Seroclearance of hepatitis B surface antigen (HBsAg) is

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1 GASTROENTEROLOGY 2008;135: HBsAg Seroclearance in Chronic Hepatitis B in Asian Patients: Replicative Level and Risk of Hepatocellular Carcinoma MAN FUNG YUEN,* DANNY KA HO WONG,* JAMES FUNG,* PHILIP IP, DAVID BUT,* IVAN HUNG,* KEVIN LAU,* JOHN CHI HANG YUEN,* and CHING LUNG LAI* *Department of Medicine, The University of Hong Kong, Queen Mary Hospital, Hong Kong; and Department of Pathology, The University of Hong Kong, Queen Mary Hospital, Hong Kong Background & Aims: Our aims were to study the virologic, histologic, and clinical outcome in chronic hepatitis B (CHB) patients with hepatitis B surface antigen (HBsAg) seroclearance. Methods: We determined the age of HBsAg seroclearance that is associated with a lower risk for hepatocellular carcinoma (HCC) in 298 CHB patients (median follow-up, 108 months). The following virologic and histologic features were also determined: liver stiffness (n 229), liver histology, serum HBV DNA levels over time (n 265), intrahepatic HBV DNA with covalently closed circular DNA (cccdna) levels, and messenger RNA (mrna) expression. Results: The median age of HBsAg seroclearance was 49.6 years. Seven (2.4%) patients developed HCC. Cumulative risk for HCC was higher in patients with HBsAg seroclearance at ages >50 years compared with those with HBsAg seroclearance at ages <50 (P.004) years. Of these 2 groups of patients, 29.5% and 7.9%, respectively, had significant fibrosis by liver stiffness measurement (P.001), and 15.4% of patients had mild histologic fibrosis. Intrahepatic total HBV DNA and cccdna were detected in 100% and 79.3% of patients, respectively. All patients had undetectable surface and precore/pregenomic RNA transcripts. One (9.1%) patient had X mrna expression. Serum HBV DNA were detectable in 13.4%, 6.1%, and 3.7% of patients within 1 year and 5 10 and >10 years after HBsAg seroclearance, respectively, and 82.1% patients had persistently normal alanine aminotransferase levels. Conclusions: HBV persisted at low replicative and transcriptional levels after HBsAg seroclearance. HBsAg seroclearance at age <50 years was associated with a lower risk for the development of HCC. Seroclearance of hepatitis B surface antigen (HBsAg) is a rare event in chronic hepatitis B (CHB) infection. Its incidence is estimated to be approximately 0.1% 0.8% per year in patients acquiring CHB early in life (ie, Asian, African, and some Mediterranean patients) and 0.4% 2% per year in patients acquiring CHB during adolescence and adulthood (ie, white patients). 1 8 HBsAg seroclearance is also observed in patients receiving treatment for CHB including interferon and nucleoside/nucleotide analogues Because of the rarity of HBsAg seroclearance, several issues concerning its clinical and virologic impacts for these patients are still largely unknown. First, it is not known whether these patients still harbor the hepatitis B virus (HBV). Several studies show that the majority of these patients has detectable HBV in the liver, although one study found no detectable DNA in the liver. 6 However, these findings remain controversial because of the limited number of patients with liver tissues for detailed virologic examination. In addition, the assays used in the past to detect extremely low viral load may not be of sufficient sensitivity. Second, data with longitudinal follow-up of the liver biochemistry and detectability of HBV DNA in the sera of these patients are lacking. Third, with respect to clinical outcome, it has been found that in general there is no reduction in the risk for development of hepatocellular carcinoma (HCC) in patients with HBsAg seroclearance compared with those who are persistently positive for HBsAg. 16,17 However, whether HBsAg seroclearance at an earlier age is associated with a lower risk for the development of HCC has not been studied. Similarly, whether earlier age of HBsAg seroclearance is associated with lower chance of significant fibrosis and cirrhosis is also unknown. All these unresolved issues need to be addressed by a longitudinal and large-scale study. We therefore conducted the present large-scale longitudinal study comprising 298 CHB patients with HBsAg seroclearance. The primary aim was to determine whether age of HBsAg seroclearance was a prognostic factor for the development of significant fibrosis and HCC. The secondary aim was to define the virologic status both in the liver and serum and determine the liver biochemical status of these patients upon long-term follow-up. Abbreviations used in this paper: cccdna, covalently closed circular DNA; CHB, chronic hepatitis B by the AGA Institute /08/$34.00 doi: /j.gastro

2 October 2008 HBsAg SEROCLEARANCE OF CHRONIC HEPATITIS B 1193 Patients and Methods Patients Patients were recruited from the Liver Clinic of Queen Mary Hospital, The University of Hong Kong, Hong Kong, during the period between January 1980 and December All CHB patients who were persistently HBsAg positive for more than 6 months with HBsAg seroclearance during subsequent follow-ups were recruited for the present study. HBsAg seroclearance was defined as loss of serum HBsAg, measured by Abbott Laboratories, Chicago, IL, on repeated testing for a period of at least 6 months and during all subsequent follow-ups until the time of analysis. Patients with cirrhosis-related complications and HCC on presentation to our Liver Clinic were excluded. Patients who had concomitant hepatitis C and D infection, evidence of Wilson s disease, autoimmune hepatitis, primary biliary cirrhosis, and significant intake of alcohol (20 g per day for female; 30 g per day for male) were also excluded. The study was approved by the Institutional Review Board, The University of Hong Kong and West Cluster of Hospital Authority, Hong Kong. Laboratory Monitoring and Surveillance for HCC Patients were followed up every 3 6 months for clinical assessment. During each follow-up, liver biochemistry, -fetoprotein (AFP) and HBV serology including HBsAg, antibody to HBsAg (anti-hbs), hepatitis B e antigen (HBeAg), and antibody to HBeAg (anti-hbe) measured by Abbott Laboratories, Chicago, IL, were checked. Ultrasounds of the liver were performed in patients with elevated AFP levels. Further imaging including computerized tomography, magnetic resonance imaging, and hepatic angiogram were performed for suspicious space-occupying lesion shown in ultrasound. HCC were diagnosed by the typical imaging features by the above imaging modalities with concomitant elevation of AFP levels or by histology. Measurement of Liver Stiffness Transient elastography was performed using the Fibroscan (EchoSens, Paris, France), which consisted of a probe with an ultrasound transducer mounted on the axis of a vibrator. A vibration of mild amplitude and low frequency was transmitted to the liver tissue via this probe, which induced an elastic shear wave. The velocity of this shear wave was measured by pulse-echo ultrasound, which is directly related to liver stiffness. Assisted by time-motion ultrasound image, measurements were taken once a segment of liver is located with thickness of over 6 cm and free of large vascular structures. At least 10 valid measurements were performed on each patient, with the median value being representative of liver stiffness. Liver stiffness scores are expressed in units of kilopascals (kpa). According to a study of 220 CHB patients correlating the liver stiffness and liver histology, 18 liver stiffness scores of 8.1 kpa and 10.3 kpa were adopted as a cut off for the presence of significant liver fibrosis ( F3 according to the Ishak scoring) and underlying cirrhosis, respectively. Measurement of Serial Serum HBV DNA Levels After HBsAg Seroclearance Serum HBV DNA levels after HBsAg seroclearance were measured within 1 year in 142 patients, at 5 10 years in 99 patients and more than 10 years in 27 patients. HBV DNA was extracted from 500 L serum using the QIAamp DSP Virus Kit (Qiagen GmbH, Hilden, Germany) according to the manufacturer s instructions, with a final elution of 26 L. HBV DNA in the extracts was then quantitatively measured by the Artus HBV RG PCR Kit (Qiagen Hamburg GmbH, Hamburg, Germany), using the Rotor-Gene 3000 Real-time Multiplexing System (Corbett Research, Mortlake, Australia), according to the manufacturer s instructions. It is a standard commercial assay for the detection of HBV DNA in human plasma samples. This internal controlled assay is calibrated with the International HBV standard (World Health Organization, Geneva, Switzerland), and it is designed for quantitative in vitro diagnostic use. This test has a lower limit of detection of 1.1 IU/mL (6.38 copies/ml) of serum. Isolation of Intrahepatic DNA and RNA All patients were interviewed, and liver biopsies were requested for virologic and histologic assessments. Twenty-nine patients agreed to have the liver biopsy. Total liver DNA and RNA were extracted from approximately 5 mg of fine-needle liver biopsy specimens using the QIAamp DNA Mini Kit and QIAamp RNA Mini Kit (Qiagen GmbH), respectively, as previously described. 19 RNase-free DNase (Qiagen GmbH) was used to remove DNA contamination in the RNA extracts, and the amount of liver RNA extracted was measured by the Quant-IT Ribogreen RNA Kit (Molecular Probes, Eugene, OR). Quantitative Measurement of Intrahepatic Total HBV DNA and Covalently Closed Circular DNA Real-time polymerase chain reaction (PCR) was performed for the detection of total intrahepatic HBV DNA, covalently closed circular DNA (cccdna), and human genomic DNA using differential primers and FRET hybridization probes. All real-time PCR was performed in the Rotor-Gene 3000 Real-time Multiplexing System (Corbett Research). For the detection of total intrahepatic HBV DNA, primers and probes targeting to the S region of the HBV genome were used. 19 The -globulin gene was used as an internal control gene for the quantitation of human genomic DNA in the liver extracts, using -globulin control primers and probes from the LightCycler Control DNA Kit (Roche Applied Science,

3 1194 YUEN ET AL GASTROENTEROLOGY Vol. 135, No. 4 Mannheim, Germany). The amount of total intrahepatic HBV DNA was expressed as HBV copies/cell. For the quantitation of HBV cccdna, aliquots of liverextracted DNA were treated with Plasmid-safe DNase (Epicentre, Madison, WI). A specific set of primers, which span across the incomplete region in the HBV relaxed circular DNA genome, was employed for the quantitation of HBV cccdna, and real-time detection was performed with the use of FRET hybridization probes as suggested. 20 Reverse Transcription and Analysis of HBV-Specific Transcripts Expression of HBV-specific transcripts was detected by reverse transcription (RT)-PCR as previously described. 19 Briefly, 0.5 g liver RNA was used for first strand complementary DNA (cdna) synthesis. Specific primers for the HBV surface (S), X, and precore/pregenomic regions, as well as primers for -actin internal reference gene, were used for the detection of RNA expression. Histologic Assessment Histologic activity index scores were assessed according to the criteria of Knodell et al. 21 Immunoperoxidase staining for HBsAg and hepatitis B core antigen (HBcAg) were performed using the respective monoclonal antibodies (Signet, Dedham, MA; dilution, 1:2). Statistical Analysis All statistical analyses were performed using the Statistical Program for Social Sciences (SPSS 14.0 for Windows; SPSS Inc, Chicago, IL). Mann Whitney U test and Kruskal Wallis test were used for continuous variables with skewed distribution for 2- and 3-group comparison, respectively, and 2 test with Yates correction factor or Fisher exact test were applied for categorical variables. Kaplan Meier method using log-rank test was applied for the cumulative rate of development of HCC. Results Demographic Data A total of 298 patients with HBsAg seroclearance were recruited. The demographic data and liver biochemistry on presentation are listed in Table 1. Of these 298 patients, 285 (95.6%) had spontaneous HBsAg seroclearance without receiving any treatment, and 10 received lamivudine and 3 received 16 weeks of conventional interferon- therapy. These 13 patients had undetectable HBV DNA levels in the serum. Lamivudine was stopped in these patients. There was no difference in the median age of HBsAg seroclearance between patients with spontaneous HBsAg seroclearance and patients with treatment-induced HBsAg seroclearance (45.3 [range, ] vs 49.9 [range, ] years, respectively, P.09). Table 1. Demographic Data and Liver Biochemistry on Presentation of the Studied Population Number of patients 298 Male/female 211/87 HBeAg/anti-HBe 44/254 a Age on presentation, y 43.1 ( ) Age of HBeAg seroconversion for HBeAg 36.9 ( ) positive-patients on presentation, y Age of HBsAg seroclearance, y 49.6 ( ) Duration of follow-up, mo ( ) Duration of follow-up after HBsAg 36.4 ( ) seroclearance, mo Liver biochemistry Albumin level, g/l 45 (39 56) Bilirubin level, mol/l 11 (3 32) Alanine aminotransferase level, U/L 30 (4 252) -Fetoprotein level, ng/ml 3 (1 194) NOTE. Continuous variables are expressed in median (range). a All HBeAg positive patients underwent HBeAg seroconversion with anti-hbe before the occurrence of HBsAg seroclearance. HBV Serologic and Biochemical Profiles After HBsAg Seroclearance In the 298 patients, 154 (51.7%) had detectable anti-hbs after HBsAg seroclearance until the time of last follow-up. Thirty-four (11.4%) and 50 (16.8%) patients developed anti-hbs at the time and within 12 months of HBsAg seroclearance, respectively. There were no differences in the cumulative rates of development of HCC between patients with and without detectable anti-hbs (P.18). The absence of this difference was also observed when we compared patients with and without detectable anti-hbs at the time of HBsAg (P.49) or within 12 months of HBsAg seroclearance (P.66). Two hundred twelve patients (71.1%) had serial liver biochemistry monitoring for at least 4 follow-ups (range, 4 19) after HBsAg seroclearance. Of these, the percentage of persistently normal value for albumin level ( 40 g/l) and bilirubin level ( 19 mol/l) were 71.2% (n 151) and 68.9% (n 146), respectively. For the alanine aminotransferase (ALT) levels, 174 (82.1%) patients had persistently normal ALT levels ( 53 U/L for male, 31 U/L for female), 18 (8.5%) had normal ALT levels in at least 80% of the follow-up visits, and 20 (9.5%) had abnormal ALT levels for more than 20% of the follow-up visits. Of the latter 2 groups (n 38) with abnormal ALT levels, 16 (42.1%) patients had ultrasonographic evidence of fatty liver. Of the remaining 22 patients, 10 had recent intake of traditional herbal medicines; 2 had HCC; 3 had biopsy-proven fatty infiltration; 1 had persistently low viremia of HBV of 30.4 copies/ml and 53.6 copies/ml at 5 and 10 years after HBsAg seroclearance, respectively; and 6 had no obvious identifiable causes. The median values for albumin level, bilirubin level, and ALT level at the last follow-up of all 298 patients were 44 g/l (range, 27 52), 11 mol/l (range, 3 218), and 21 U/L (range, 7 136), respectively.

4 October 2008 HBsAg SEROCLEARANCE OF CHRONIC HEPATITIS B 1195 Recently, a stricter criteria of normal ALT levels ( 30 U/L for male, 19 U/L for female) has been proposed. 22 Using these criteria, 134 (63.2%) patients had persistently normal ALT levels throughout their entire follow-up periods. HBV DNA Detectability in Serum To determine whether the HBV DNA was detectable in the serum over long-term follow-up in patients with HBsAg seroclearance, 265 (88.9%) patients available stored serum taken within 1 year and 5 10 years and more than 10 years after HBsAg seroclearance were measured for HBV DNA levels. Of the 142 samples taken within 1 year after HBsAg seroclearance, 19 (13.4%) had detectable HBV DNA at the median level of 7.0 IU/mL (40.6 copies/ml) (range, IU/mL). Of the 99 samples taken between 5 and 10 years after HBsAg seroclearance, 6 (6.1%) had detectable HBV DNA at the median level of 13.9 IU/mL (80.6 copies/ml) (range, IU/mL). Of the 27 samples taken more than 10 years after HBsAg seroclearance, only 1 (3.7%) had detectable HBV DNA at the level of 53.6 IU/mL (310.9 copies/ ml). There was an increasing proportion of patients with undetectable HBV DNA levels with longer duration after HBsAg seroclearance. Eleven patients had paired serum taken within 1 year and after 5 10 years of HBsAg seroclearance. Eight patients (72.7%) had persistent undetectable HBV DNA levels at both time points. Of the remaining 3 patients with detectable HBV DNA levels (7.2, 1.5, and 1.5 IU/mL, respectively) within 1 year after HBsAg seroclearance, the HBV DNA of 2 patients became undetectable 5 10 years after HBsAg seroclearance; the third patient had persistent detectable HBV DNA of 4.6 IU/mL at 5 10 years after HBsAg seroclearance. Another 19 patients had paired samples for HBV DNA measurement at 5 10 years and more than 10 years after HBsAg seroclearance. Only 1 patient had persistently detectable HBV DNA levels of 30.4 and 53.6 IU/mL, respectively, at both time points. The remaining 18 (94.7%) patients had persistent undetectable HBV DNA at both time points. Therefore, the majority of patients had persistently undetectable HBV DNA level during follow-up. Liver Histology Of the 29 patients who underwent liver biopsies, 26 had the biopsy tissues with length longer than 1.5 cm with more than 4 portal tracts for reliable assessment of necroinflammation and fibrosis. The median age at the time of liver biopsy and the duration after HBsAg seroclearance were 40.5 years (range, ) and 47.8 months (range, ), respectively. Twenty-three (88.5%) patients had normal ALT levels at the time of liver biopsy. Twelve (46.2%) patients had mild to moderate steatosis (5 also had ultrasonographic evidence of fatty change). Seventeen (65.4%) patients had no evidence of necroinflammation and fibrosis. The remaining 9 (34.6%) patients had inflammation and/or fibrosis (7 had mild intralobular degeneration and focal necrosis and/or portal inflammation; 4 had F1 fibrosis). Of the 4 patients with F1 fibrosis, the liver stiffness scores were 5.9, 8.9, 8.8, and 9.6 kpa, respectively. The details of histologic grading of these 9 patients with inflammation and/or fibrosis are listed in Table 2. Immunostaining for HBsAg and HBcAg was negative in all patients. HBV DNA and Messenger RNA in Liver All 29 liver biopsies were assessed for intrahepatic total HBV DNA and cccdna. All patients (100%) had detectable intrahepatic HBV DNA. Twenty-three (79.3%) patients had detectable cccdna. The median total HBV DNA and cccdna levels were copies/cell (range, ) and copies/cell (range, ), respectively. Thirteen (44.8%) of these patients had detectable anti-hbs before the time of liver biopsy. Table 2. Details of Patients With Abnormal Liver Histology Necroinflammation Patients Age of HBsAg seroclearance, y Time between HBsAg seroclearance and histologic assessment, mo Periportal bridging necrosis (score, 0 10) Interlobular degeneration and focal necrosis (score, 0 4) Portal inflammation (score, 0 4) Fibrosis (score, 0 4) Total HAI (score, 0 22) HAI, histologic activity index.

5 1196 YUEN ET AL GASTROENTEROLOGY Vol. 135, No. 4 Figure 1. Analysis of the expression of HBV X mrna and the -actin mrna in the liver tissues. Expressions of precore/pregenomic and S mrna were not detectable in the liver tissues of all 11 patients (lanes 1 11) and hence not shown. Top panel: expression of X mrna. Bottom panel: expression of -actin mrna. M, 50-bp DNA molecular markers; lanes 1 11: patients 1 11;, negative controls, with HBV DNA cloned in a plasmid and digested with RNase-free DNase;, positive controls, with mrna extracted from a chronic hepatitis B patient. Adequate liver tissues for messenger RNA (mrna) measurement were available for 11 patients. All patients had undetectable mrna expression of the surface and precore/pregenomic genomes. Only 1 (9.1%) patient had detectable mrna expression of the X gene (Figure 1). Liver Stiffness After HBsAg Seroclearance Liver stiffness measurement was performed in 229 (76.8%) patients with the median duration of 35.2 months (range, ) after HBsAg seroclearance and the median age of 52.9 years (range, ). The median liver stiffness of these patients was 5.8 kpa (range, ) (the cut-off values for significant fibrosis and cirrhosis are 8.1 and 10.3 kpa, respectively). Of these patients, 154 had the liver stiffness measurement within 5 years and 56 patients between 5 and 10 years after HBsAg seroclearance. The median liver stiffness scores for these 2 groups of patients were 5.9 kpa (range, ) and 5.8 kpa (range, ), respectively. Because of the limited number of patients (n 56) with liver stiffness measurement at 5 10 years after HBsAg seroclearance, the relationship between the age of HBsAg seroclearance and the degree of fibrosis was examined only in the 154 patients with liver stiffness measurement within 5 years of HBsAg seroclearance. As shown in Table 3, there was a significant trend of increasing liver stiffness in patients with HBsAg seroclearance before ages 40, 40 49, and 50 years (P.039). In addition, 6 out of 76 (7.9%) patients with HBsAg seroclearance at age 50 (median age of HBsAg seroclearance was 49.6 years in the present study, see Table 1) years had significant fibrosis compared with 23 out of 78 (29.5%) patients with HBsAg seroclearance at age 50 (P.001; odds ratio [OR] 4.9; 95% confidence interval [CI], ). Meaningful comparison for cirrhosis was difficult to perform because only 12 patients had liver stiffness 10.3 kpa (3 and 9 for patients with HBsAg seroclearance at ages 50 and 50 years, respectively). Development of HCC and Clinical Complications of Cirrhosis Seven patients (all male) developed HCC at a median age of 69.3 years (range, ). All 7 patients had HBsAg seroclearance (4 had detectable anti-hbs) after the age of 50 years. The median duration between HBsAg seroclearance and development of HCC was 43.5 months (range, ). The cumulative rate of development of HCC was significantly lower in patients with HBsAg seroclearance at age 50 years compared with those with HBsAg seroclearance at age 50 (Figure 2, P.004) years. Six of these 7 patients had ultrasonographic evidence of cirrhosis (small sized liver with nodular surface) taken within 1 year before or at the time of HBsAg seroclearance. Five patients had serum HBV DNA levels measured at the time of development of HCC. Of these, 2 had detectable HBV DNA of 23.3 and copies/ml, respectively. One patient underwent surgical resection, and there was no evidence of cirrhosis (he had the HBV DNA level of 23.3 copies/ml). These suggest that most of the patients who had HBsAg seroclearance after the age of 50 years with HCC development had established cirrhosis before the time of HBsAg seroclearance. A minority of patients who had no cirrhosis could develop HCC after HBsAg seroclearance probably because of the direct oncogenic effect of the virus even at very low HBV DNA levels of 50 copies/ml. Nine patients developed clinical complications of cirrhosis including ascites and bleeding esophageal varices. Four of these patients developed these complications before HBsAg seroclearance (time ranging from 35.2 to months). The remaining 5 patients developed the complications after HBsAg seroclearance (time ranging from 2 to 40.1 months). Of these, 4 had HBsAg seroclearance at age 50 years (at the age of 54.8, 62.2, 63.5, 63.9 years, respectively) and the remaining patient had HBsAg seroclearance at the age of 47.6 years. Three had abnormal ALT levels in which 2 had HBV DNA levels of 233 and 113 copies/ml, respectively, taken within 1 year after HBsAg seroclearance, and 1 had fatty liver diagnosed by ultrasound. Fibroscan was performed in 3 patients within 1 year after HBsAg seroclearance: all had abnormal readings of 12.1, 17.5, and 18.8 kpa, respectively. All these findings suggest that cirrhosis was likely to be present before HBsAg seroclearance, and the continuing Table 3. Liver Stiffness of Patients With HBsAg Seroclearance at Different Age Age of HBsAg seroclearance, y Liver stiffness, kpa P value 40 (n 26) 5.2 ( ) (n 50) 5.9 ( ) (n 78) 6.2 (3 22.8) NOTE. Continuous values expressed in median (range). P value refers to the Kruskal Wallis test comparing the median values of 3 groups.

6 October 2008 HBsAg SEROCLEARANCE OF CHRONIC HEPATITIS B 1197 Figure 2. Cumulative risk for the development of HCC in patients with HBsAg seroclearance at age 50 and 50 years. viremia, even at levels below 300 copies/ml, might cause continuous liver inflammation after HBsAg seroclearance in these patients. This would lead to progression of the cirrhosis resulting in clinical decompensation. Discussion To our knowledge, the present study was the largest longitudinal study examining the clinical significance of HBsAg seroclearance. The possibility that the HBsAg seroclearance in the patients was due to viral escape mutation in the a determinant of envelope protein had not been studied but is very unlikely. It has been shown that most of the serologic assays used are able to detect HBsAg mutants. 23 In addition, most of the previous studies have documented that the majority of HBsAg seroclearance is due to decrease in HBsAg production secondary to extremely low viral replication rather than the occurrence of escape mutation 24,25 evading the HBsAg detection by commercially available assays. After HBsAg seroclearance, most of the patients (82.1%) had persistently normal ALT levels, and the median values of albumin, bilirubin, and ALT levels in the last follow-up for the study population were all within the normal range, which suggests that HBsAg seroclearance is usually associated with improvement of liver biochemistry in the majority of patients as shown in our previous study. 16 However, whether there is a regression of fibrosis remains to be defined in future studies because we have not performed serial fibrosis measurements in this study population. The present study showed that 86.6% of patients had undetectable serum HBV DNA taken within 6 12 months after HBsAg seroclearance using an extremely sensitive assay (lower limit of detection, 1.1 IU/mL). For the patients with detectable serum HBV DNA, the levels were indeed very low (median level, 7.0 IU/mL [40.6 copies/ml]). This probably reflects the extremely low viral replication leading to undetectable HBsAg. However, from the virologic studies of the 29 liver biopsy specimens, all the patients still harbored the HBV inside the liver after HBsAg seroclearance, a finding also consistently shown in other studies ,26,27 However, the virus was in a very low replicative and transcriptionally inactive phase. This is supported by several findings. First, the median total intrahepatic HBV DNA was only copies/cell. Approximately 20% of patients had undetectable cccdna in the present study. For those with detectable cccdna, the median level was only copies/ml, again indicating a very small number of viral templates for HBV replication. According to previous studies, the median total intrahepatic HBV DNA levels for anti-hbe-positive patients without HBsAg seroclearance is 100 copies/cell, 16,20 and the median cccdna level is 0.71 copies/cell. 16 Second, the transcriptional activity was also very low because all had undetectable mrna expression for surface and precore/pregenomic genomes, and only 1 out of 11 patients had detectable mrna for X genome (Figure 1). The failure of detection of the mrna expression for surface and precore/pregenomic mrna may be due to the inadequate sensitivity of the primers used for these 2 regions especially in conditions with extremely low transcriptional activity. Kuhns et al 13 have documented that surface gene mrna is not detectable in patients with HBsAg seroclearance. However, the finding of mrna expression for X gene despite the absence of mrna expression of surface and

7 1198 YUEN ET AL GASTROENTEROLOGY Vol. 135, No. 4 precore/pregenomic genomes may explain the possibility of development of HCC even after HBsAg seroclearance. Third, 93.9% and 96.3% of patients had undetectable serum HBV DNA levels 5 10 and 10 years after HBsAg seroclearance, respectively. For those with detectable serum HBV DNA levels, the median HBV DNA levels were 13.9 IU/mL at 5 10 years of HBsAg seroclearance (only 1 out of 27 patients had detectable HBV DNA at more than 10 years after HBsAg seroclearance). With this extremely low viremic state, more than 82% of patients had persistently normal ALT levels after HBsAg seroclearance. In fact, at least 42% of remaining patients with abnormal ALT had ultrasonographic evidence of fatty liver. In addition, most of the patients with liver histologic assessment had minimal necroinflammation and no significant fibrosis, although it should be noted that the median age of patients with liver biopsy was 40.5 years. In spite of all these favorable virologic, biochemical, and histologic parameters, HCC could still develop in some patients, as also documented in previous studies. 16,28 31 Several studies have shown that there is no reduced risk for the development of HCC in patients with established cirrhosis at the time of HBsAg seroclearance. 18,32,33 However, to define asymptomatic cirrhosis is difficult in clinical settings. The present study had identified a major determinant for chance of development of HCC in patients with HBsAg seroclearance. If the HBsAg seroclearance occurred before the age of 50 years, these patients would have a significantly lower risk for the development of HCC (Figure 2). This was in turn related to the finding that only 7.9% of patients with HBsAg seroclearance at age 50 years had significant fibrosis as measured by transient elasticity compared with 29.5% in patients with HBsAg seroclearance at age 50 years (P.001). It was also shown that the age of HBsAg seroclearance directly correlated with liver stiffness (Table 3). Finally, the present study showed that there was no difference in the cumulative risk for the development of HCC between patients with and without anti-hbs. This was true whether the anti-hbs became detectable at the time of HBsAg seroclearance or during subsequent follow-up. In conclusion, HBsAg seroclearance did not signify eradication of HBV. It was, however, associated with a very low viremic phase with minimal replication and transcriptional activities, resulting in improvement of liver histology and liver biochemistry. Patients who had HBsAg seroclearance at the age 50 years were at a higher risk for the subsequent development of HCC compared with patients who had HBsAg seroclearance before the age of 50 years. References 1. Sampliner RE, Hamilton FA, Iseri OA, et al. The liver histology and frequency and clearance of the hepatitis B surface antigen (HBsAg) in chronic carriers. Am J Med Sci 1979;277: Alward WLM, McMahon BJ, Hall DB, et al. The long-term serological course of asymptomatic hepatitis B virus carriers and the development of primary hepatocellular carcinoma. J Infect Dis 1985;151: Wu TT, Hsu HC, Chen DS, et al. Clearance of hepatitis B surface antigen (HBsAg) after surgical resection of hepatocellular carcinoma. J Hepatol 1987;4: McMahon BJ, Alberts SR, Wainwright RB, et al. Hepatitis B- related sequelae. Prospective study in 1400 hepatitis B surface antigen-positive Alaska Native carriers. Arch Intern Med 1990; 150: Liaw YF, Sheen IS, Chen TJ, et al. Incidence, determinants and significance of delayed clearance of serum HBsAg in chronic hepatitis B virus infection: a prospective study. Hepatology 1991; 13: Hsu HY, Chang MH, Lee CY, et al. Spontaneous loss of HBsAg in children with chronic hepatitis B virus infection. 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