Long-term clinical and histological outcomes in patients with spontaneous hepatitis B surface antigen seroclearance

Journal of Hepatology 42 (2005) 188–194 www.elsevier.com/locate/jhep

Long-term clinical and histological outcomes in patients with spontaneous hepatitis B surface antigen seroclearance Sang Hoon Ahn1,3,†, Young Nyun Park2,3,4,5,†, Jun Yong Park1, Hye-Young Chang1, Jung Min Lee1, Ji Eun Shin1,5, Kwang-Hyub Han1,3,5, Chanil Park2,3, Young Myoung Moon1,3, Chae Yoon Chon1,3,* 1

Department of Internal Medicine, Yonsei University College of Medicine, C.P.O. Box 8044, Seoul, South Korea 2 Department of Pathology, Yonsei University College of Medicine, Seoul, South Korea 3 Yonsei Institute of Gastroenterology, Yonsei University College of Medicine, Seoul, South Korea 4 Center for Chronic Metabolic Disease, Yonsei University College of Medicine, Seoul, South Korea 5 Brain Korea 21 Project for Medical Science, Yonsei University College of Medicine, Seoul, South Korea

Background/Aims: During the natural course of hepatitis B virus (HBV) infection, the long-term clinical and histological outcomes following spontaneous hepatitis B surface antigen (HBsAg) seroclearance remain unclear. Methods: Between 1984 and 2003, 49 (9.5%) out of 432 inactive HBsAg carriers had no detectable level of circulating HBsAg. Fifteen of 49 patients had undergone paired peritoneoscopic liver biopsies. Results: During a mean follow-up period of 19.6 months after HBsAg seroclearance, 5 of 49 (10.2%) patients were noted to have HCC. Liver cirrhosis (PZ0.040), a history of perinatal infection (PZ0.005) and long-standing duration (at least 30 years) of HBsAg positivity (PZ0.002) were associated with a significantly higher risk of developing HCC. Despite HBsAg seroclearance, HBV DNA was detected in the liver tissues from all 15 patients who underwent paired liver biopsies. Necroinflammation was significantly ameliorated (P!0.0001). On the other hand, amelioration of the fibrosis score did not reach a statistically significant level (PZ0.072). Interestingly, aggravation of liver fibrosis was evident in 2 patients (13.3%) including one who had rapidly progressed to overt cirrhosis. Conclusions: In patients with spontaneous HBsAg seroclearance, necroinflammation was markedly improved and liver fibrosis was unchanged or regressed despite occult HBV infection. However, HCC developed in a minority of cases. q 2004 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved. Keywords: Hepatitis B virus; Hepatitis B Surface Antigen; Histology; Fibrosis; Outcomes

1. Introduction Hepatitis B virus (HBV) infection is the most common cause of acute and chronic liver disease worldwide, and it is Received 27 May 2004; received in revised form 15 October 2004; accepted 19 October 2004; available online 20 November 2004 * Corresponding author. Address: Department of Internal Medicine, Yonsei University College of Medicine, C.P.O. Box 8044, Seoul, South Korea. Tel.: C82 2 361 5426; fax: C82 2 393 6884. E-mail address: [email protected] (C.Y. Chon). † Both authors have contributed equally to this work. Abbreviations HBV, hepatitis B virus; HBsAg, hepatitis B surface antigen; anti-HBs, antibody to HBsAg; HBeAg, hepatitis B e antigen; anti-HBe, antibody to HBeAg.

a major global health problem [1]. The natural history of chronic HBV infection ranges from the replicative phase with active liver disease (hepatitis B e antigen [HBeAg]positive hepatitis) to low or non-replicative phase with HBeAg seroconversion and remission of liver disease (inactive carriers) [2,3]. Subsequently in some cases, spontaneous hepatitis B surface antigen (HBsAg) seroclearance, which is regarded as a surrogate marker of resolved hepatitis B, may occur with an estimated annual incidence of 0.1–2% with geographic variations [4–6]. Albeit previous reports of the long-term outcomes of patients with chronic hepatitis B after HBeAg seroconversion by antiviral treatment [7,8], the information about the clinical spectrum and the histologic activity after

0168-8278/$30.00 q 2004 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.jhep.2004.10.026

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spontaneous HBeAg and subsequently HBsAg seroclearance is scarce. To date, HBsAg seroclearance has been associated with a good prognosis, including histologic and liver function improvement that suggests a prolonged patient survival. However, progression towards liver cirrhosis and the development of hepatocellular carcinoma (HCC) has been reported to occur even after HBsAg seroclearance [9,10]. The relative risk of HCC for patients who have lost serum HBsAg is also highly controversial. It has been shown that cirrhotic patients who achieved clearance of HBsAg display a significantly lower risk of HCC than those with persistent HBs antigenemia [11,12]. On the other hand, an extraordinarily high incidence of HCC has also been reported for those patients with HBsAg seroclearance [9]. HBV DNA can be present in serum or liver tissue after HBsAg seroclearance [13–15]. Moreover, Yuki et al. [16] have reported the possibility of occult HBV infection after complete clinical recovery from acute self-limited hepatitis B. These findings suggest that HBsAg seroclearance does not necessarily imply HBV eradication. In the clinical spectrum and natural course of HBV infection, the histological changes involved with the active replicative phase to time of resolution have not been reported. The clinical significance of persistent occult HBV infection after HBsAg seroclearance remains unclear and it is still in question whether the patients with HBsAg seroclearance are really cleared of the disease. To further investigate the issue, we conducted this study on the longterm virological and histological outcomes in chronic hepatitis B patients who spontaneously achieved clearance of HBeAg and HBsAg.

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2.1. Hepatitis B virus DNA analyses from liver tissues or serum HBV DNA was extracted from the biopsy tissues or serum samples with slight modification [20,21]. The S gene of HBV was amplified by PCR as previously described with slight modification [10,21,22]. To amplify the entire HBV surface gene, the primers sets used were sense: 5 0 -GGTAAGCTTATGGAGAACACAACATCAGGA-3 0 (underlined: HindIII, 29–49), and antisense: 5 0 -TCGGAATTCTCAAATGTATACCCAAAGACAAAAGAA-3 0 (underlined: EcoRI, 683–709). The PCR product was separated on agarose gel and 680 base pair DNA band was verified on the gel. Negative and positive controls confirmed the HBV DNA band in parallel. Following cloning into pGEM-T vector, the HBV insert was released by double digestion with EcoRI and HindIII, purified by gel extraction and subcloned to pcDNA3.1, a mammalian expression vector (Invitrogen, Groningen, Netherlands). HBV serotype and genotype of all clones were determined by the direct sequence method.

2.2. Histological evaluation A target liver biopsy by peritoneoscopsy was performed on all patients. Liver specimens for histological evaluation were routinely processed and stained with hematoxylin-eosin and Masson’s trichrome. Liver histology was graded according to the Ishak et al. [23] scoring system that includes a fibrosis score (0–6) and a necroinflammatory score (0–18). The latter is the sum of four scores, piecemeal necrosis (0–4); confluent necrosis (0–6); focal lytic necrosis, apoptosis and focal inflammation (0–4); portal inflammation (0–4). Immunohistochemical staining was performed using the streptavidin-biotin amplified method (Dako, CA, USA) and the primary antibodies were HBsAg and HBcAg (Dako, CA, USA). Negative control experiments were carried out by substituting the primary antibodies for phosphate-buffered saline.

2.3. Statistical analysis All statistical analyses were performed using the Statistical Program for Social Sciences (SPSS 10.0 for Windows; SPSS Inc., Chicago, IL). All continuous variables were tested for normality using the Kolmogorov– Smirnov test. Comparison between continuous variables with normal distribution was tested by Student t test. Continuous skewed variables were tested by Mann–Whitney U-test. Wilcoxon signed rank test was used to assess histological improvement of liver fibrosis on the paired biopsies. A two-sided P value of less than 0.05 was considered significant.

2. Materials and methods Between August 1984 and February 2003, 49 (9.5%) out of 432 inactive HBsAg carriers who had visited Severance Hospital, Yonsei University College of Medicine were spontaneously cleared of circulating HBsAg. HBeAg and HBsAg seroclearance was defined as a persistent absence of antigenemia on two consecutive occasions at 6 months apart. To confirm an occult HBV infection, 15 patients agreed to receive repeated liver biopsies. We received an informed consent from all patients and the study protocol was approved by the institutional review boards. Since blind percutaneous liver biopsies have been shown to be unreliable [17–19], we included only the patients who had undergone paired peritoneoscopic biopsies, and we compared the discrepancy between the gross findings of the liver surface and the histologic or ultrasonographic findings. Besides Child-Pugh scoring system, ultrasonography complemented with splenomegaly and thrombocytopenia provided evidence of liver cirrhosis. Liver biochemistry, HBV serological tests for HBeAg and anti-HBe, and testing for serum HBV-DNA levels were checked every 6–12 months. However, serum HBsAg and anti-HBs were tested at an interval of 24 months. Ultrasonography and/or liver spiral computed tomography to detect HCC were performed every 6–12 months. All patients included in this study fulfilled the following criteria: (1) no evidence of concurrent infection with hepatitis C virus and/or anti-human immunodeficiency virus and/or hepatitis delta virus, and (2) exclusion of other causes of chronic liver disease (alcoholism, hepatotoxic drugs, autoimmune chronic hepatitis, hemochromatosis, Wilson’s disease and a1-antitrypsin deficiency).

3. Results 3.1. Clinical characteristics and outcomes of the patients with hepatitis B surface antigen (HBsAg) seroclearance During a period of 223 months, 49 (9.5%) of the 432 Korean inactive HBsAg carriers had spontaneously cleared of HBsAg as compared to an estimated annual incidence of 0.5% spontaneous seroclearance. Table 1 shows clinical characteristics of the 49 patients with HBsAg seroclearance. The mean age of the 49 patients (male 36, female 13) was 50.0 years (range:27–72). Nineteen patients had a family history of perinatal transmission. At the time of HBsAg seroclearance, 17 (34.7%) of 49 patients showed evidence of liver cirrhosis and 1 patients (2.0%) went on to further develop cirrhosis after 27 months. Seroconversion to antiHBe and anti-HBs were observed in 29 and 28 patients, respectively. All patients showed undetectable serum HBV DNA by direct hybridization assay.

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Table 1 Clinical characteristics and outcomes of 49 patients with HBsAg seroclearance

Demographic data Age in years (range) O40 years %40 years Sex (male/female) History of perinatal infection Clinical Status Liver cirrhosis (Child-Pugh score A/B/C) Splenomegaly with thrombocytopenia Virological status Positive antiHBe/antiHBs Positive HBV-DNAa Median duration with HBsAg (months, range) Median duration from HBsAg seroclearance to development of HCC (months, range)

Without development of HCC (nZ44)

With development of HCC (nZ5)

P-value

49.5G9.4 (27–67) 39 (88.6%) 5 (11.4%) 31/13 (70.5/29.5%) 14 (31.8%)

50.0G15.8 (30–72) 4 (80.0%) 1 (20.0%) 5/0 (100/0%) 5 (100%)

NS

NS 0.005

11/2/0 (25/5/0%) 9 (20.5%)

4/0/0 (80/0/0%) 4 (80.0%)

0.040 0.014

25/24 0 192 (96–792) 0

4/4 0 600 (360–864) 20 (5–37)

NS NS 0.002 –

NS; not significant, HCC; hepatocellular carcinoma. a Hybridization assay of serum HBV-DNA; sensitivity !0.5 pg/ml.

During a mean follow-up period of 19.6 months (range: 5–37 months) after HBsAg seroclearance, 5 of 49 (10.2%) patients were noted to have HCC and these patients were compared to the remaining 44 patients. All the HCC patients were diagnosed at an early stage and they underwent successful curative treatment (surgery for 3 patients and local ablation for 2 patients). Loss of HBsAg was seen more frequently in carriers aged at least 40 years or more than in those patients aged less than 40 years. However, the age factor cannot be the explanation for developing HCC as it was without a statistical differences. All the HCC patients were male subjects, but no statistical differences were noted, and this was probably due to small number of patients in the sample size. A history of perinatal infection and a longstanding duration of HBsAg positivity were associated with a significant higher risk of developing HCC (PZ0.005 and PZ0.002, respectively). HCC developed in 4 of the 17 cirrhotic patients but only in 1 of the 32 noncirrhotic patients and incidence of HCC was significantly higher in cirrhotic patients than in non-cirrhotic patients (23.5 vs. 3.1%, PZ0.040). 3.2. Histological and virological comparisons before and after HBsAg seroclearance Fifteen (11 males and 4 females) out of 49 patients had undergone paired peritoneoscopic liver biopsies before and after spontaneous loss of HBsAg. At the time of initial biopsy of the 15 HBsAg seroclearance patients, their mean age was 36.0G8.0 years (range, 23–51). The time from baseline to the follow-up liver biopsy ranged between 45 and 220 months (median 124G61.3) (Fig. 1). The median duration of the follow-up liver biopsy after HBeAg and HBsAg seroclearance were 77.0 (range, 36–220) and 2.3 (range, 0.3–35) months, respectively. Seven of 15 patients

achieved clearance of serum HBeAg that was accompanied by an elevation in serum alanine aminotransferase (ALT) levels at the time of first biopsy, and they maintained HBeAg negativity with normalization of ALT levels until HBsAg seroclearance. Six of 15 patients had been HBeAg negative with normal ALT levels throughout the follow-up periods. However, the remaining two patients (patient 2 and 7) achieved HBeAg seroconversion and subsequent normalization of ALT levels long (159 and 108 months, respectively) after their first biopsy. By the end of the follow-up, seroconversion to anti-HBs was observed in 6 (40%) patients. All the patients showed HBeAg seroclearance, while production of anti-HBe was noted in 11 (73%) patients by the end of the follow-up. For all the patients, ALT activity was persistently normal from the time of HBeAg seroclearance to the time of the follow-up liver biopsies. The histological grading and staging of liver biopsies before and after the HBsAg seroclerance was summarized in Table 3. The total grading score was significantly ameliorated from 2–11 (median 10) to 1–4 (median 2) (P!0.0001). As for changes in each inflammatory score, amelioration was observed for piecemeal necrosis (PZ 0.000) and focal lytic necrosis (PZ0.000). Piecemeal necrosis and portal inflammation were initially present in 14 (93.3%) and 15 (100%) patients, but this inflammation had disappeared by the second biopsies in 12 (85.7%) and 8 (53.3%) patients, respectively. Focal lytic necrosis was also ameliorated (P!0.0001), although mild activity has been sustained in all patients. Confluent necrosis was not evident in all of the first and second biopsies. On the other hand, amelioration of the fibrosis score did not reach a statistically significant level (PZ0.072). Reduction of the fibrosis score was observed for nine patients including two patients having a statistically significant difference

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Fig. 1. The time from baseline to the follow-up liver biopsy. The closed bars represent the period between the first and the follow-up liver biopsy. The time of HBeAg and HBsAg seroclearance are shown on the bars (;, HBeAg seroclearance points; 7, HBsAg seroclearance points). The duration from the time of HBeAg and HBsAg seroclearance to the time of the follow-up liver biopsy is shown at the right side of the figure. Liver biopsy specimens obtained at the end of follow-up were subjected to HBV DNA PCR and histological evaluation.

(improvementR2 score). Four patients did not show any difference on the paired biopsies. No histological improvement was seen in the one definitely diagnosed cirrhotic patient. Interestingly, aggravation of liver fibrosis was evident two patients including one who had rapidly progressed to overt cirrhosis. Thus, a total of 9 patients (60%) showed regression of liver fibrosis despite a persistent occult HBV infection in the liver, while liver fibrosis was unchanged in 4 patients (26.7%) and liver cirrhosis showed progression in two patients (13.3%) (Fig. 2). Although it was not perfect, peritoneoscopic diagnosis was representative of the histologic findings in most patients (Tables 2 and 3). However, the ultrasonographic findings did not correlate with the peritoneoscopic findings. Abdominal ultrasonography could not differentiate liver cirrhosis from chronic hepatitis, and this resulted in the underestimation of the status of liver disease. Only patient 7 showed the typical ultrasonographic findings of liver cirrhosis such as nodular surface and coarse parenchymal echogenecity with regenerative nodules. After the HBsAg seroclearance, serum HBV-DNA remained undetectable by conventional hybridization assay (Digene Diagnostics Inc., Maryland, USA) in all patients, while serum HBV-DNA was detected in 9 out of 15 patients by the PCR method (Table 1). Interestingly, albeit HBsAg seroclearance and undetectable serum HBV-DNA by hybridization assay, persistent HBV infection in the liver was confirmed. Liver biopsy specimens obtained from all

patients after HBsAg seroclearance were subjected to PCR test to detect the surface region of HBV genome. PCR test revealed that HBV DNA was present in the liver biopsy specimens from all of the 15 patients examined. Whereas 14 of 15 patients were infected with subtype ayw (genotype D), only patient 7 were infected with subtype adr (genotype C) in presence of the D144E mutation.

Fig. 2. The first liver biopsy feature of patient 6 showing bridging necrosis and fibrosis (A, B). In second liver biopsy, the necroinflammatory activity is markedly decreased with occasional focal inflammation and apoptosis (C). Fibrosis is also decreased with portal expansion and short fibrous septa (D). (A, H–E !40; B, C, H–E !100; D, Masson’s trichrome !100).

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a

b

36 39 33 33 51 27 46 46 28 29 38 23 32 36 44

Dx; Peritoneoscopic diagnosis, CH, min;chronic hepatitis, minimal, CH, mod;chronic hepatitis, moderate, LC, min;liver cirrhosis, minimal, LC, mod;liver cirrhosis, moderate. Hybridization assay of serum HBV-DNA; sensitivity !0.5 pg/ml, NT; not tested.

K K K K K K D144E K K K K K K K K ayw/D ayw/D ayw/D ayw/D ayw/D ayw/D adr/C ayw/D ayw/D ayw/D ayw/D ayw/D ayw/D ayw/D ayw/D C/C C/C K/C C/C C/C C/C C/C K/C C/C K/C K/C C/C K/C C/C K/C K K K K K K K K K K K K K K K K/C K/K K/C K/K K/C K/K K/C K/C K/C K/C K/C K/K K/C K/C K/C K/C K/K K/K K/K K/K K/K K/K K/C K/K K/K K/C K/K K/C K/C K/C CH,min CH,min LC,min CH,min CH,min CH,min LC,min CH,min CH,min CH,min CH,min CH,min CH,min LC,min CH,min NT NT NT NT NT NT NT NT C K K C C K K

HBV-DNAb HBeAg/ antiHBe

K/C C/K C/K C/K K/C C/K K/C K/C C/K K/C K/C C/K C/K K/C K/C CH,min CH,mod CH,mod CH,mod CH,mod CH,mod LC,mod CH,min LC,min CH,mod CH,mod CH,mod CH,mod LC,min CH,mod Yes Yes Yes Yes Yes Yes No No No No No Yes Yes Yes No

Dxa Dxa

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

F M M M M M M F M M F M F M F

(After HBsAg seroclearance) (Before HBsAg seroclearance)

HBsAg/ antiHBs

At follow-up liver biopsy At baseline liver biopsy

Family history Age

Sex

4. Discussion

Patient No

Table 2 Clinical and virological comparisons between before and after HBsAg seroclearance

HBeAg/ antiHBe

HBVDNAb

HBV-PCR (serum/tissue)

HBV serotype/ genotype

‘a’-determinant mutations

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The clinical significance of occult HBV infection after seroclearance of circulating HBsAg, and especially in cases of spontaneous clearance, is largely unknown in patients with chronic hepatitis B. Moreover its impact on liver histology particularly remains to be resolved. To address these issues, we conducted this study on the clinical outcomes of 49 patients with HBsAg seroclearence and the histological and virological characteristics in 15 patients with paired liver biopsies before and after HBsAg seroclearence. Previous reports [17–19] on sampling variability and error by blind percutaneous liver biopsy led us to select the patients who have received peritoneoscopic liver biopsy. Even after clearance of serum HBsAg, HBV DNA was persistently detected in the liver tissues from all the patients we examined. Thus, serologic recovery from chronic hepatitis B, which is marked by the absence of HBsAg does not mean there has been HBV eradication in the liver and it seems to merely represent the immunologic control of viral replication. In this study, undetectable HBsAg in the serum was due to a decrease in viremia rather than the emergence of HBsAg mutants. Only 1 of 15 (6.7%) patients harbored mutations in the major hydrophilic region of the S gene (Table 2). Functional analysis through transfection experiments in HepG2 cells indicated that the D144E mutations drastically reduced HBsAg antigenecity. All patients showed low levels of HBV covalently closed circular (ccc) DNA in the liver including patient 7 with the D144E mutations (data not shown). However, maintenance of viral replication and role of integrated HBV DNA is not yet clear. Despite of the persistence of HBV in the liver, the paired liver biopsies revealed significant amelioration of necroinflammatory scores after HBsAg seroclearance (Table 3), although there was a sustained necroinflammatory activity at very low level. Liver fibrosis was also improved in 13 (60.0%) patients, with only 2 (13.3%) patients experiencing aggravated liver cirrhosis. Thirteen of the 15 patients had HBeAg seroconversion within 1 year after the baseline liver biopsy and they had been in an inactive carrier state throughout the follow-up periods. Twelve of the 15 patients had follow-up biopsies within 6 months following HBsAg seroclearance. Therefore, the periods between the first and second liver biopsies included the inactive carrier state (after HBeAg seroclearance) and the duration after HBsAg seroclearance might not be long enough for histological changes. However, there is the possibility that HBsAg seroclearance might have occurred earlier than that suggested by the results, because HBsAg was checked at an interval of 24 months in this study. Most cases revealed no correlation between liver histology and the interval from HBeAg/HBsAg clearance, or the detection of HBV in serum or liver tissues. However, in two patients, the first biopsies were taken during

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Table 3 Histological grading and staging of liver biopsies before and after HBsAg seroclearance Patient No

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

HAI Gradea

Stageb

Baseline

Follow-up

Baseline

Follow-up

2 (0,0,1,1) 10 (4,0,2,4) 9 (3,0,3,3) 11 (4,0,3,4) 10 (3,0,3,4) 11 (4,0,3,4) 11 (4,0,3,4) 3 (1,0,1,1) 11 (4,0,3,4) 9 (3,0,3,3) 10 (4,0,3,3) 11 (4,0,3,4) 11 (4,0,3,4) 3 (1,0,1,1) 11 (4,0,3,4)

1 (0,0,1,0) 1 (0,0,1,0) 4 (1,0,2,1) 2 (0,0,2,0) 1 (0,0,1,0) 1 (0,0,1,0) 2 (0,0,1,1) 1 (0,0,1,0) 3 (1,0,1,1) 3 (0,0,2,1) 1 (0,0,1,0) 2 (0,0,1,1) 2 (0,0,1,1) 1 (0,0,1,0) 2 (0,0,1,1)

1 3 2 4 4 4 6 3 5 2 3 3 3 4 3

1 2 3 2 3 2 6 3 5 1 2 2 2 6 2

Interval A (months)c

Interval B (months)d

220 204 195 174 171 151 146 124 119 78 71 57 56 48 45

220 192 36 168 171 134 38 124 116 77 71 46 43 60 45

a P!0.0001, HAI, Hepatitis Activity Index; The activity of piecemeal necrosis, confluent necrosis, focal lytic necrosis/inflammation and portal inflammation are recorded in order in the parentheses. b PZ0.072. c Interval A; duration between baseline and the follow-up liver biopsy. d Interval B; duration between the time of HBeAg seroclearance and follow-up liver biopsy.

the HBeAg positive phase and there was long duration before HBeAg seroclearance (159 months in patient 3 and 108 months in patient 7), with mild fluctuations (no more than two times normal) of ALT levels. Therefore, fibrosis could have further progressed during the highly replicative phase preceding anti-HBe seroconversion and the follow-up biopsy period might have been too short after HBeAg clearance for noting any histological improvement in patient 3 (Fig. 1). Patient 7 that showed cirrhosis at first biopsy before HBeAg seroclearance was prone to maintain this condition even after HBsAg seroclearance. However, in patient 14, the gross and histological findings indicated that fibrosis had rapidly progressed to cirrhosis within 4 years regardless of HBsAg seroclearance, in whom the normal ALT levels and undetectable serum HBV DNA level by hybridization method, were maintained throughout the periods of two biopsies. Rapid progression, despite the full control of viral replication and inflammation in patient 14, suggests that other factors aside from necroinflammation are involved in directly influencing liver fibrogenesis. Studies on the long-term impact of occult HBV infection on liver histology are very limited, and controversy still remains. Recently Suzuki et al. [24] have recently demonstrated that mild liver inflammation, with the HBV replication fully controlled by Lamivudine, may not exert any influence on the liver fibrosis stage. The sustained strong suppression of HBV replication by lamivudine therapy led to the regression of liver fibrosis although very mild inflammation can still persist in the liver. Nevertheless, the above study population was small, and the data obtained was preliminary. Likewise, overall biochemical and histological resolutions of chronic

hepatitis B were observed in spite of the persistence of occult HBV infection in our study. These findings do suggest that there was no significant difference for the clinical and histological outcomes after the full control of HBV replication either by spontaneous immune response or by antiviral therapy. However, as was demonstrated by patient 14, occult HBV infection may influence the progression of liver fibrosis by an unknown mechanism. The majority of patients with HBsAg seroclearance may eventually show clinical remission, but such an event does not exclude the risk of HCC as a late complication, and especially for patients in the advanced stages of liver fibrosis. Very recently, Pollicino et al. [10] have provided strong evidence of an association between occult HBV infection and HCC. The relative risk of HCC for patients who have lost HBsAg is highly controversial. It has been shown that cirrhotic patients who have lost HBsAg display a significantly lower risk of HCC than those with persistent HBs antigenemia [25]. On the other hand, Huo et al. [9] have reported in a study from Taiwan an extraordinarily high incidence of HCC. Seventeen of 55 patients with HBsAg seroclearance had still developed liver cirrhosis (10.9%) and HCC (20.0%), in a mean follow-up period of 23 months. In the present study, 5 out of the 49 (10.2%) patients developed HCC after HBsAg seroclearance (Table 1). The significant higher risk for patients with liver cirrhosis, a history of perinatal infection and a longstanding duration (at least 360 months in this study) of HBsAg positivity suggest that long-standing HBV related liver injury or the viral genome integration into the liver may have initiated the carcinogenesis before or even after HBsAg seroclearance. Liver cirrhosis seems to have an

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oncogenic potential and occult HBV infection may also contribute to it. Full control of HBV replication with traceamount viral load in the liver along with the loss of HBsAg may not preclude the development of HCC at this stage. Regular screening for early detection of HCC may be required even after HBsAg seroclearance, and especially for patients with liver cirrhosis. A median period of 74.8 months (range, 25–202) from HBeAg to HBsAg seroclearance may suggest the necessity for a check-up of serum HBsAg/anti-HBs levels at a more-than-2 year interval in patients with spontaneous HBeAg seroclearance. In conclusion, we demonstrated that spontaneous HBeAg seroclearance and subsequently HBsAg seroclearance with sustained normalization of ALT levels markedly improved necro-inflammation and liver fibrosis was unchanged or regressed in spite of occult HBV infection in the liver. However, progression of fibrosis to cirrhosis and subsequently, the development of HCC cannot be totally eliminated. Practical clinical guidelines are needed to manage patients with HBsAg seroclearance, and especially for those patients with prolonged HBsAg positivity or liver cirrhosis.

Acknowledgements This work is partly supported by a grant from the Brain Korea 21 Project for Medical Science and R13-2002-05401005-0 from the Basic Research Program of the Korea Science and Engineering Foundation.

References [1] Maddrey WC. Hepatitis B: an important public health issue. J Med Virol 2000;61:326–362. [2] Lok AS, McMahon BJ. Chronic hepatitis B. Hepatology 2001;34: 1225–1241. [3] Lok AS. Chronic hepatitis B. N Engl J Med 2002;346:1682–1683. [4] Lindsay KL, Redeker AG, Ashcavai M. Delayed HBsAg clearance in chronic hepatitis B viral infection. Hepatology 1981;1:586–589. [5] Liaw YF, Sheen IS, Chen TJ, Chu CM, Pao CC. Incidence, determinants and significance of delayed clearance of serum HBsAg in chronic hepatitis B virus infection: a prospective study. Hepatology 1991;13:627–631. [6] Marrero JA, Lok AS. Occult hepatitis B virus infection in patients with hepatocellular carcinoma: Innocent bystander, cofactor, or culprit? Gastroenterology 2004;126:347–350. [7] Ruiz-Moreno M, Otero M, Millan A, Castillo I, Cabrerizo M, Jimenez FJ, et al. Clinical and histological outcome after hepatitis B e antigen to antibody seroconversion in children with chronic hepatitis B. Hepatology 1999;29:572–575. [8] Perrillo RP, Brunt EM. Hepatic histologic and immunohistochemical changes in chronic hepatitis B after prolonged clearance of hepatitis B e antigen and hepatitis B surface antigen. Ann Int Med 1991;115: 113–115.

[9] Huo TI, Wu JC, Lee PC, Chau GY, Lui WY, Tsay SH, et al. Sero-clearance of hepatitis B surface antigen in chronic carriers does not necessarily imply a good prognosis. Hepatology 1998;28: 231–236. [10] Pollicino T, Squadrito G, Cerenzia G, Cacciola I, Raffa G, Crax A, et al. Hepatitis B virus maintains its pro-oncogenic properties in the case of occult HBV infection. Gastroenterology 2004;126:102–110. [11] Fattovich G, Giustina G, Sanchez-Tapias J, Quero C, Mas A, Olivotto PG, et al. Delayed clearance of serum HBsAg in compensated cirrhosis B: relation to interferon alpha therapy and disease prognosis. European Concerted Action on Viral Hepatitis (EUROHEP). Am J Gastroenterol 1998;93:896–900. [12] Chen YC, Sheen IS, Chu CM, Liaw YF. Prognosis following spontaneous HBsAg seroclearance in chronic hepatitis B patients with or without concurrent infection. Gastroenterology 2002;123: 1084–1089. [13] Marcellin P, Martinot-Peignoux M, Loriot MA, Giostra E, Boyer N, Thiers V, et al. Persistence of hepatitis B virus DNA demonstrated by polymerase chain reaction in serum and liver after loss of HBsAg induced by antiviral therapy. Ann Int Med 1990;112:227–228. [14] Loriot MA, Marcellin P, Walker F, Boyer N, Degott C, Randrianatoavina I, et al. Persistence of hepatitis B virus DNA in serum and liver from patients with chronic hepatitis B after loss of HBsAg. J Hepatol 1997;27:251–258. [15] Chung HT, Lai CL, Lok AS. Pathogenic role of hepatitis B virus in hepatitis B surface antigen-negative decompensated cirrhosis. Hepatology 1995;22:25–29. [16] Yuki N, Nagaoka T, Yamashiro M, Mochizuki K, Kaneko A, Yamamoto K, et al. Long-term histologic and virologic outcomes of acute self-limited hepatitis B. Hepatology 2003;37:1172–1179. [17] Soloway RD, Baggenstoss AH, Schoenfield LJ, Summerskill WH. Observer error and sampling variability tested in evaluation of hepatitis and cirrhosis by liver biopsy. Am J Dig Dis 1971;16:1082–1086. [18] Abdi W, Millan JC, Mezey E. Sampling variability on percutaneous liver biopsy. Arch Int Med 1979;139:667–669. [19] Maharaj B, Maharaj RJ, Leary WP, Cooppan RM, Naran AD, Pirie D, et al. Sampling variability and its influence on the diagnostic yield of percutaneous needle biopsy of the liver. Lancet 1986;1:523–525. [20] Ahn SH, Kramvis A, Kawai S, Spangenberg HC, Li J, Kimbi G, et al. Sequence variation upstream of precore translation initiation codon reduces hepatitis B virus e antigen production. Gastroenterology 2003;125:1370–1378. [21] Kim KH, Ahn SH, Chung HY, Paik YH, Lee KS, Kim YS, et al. Hepatitis B virus infection after renal transplantation in the presence of antibody to hepatitis B surface antigen immunity. J Gastroenterol Hepatol 2004;19:847–853. [22] Kim KH, Lee KH, Chang HY, Ahn SH, Tong S, Yoon YJ, et al. Evolution of hepatitis B virus sequence from a liver transplant recipient with rapid breakthrough despite hepatitis B immune globulin prophylaxis and lamivudine therapy. J Med Virol 2003;71: 367–375. [23] Ishak K, Baptista A, Bianchi L, Callea F, DeGroote J, Gudat F, et al. Histological grading and staging of chronic hepatitis. J Hepatol 1995; 22:696–699. [24] Suzuki Y, Kumada H, Ikeda K, Chayama K, Arase Y, Saitoh S, et al. Histological changes in liver biopsies after one year of lamivudine treatment in patients with chronic hepatitis B infection. J Hepatol 1999;30:743–748. [25] Fattovich G. Natural history and prognosis of hepatitis B. Semin Liver Dis 2003;23:47–58.