated ALT level at the end of therapy. Direct sequence analysis of the region of the DNA polymerase gene encoding the YMDD motif showed a mutation resulting in a coding change to YIDD (four patients) or YVDD (one patient) [15]. Normalization of ALT was observed in 12/19 (63%). Of the 15 HBeAg-positive patients, anti-HBe appeared in 9 (60%) and seroconversion to anti-HBe (HBeAg-negative and anti-HBe positive) occurred in 4 (27%) when therapy was completed. After the cessation of the treatment, 17 (90%) had an elevation in ALT values due to reactivation of HBV DNA during the period of 2/10 months after treatment
15
(Fig. 2A and B). The mean interval after the cessation of LMV treatment to the peak of ALT was 5.1 months. No patients showed signs of jaundice or hepatic failure. The maximum value of total bilirubin among the patients was 2.4 mg/dl. Two patients (patient 12 and 15 in Fig. 1) did not show rebound of ALT values. Patient 15 (the youngest, 18 years-old), whose HBV DNA and ALT were 2500 pg/ ml and 171 IU/l, respectively, achieved complete seroconversion to a high titer of anti-HBe, with normalization of ALT values and negativity for HBV DNA, during the follow-up period. ALT and HBV DNA values decreased in all the patients after the flare-up. YMDD mutants were no longer detected at 1 year after the end of treatment in any of the five patients. The mean values of ALT were elevated during the follow-up in the 14 patients, who were followed-up for a long term without antivirals (Fig. 2A). The mean titer of HBV DNA (Fig. 2A) and the rate of seroconversion to anti-HBe in the 11 HBeAg-positive patients (Fig. 3) fluctuated during the course. Four of the 11 (36%) patients who initially had been positive for HBeAg seroconverted to anti-HBe at the end of the follow-up and none of them were negative for HBV DNA by TMA (Fig. 1, Group B). All the three antiHBe-positive patients remained anti-HBe-positive throughout the course (Group C). Patient 17 (anti-HBe-positive) was re-treated with LMV at the time of ALT rebound and continued. He had normalized ALT values for 33 months until YMDD mutant appeared. He was later treated with natural IFN a, but HBV DNA titer and ALT value remained high (6.1 LGE/ml and 192 IU/l, respectively) at the end of the follow-up. Patient 18 (HBeAg-positive) was treated with three MU of IFN b every day for 4 weeks against the elevation of ALT value and HBV DNA titer due to YMDD mutant, but was not seroconverted to anti-HBe. He was later treated with six MU of recombinant IFN a2a at the time of ALT elevation. He was seroconverted to anti-HBe soon after this treatment. However, he remained positive for HBV DNA (6.2 LGE/ml) and had an elevated ALT value (57 IU/l) at the end of the followup. Patient 20 (HBeAg-positive) was re-treated with LMV at the time of ALT rebound and continued thereafter. Despite the three-courses of IFN treatment, he remained positive for HBeAg with HBV DNA titer (8.0 LGE/ml) and high ALT value (171 IU/ml) at the end of the follow-up. Both patients (one and two) who were re-treated with LMV 4.5 years after the end of the first treatment did not obtain YMDD mutants and became negative for HBV DNA by TMA.
S. Ohkoshi et al. / Hepatology Research 27 (2003) 13 /17
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Fig. 3. Changes in seroconversion rate of the 11 patients who initially were positive for HBeAg and completed the follow-up.
4. Discussion LMV is an effective anti-HBV agent [1 /3]. In this study, serum HBV DNA decreased rapidly and became negative in all 19 patients. In addition, seroconversion from HBeAg to anti-HBe occurred in four of 15 (27%) patients at the end of the treatment. This effect is similar to that of past reports of 52 weeks treatment [4,5]. However, as was reported in a previous report on this trial [15], the mutation of the YMDD motif in the reverse transcriptase domain of the HBV DNA polymerase occurred in five of 19 (26%) patients at the earliest after 28 weeks. The emergence of mutant virus was always accompanied by an elevation of ALT levels, with 233 IU/l at maximum. However, because of the mandatory cessation of LMV according to the trial’s protocol, rebounding flare-ups of the ALT and HBV DNA levels overcame liver dysfunction attributable to the mutant virus. In total, 17 of the 19 patients including these five experienced a flare of ALT accompanied by an upsurge of HBV DNA. A relapse of the disease due to reactivation of HBV DNA has been noted in previous clinical trials with 26 /52 weeks of LMV treatment [3 / 6]. An incidental clinical benefit caused by the withdrawal of LMV followed by the elevation of HBV DNA could be an establishment of sustained HBe seroconversion, which results from the reconstitution of the immune response against HBV. However, the seroconversion rate to anti-HBe at 1-year after the end of the treatment, that is, after the flare of ALT due to LMV withdrawal was lower than that at the end of the treatment (Fig. 3). Thus, successful seroconversion because of the rebounding ALT elevation after LMV withdrawal is unlikely. A therapeutic choice for the patients who had YMDD mutants and the elevation of ALT values is IFN [17].
However, the effect is still controversial in general [18]. Although patients 17 /19 were actively treated with IFN when the mutants appeared and ALT elevated, none of the 3 patients achieved PR or CR at the end of the follow-up. Thus, IFN did not seem to be an effective therapy for the patients in this study. This study showed that a 1-year treatment with LMV did not lead to clinical remission in most of the patients who had been positive for HBeAg. Of the 11 HBeAgpositive patients who completed the follow-up without any active anti-viral treatment, only 2 (18%) achieved PR and none of them obtained CR. Considering the clinical remission that may occur during the natural course of chronic HBV infection, this result does not reveal any beneficial effect of 1-year treatment of LMV for HBeAg-positive patients. Recent reports have shown a good clinical outcome following an extended LMV treatment of Asian patients for more than 2 years [19,20]. Two years of LMV therapy resulted in incremental HBeAg seroconversion from 17% at week 52 to 27% at week 104 [19]. Forty (40%) of the patients who received 3 years of treatment with LMV achieved HBeAg seroconversion [20]. Although YMDD mutations appeared in 38/57% of the patients, they continued to clear serum HBeAg and did not show a major deterioration of liver function. However, because liver dysfunction caused by the mutants can be very severe, similar to that after withdrawal of LMV [12 /14], it remains controversial how long patients should be treated and how they should be managed when the mutants emerge. Serfaty et al. reported that sequential treatment with LMV and IFN result in a high rate of seroconversion to anti-HBe and negativity of HBV DNA [21]. Considering that high HBV titers may be one of the predictive factors for the appearance of YMDD mutant [22], pretreatment of IFN before LMV
S. Ohkoshi et al. / Hepatology Research 27 (2003) 13 /17
administration to lower HBV DNA level can be another choice of treatment.
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