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Long-term clinical and histological outcomes in patients with spontaneous hepatitis B surface antigen seroclearance
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Journal of Hepatology (February 2005)
Sang Hoon Ahnac†, Young Nyun Parkbcde†, Jun Yong Parka, Hye-Young Changa, Jung Min Leea, Ji Eun Shinae, Kwang-Hyub Hanace, Chanil Parkbc, Young Myoung Moonac, Chae Yoon Chonac*
a Department of Internal Medicine, Yonsei University College of Medicine, C.P.O. Box 8044, Seoul, South Korea
b Department of Pathology, Yonsei University College of Medicine, Seoul, South Korea
c Yonsei Institute of Gastroenterology, Yonsei University College of Medicine, Seoul, South Korea
d Center for Chronic Metabolic Disease, Yonsei University College of Medicine, Seoul, South Korea
e Brain Korea 21 Project for Medical Science, Yonsei University College of Medicine, Seoul, South Korea
"...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..."
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
ABSTRACT
Background: 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 (P=0.040), a history of perinatal infection (P=0.005) and long-standing duration (at least 30 years) of HBsAg positivity (P=0.002) 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 (P=0.072). 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.
1. Introduction
2. Materials and methods
2.1. Hepatitis B virus DNA analyses from liver tissues or serum
2.2. Histological evaluation
2.3. Statistical analysis
3. Results
3.1. Clinical characteristics and outcomes of the patients with hepatitis B surface antigen (HBsAg) seroclearance
3.2. Histological and virological comparisons before and after HBsAg seroclearance
4. Discussion
AUTHOR DISCUSSION
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 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 long-standing 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 oncogenic potential and occult HBV infection may also contribute to it. Full control of HBV replication with trace-amount 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.
1. Introduction
Hepatitis B virus (HBV) infection is the most common cause of acute and chronic liver disease worldwide, and it is 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 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 long-term virological and histological outcomes in chronic hepatitis B patients who spontaneously achieved clearance of HBeAg and HBsAg.
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 anti-HBe and anti-HBs were observed in 29 and 28 patients, respectively. All patients showed undetectable serum HBV DNA by direct hybridization assay.
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 long-standing duration of HBsAg positivity were associated with a significant higher risk of developing HCC (P=0.005 and P=0.002, 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%, P=0.040).
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 36.0±8.0 years (range, 23--51). The time from baseline to the follow-up liver biopsy ranged between 45 and 220 months (median 124±61.3) (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 (P=0.000) and focal lytic necrosis (P=0.000). 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 (P=0.072). Reduction of the fibrosis score was observed for nine patients including two patients having a statistically significant difference (improvement>=2 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%).
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.
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 α1-antitrypsin deficiency).
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′-GGTAAGCTTATGGAGAACACAACATCAGGA-3′ (underlined: HindIII, 29--49), and antisense: 5′-TCGGAATTCTCAAATGTATACCCAAAGACAAAAGAA-3′ (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.
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