Long-term outcome after spontaneous HBeAg seroconversion in patients with chronic hepatitis B

Long-Term Outcome After Spontaneous HBeAg Seroconversion in Patients With Chronic Hepatitis B Yao-Shih Hsu,1 Rong-Nan Chien,1 Chau-Ting Yeh,1 I-Shyan Sheen,1 Hung-Yi Chiou,2 Chia-Ming Chu,1 and Yun-Fan Liaw,1 During the course of chronic hepatitis B virus (HBV) infection, hepatitis B e antigen (HBeAg) seroconversion to its antibody (anti-HBe) often coincides with normalization of liver biochemical test and clinical remission, but data regarding long-term outcome after spontaneous seroconversion are still scarce. Excluding patients with other virus(es) concurrent infection, 283 patients with chronic HBV infection were followed up for at least 1 year after spontaneous HBeAg seroconversion to anti-HBe. Follow-up studies included clinical, biochemical, and virologic evaluation and hepatocellular carcinoma (HCC) screening with ultrasonography and ␣-fetoprotein assay. During a median follow-up period of 8.6 years (range, 1 to 18.4 years) after HBeAg seroconversion in 283 patients, 189 (66.8%) showed sustained remission, whereas the remaining 94 (33.2%) experienced alanine aminotransferase (ALT) elevation over twice the upper limit of normal: 12 (4.2%) associated with HBeAg reversion, 68 (24%) with detectable serum HBV DNA but HBeAg negative, and 14 (4.9%) of undetermined causes. Of the 269 patients without evidence of cirrhosis at the time of HBeAg seroconversion, 21 (7.8%) developed cirrhosis with a cumulative incidence and relative risk significantly higher in patients developing active hepatitis than in patients with sustained remission (P < .05). HCC developed in 6 (2.2%) of the 283 patients, also with a significantly higher cumulative incidence in patients developing active hepatitis after HBeAg seroconversion (P < .005). In conclusion, the results suggest that spontaneous HBeAg seroconversion confers favorable long-term outcomes. However, active hepatitis still may develop and lead to cirrhosis and HCC. (HEPATOLOGY 2002;35:1522-1527.)

D

uring the course of chronic hepatitis B virus (HBV) infection, presence of hepatitis B e antigen (HBeAg) often is associated with active and usually continuing liver disease, whereas HBeAg seroconversion to its antibody (anti-HBe) often coincides with loss of serum HBV DNA, normalization of liver biochemical test, clinical remission and subsidence of hepatic

Abbreviations: HBV, hepatitis B virus; HBeAg, hepatitis B e antigen; HCC, hepatocellular carcinoma; HCV, hepatitis C virus; HDV, hepatitis D virus; ALT, alanine aminotransferase; HBsAg, hepatitis B surface antigen. From the 1Liver Research Unit, Chang Gung Memorial Hospital, Chang Gung University; and the 2School of Public Health, Taipei Medical University, Taipei, Taiwan. Received August 2, 2001; accepted March 16, 2002. Y.-S.H. is currently affiliated with the Department of Internal Medicine, Taipei Medical University Hospital, Taipei, Taiwan. Supported by a grant from the Chang Gung Medical Research Fund (CMRP800). Address reprint requests to: Yun-Fan Liaw, M.D., Liver Research Unit, Chang Gung Memorial Hospital, 199, Tung Hwa North Rd, Taipei, Taiwan 105. E-mail: [email protected]; fax: (886) 3-3282824. Copyright © 2002 by the American Association for the Study of Liver Diseases. 0270-9139/02/3506-0028$35.00/0 doi:10.1053/jhep.2002.33638 1522

inflammatory activity,1-4 and may even be followed by spontaneous HBV surface antigen (HBsAg) seroclearance.5 Several studies have shown that the long-term outcome of the patients after interferon-related HBeAg seroconversion is favorable.6-10 However, few studies have addressed the issue of long-term outcome after spontaneous HBeAg seroconversion.11 Given the fact that most patients with liver cirrhosis and hepatocellular carcinoma (HCC) are seropositive for anti-HBe, it has even been suggested that the risk of HCC might increase when HBV infection became quiescent.12 Therefore, we conducted this study to elucidate the long-term outcome after spontaneous HBeAg seroconversion in a large series of patients.

Patients and Methods Patients. The long-term follow-up study of our patients with chronic hepatitis B, as described previously,5,10,13 has continued. By the end of 1998, a total of 1,345 HBeAg seropositive patients were confirmed to have chronic hepatitis B by liver biopsy at entry to our

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follow-up program. Excluding 361 patients who received anti-HBV therapy (interferon, thmosin-␣1 or lamivudine) and others with concurrent hepatitis C virus (HCV), hepatitis delta virus (HDV) infection, or other liver diseases (cirrhosis, alcoholic, autoimmune), 283 patients had well-documented spontaneous HBeAg seroconversion to anti-HBe 1 to 203 (median, 32) months after entry and were followed-up for more than 1 year after HBeAg seroconversion. They formed the basis of this study. Methods. After HBeAg seroconversion, the patients were followed up every 6 months or more frequently when clinically indicated, such as for alanine aminotransferase (ALT) elevation. Follow-up studies included clinical, biochemical, and virologic aspects as well as HCC screening using ultrasonography and alfa-fetoprotein. Liver biopsy was routinely advised when it was indicated, such as ALT elevation and when liver cirrhosis or HCC was suspected. A total of 74 biopsies were performed in 61 patients who gave written, informed consent. The biochemical tests were measured using routine automated techniques and carried out in the clinical pathology laboratories of Chang Gung Memorial Hospital. HBsAg, antibody to HDV, HBeAg, and anti-HBe were all assayed using commercially available radioimmunoassay kits (Ausria II, and anti-delta, Abbott Laboratories, North Chicago, IL; HBeAg/anti-HBe test, Diasorin, Saluggia, Italy). Serum samples also were assayed for anti-HCV with a second- or third-generation enzyme immunoassay kit (Abbott Laboratories). Stored serum specimens of the patients having ALT elevation during follow-up were assayed for HBV DNA retrospectively with a signal amplification hybridization assay (Digene HBV test, Hybrid Capture II, Digene Corp., MD). The detection sensitivity of this assay is 0.5 pg/mL. The first available serum specimens during ALT elevation in 47 patients with HBeAg negative hepatitis were further processed by an amplification-created restriction site method14 to determine whether there was a stop codon in the precore region. Real-time ultrasonography (Aloka SSD, 620; Aloka Co, Ltd, Tokyo, Japan) was performed every 6 months, or every 3 months if the patients had developed cirrhosis. The development of cirrhosis was diagnosed by histology or imaging features suggestive of cirrhosis supplemented with the presence of esophageal varices, encephalopathy, or ascites. The diagnostic accuracy of ultrasonography for liver cirrhosis was at least 80%.15,16 HCC was diagnosed by histology, cytology, or tumors found by ultrasonography or other diagnostic imaging methods with an elevated alfafetoprotein level greater than 400 ng/mL.17 Patients that remained seropositive for anti-HBe with normal serum

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ALT through the course of follow-up were defined as sustained remission. Active hepatitis was defined as ALT elevation over 2 times the upper limit of normal on 2 occasions at least 2 weeks apart. Of the patients with active hepatitis, those who became seropositive for HBeAg again were classified as HBeAg reversion, those seropositive for HBV-DNA but seronegative for HBeAg were considered to have HBeAg negative hepatitis, whereas those seronegative for both HBeAg and HBVDNA were classified as cause undetermined. Acute exacerbation was defined as an abrupt increase of ALT by 2-fold and to a level greater than 5 times the upper limit of normal, or to a level greater than 300 IU/L (n ⬍ 40 IU/L), as described elsewhere.13 Statistical Analysis. Statistical analysis was performed with the SAS software version 6.12 (SAS Institute Inc, Cary, NC). A logistic regression model was used to calculate univariate and multivariate odds ratios and their 95% CI. Statistical significance was tested by maximum likelihood function. Cumulative incidence rate was calculated with life table method. Kaplan-Meier survival analysis and log rank test were performed to test the cumulative incidence difference among these 3 groups (HBeAg reversion group, HBeAg-negative hepatitis group, and sustained remission group). Normal approximation method was used to examine the difference between the slope of cumulative incidence among various groups. Differences with a P value of less than .05 were considered significant.

Results A total of 283 patients (231 male, 52 female) with spontaneous HBeAg seroconversion to anti-HBe during a median follow-up period of 32 months (range from 1 to 203 months) met our selection criteria and were included in this study. At the time of seroconversion, their median age was 32 years (16-58 years) and 14 patients (4.9%) had evidence of cirrhosis (9 confirmed histologically). After spontaneous HBeAg seroconversion, 74 follow-up liver biopsies were performed in 61 (22%) patients during a median period of 37 months (range, 1-87 months). Of these, 1 of 15 patients with sustained remission, 2 of 3 patients with HBeAg reversion, and 12 of 42 patients with HBeAg negative hepatitis showed liver cirrhosis (P ⬎ .05). After seroconversion from HBeAg to anti-HBe, the majority of patients were asymptomatic with normal serum ALT. In a subsequent 9-year follow-up (1-18.4 years; median, 8.6 years), 189 (66.8%) of the 283 patients showed sustained remission, whereas the remaining 94 (33.2%) experienced ALT elevation greater than twice the upper limit of normal or active hepatitis by definition. Of

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these 94 patients with active hepatitis, 12 (12.8%) were seropositive for both HBeAg and HBV DNA (HBeAg reversion), and 68 (72.3%) were seropositive for HBV DNA but seronegative for HBeAg (HBeAg negative hepatitis). The remaining 14 (14.9%) were seronegative for both HBeAg and HBV DNA in all available serum specimens during hepatitis (cause undetermined). Seven (58.3%) patients with HBeAg reversion, 48 (70.6%) patients with HBeAg negative hepatitis, and 6 (42.9%) patients with undetermined cause showed persistent or intermittent ALT elevation, whereas the remaining patients showed transient ALT elevation. Stepwise logistic regression analysis on baseline variables including age, gender, ALT, HBV-DNA, and cirrhosis at the time of HBeAg seroconversion did not correlate to the development of new cirrhosis and HCC. Moreover, the logistic regression model showed no risk factors to predict developing acute exacerbation after HBeAg seroconversion. The pattern of ALT elevation did not correlate to the development of new cirrhosis in these 3 groups of patients. HBeAg reversion occurred 0.3 to 3.3 years (median, 1.1 years) after HBeAg seroconversion. Acute exacerbation was documented in 8 (67%) patients on or after HBeAg reversion with 1(13%) developing hepatic decompensation with prolongation of prothrombin time and marked jaundice. The HBe antigenemia was then maintained for a median period of 4 months (range, 3 to 45 months). Multivariate analysis showed that patients with more frequent acute exacerbation before HBeAg seroconversion or patients with liver cirrhosis at the time of HBeAg seroconversion had a significantly higher risk of developing HBeAg negative hepatitis (odds ratios of 10.37; 95% CI: 1.87-57.66 and 3.56; 95% CI: 1.0512.08, respectively), and patients with liver cirrhosis at the time of HBeAg seroconversion also were significantly associated with the risk of HBeAg reversion (odds ratio of 12.28; 95% CI: 2.53-59.56). Of the 68 patients with HBeAg negative hepatitis, 16 (23.5%) were seropositive for HBV DNA at the time of seroconversion, whereas the remaining 52 patients became HBV DNA seropositive in the following years (Fig. 1).There were no differences in the ALT levels, HBV DNA levels, and incidence of new cirrhosis or HCC between those with early and late HBV viremia. Of the serum specimens from 47 patients with HBeAg negative hepatitis, 41 (87%) showed mixed or pure precore mutants. Development of Cirrhosis. Of the 269 patients without evidence of cirrhosis at the time of HBeAg seroconversion, 21 (7.8%) patients developed new cirrhosis (15 with histologic evidence, 6 diagnosed by clinical and imaging features) during follow-up with an estimated annual incidence of 0.9%. Breakdown data showed that 14

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Fig. 1. The cumulative incidence of HBeAg-negative hepatitis over time after spontaneous HBeAg seroconversion.

(23%) of the 62 patients with HBeAg-negative hepatitis, 5 (55%) of the 9 patients with HBeAg reversion, 1 (7%) of the 14 patients with active hepatitis of undetermined causes, and one (0.5%) of the 184 patients with sustained remission developed cirrhosis during the follow-up period (Table 1). The median time to development of cirrhosis was 204 and 127 months for the HBeAg negative hepatitis group and the HBeAg reversion group, respectively (Fig. 2).The cumulative incidence of cirrhosis development was highest in the HBeAg reversion group, followed by the HBeAg-negative hepatitis group, and then the sustained remission group. The differences were statistically significant among these 3 groups (P ⫽ .0129) and between groups (all P ⬍ .01) by log rank tests. Age/sexadjusted multivariate analysis showed that patients with HBeAg-negative hepatitis were significantly associated with new cirrhosis development, whereas age at the time of HBeAg seroconversion and male sex did not correlate with the risk of new cirrhosis development (Table 2). Development of HCC. HCC was detected 5.3 to 14.3 years after spontaneous HBeAg seroconversion in 6 patients (2.2%; 5 with histologic confirmation, 1 diagnosed by imaging features plus high alfa-fetoprotein level) with an estimated annual incidence of 0.2%. It occurred in 3 (1.6%) of the 189 patients with sustained remission and in 3 (4.4%) of the 68 patients with HBeAg-negative hepatitis. One (1.5%) patient in the latter group died of esophageal varices bleeding 1 year after diagnosis of HCC. The difference between the slope of cumulative incidence of HCC development was significantly higher in patients with HBeAg-negative hepatitis as compared with patients with sustained remission (P ⬍ .005). However, age/sex-

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Table 1. Long-Term Outcomes After Spontaneous HBeAg Seroconversion Active Hepatitis

No. of patients Hepatitis onset age (median) Male (%) Liver biopsy Prior to HBeAg loss No. Timing* After HBeAg loss No. Timing* Follow-up* Cirrhosis at entry† New cirrhosis† HCC† HBsAg seroclearance†

Sustained Remission

HBeAg (ⴙ) HBV-DNA (ⴙ)

HBeAg (ⴚ) HBV-DNA (ⴙ)

HBeAg (ⴚ) HBV-DNA (ⴚ)

189 — 79

12 35 100

68 37 85

14 42 78

189 19 (1-185)

12 5 (1-79)

68 8 (1-175)

14 13.5 (1-123)

15 12 (1-145) 99 (24-218) 5 (2.6%) 1 (0.5%) 3 (1.6%) 9 (4.8%)

3 26 (17-103) 92 (14-130) 3 (2.5%) 5 (55%) 0 0

42 44 (1-187) 108 (12-221) 6 (8.8%) 14 (23%) 3 (4.4%) 3 (4.4%)

1 7 103 (34-196) 0 1 (7.1%) 0 0

NOTE. Entry: time of HBeAg seroconversion. *Data expressed as median (range) months. †Data expressed as No. (%).

adjusted multivariate analysis showed no difference in development of HCC among the 4 groups and the relative risk of HCC development was not significantly higher (odds ratio: 2.9; 95% CI: 0.6⬃14) (Table 3). Loss of HBsAg. Nine (4.8%) of the 189 patients with sustained remission and 3 (4.4%) of the 68 patients with HBeAg-negative hepatitis underwent spontaneous HBsAg seroclearance 4.9 to 14.3 years after spontaneous HBeAg seroconversion (P ⬎ .05) (Table 1).

Discussion The results of the present study have shown that the majority of the patients had sustained remission whereas only 7.8% developed cirrhosis (0.9% per year) and 2.2% developed HCC (0.2% per year) during a median follow-up period of 9 years after spontaneous HBeAg seroconversion. These long-term results are far better than those of HBeAg-positive patients16,17 and have confirmed the short-term observation that spontaneous HBeAg seroconversion confers favorable outcome (1⬃5). Given the fact that follow-up liver biopsies were not performed in all patients, it is possible that progression to compensated liver cirrhosis will be missed. However, the possible underestimation has been minimized by regular follow-up ultrasonography, which has a diagnostic accuracy of at least 80%.15,16

Table 2. Relative Risk of Cirrhosis Development

Variable Age at entry (y)

Sex

Fig. 2. The cumulative incidence of cirrhosis development after spontaneous HBeAg seroconversion in patients with chronic hepatitis B. The incidence of cirrhosis development is significantly higher in patients with HBeAg reversion than in patients with HBeAg-negative hepatitis and also significantly higher in patients with HBeAg-negative hepatitis than patients with sustained remission (P ⫽ .0129) by Kaplan-Meier survival analysis and log rank test.

Sustained Remission HBeAg(⫹) HBV-DNA(⫹)* HBeAg(⫺) HBV-DNA(⫹)* HBeAg(⫺) HBV-DNA(⫺)*

Group ⬉40 40 ⬃ 50 ⬎50 Woman Man Yes No

Number at New Odds Entry Cirrhosis Ratio 233 27 9 50 219 184 85 9 62 14

*Compared with sustained remission.

18 1 2 3 18 1 20 5 14 1

1 0.5 3.4 1 1.4 1 56 999 44 67

95% CI

P

0.06 ⬃ 3.6 0.66 ⬃ 17.7

.41 .12

0.4 ⬃ 4.9

.59

7.4 ⬃ 428 0 ⬃ 999 5.55 ⬃ 348.82 0.81 ⬃ 999

.001 .93 .001 .062

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Table 3. Relative Risk of HCC Development Variable Age at entry (y)

Sex Sustained remission HBeAg(⫺) HBV-DNA(⫹)*

Group ⬉40 40 ⬃ 50 ⬎50 Woman Man Yes No

Number at Entry

HCC

244 29 10 52 231 189 94 68

5 1 0 0 6 3 3 3

Odds Ratio

95% CI

P

1 1.7

0.19 ⬃ 15

.65

0.4 ⬃ 10 0.6 ⬃ 14

.39 .21

1 1 2 2.9

*Compared with sustained remission.

Because many acute exacerbations of chronic HBV infection are subclinical or asymptomatic,13 the single patient with sustained remission who developed cirrhosis after spontaneous HBeAg seroconversion might have experienced undetected subclinical episodes of hepatitis during the follow-up interval. Of note is that HCC developed in 3 (1.6%) patients with sustained remission, indicating the importance of HCC screening in asymptomatic HBsAg carriers, even in noncirrhotics.17,18 Nevertheless, the relative risk of developing HCC and cirrhosis in the patients with sustained remission was significantly lower than that of the patients with hepatitis after spontaneous HBeAg seroconversion. Of note is that 33% of our patients experienced ALT elevation during long-term follow-up after spontaneous HBeAg seroconversion. This is consistent with the observation of Niederau et al.9 that 2 (29%) of their untreated patients with spontaneous HBeAg clearance experienced continuing viral replication and inflammation during a mean follow-up period of 50 months. However, the HBeAg reversion rate after spontaneous HBeAg seroconversion in the present study was only 4.2%, which is lower than a rate over 10% after interferon-treated HBeAg seroconversion.7-10 In contrast, HBeAg-negative hepatitis was rare after interferon-treated HBeAg seroconversion,7-9 whereas it occurred in a much higher proportion (24%) of our patients with spontaneous HBeAg seroconversion. The reasons for these discrepancies are not clear and require further studies. Consistent with earlier observations, the present study also has shown that most reversions occurred within 1.5 years after HBeAg seroconversion and were often severe.8,11,19 This may explain the high risk (5 in 9) of cirrhosis development in our patients with HBeAg reversion (Fig. 2), as was shown in our early study.16 The most important finding in this study was that HBeAg-negative hepatitis accumulated over time after HBeAg seroconversion in 24% of the patients, and 87% of these patients had HBV strains with a point mutation at nucleotide 1896 in the precore region, or so-called

precore mutant.20,21 In general, patients with chronic HBeAg-negative hepatitis usually run a progressively deteriorating course.22 Our earlier study did show that HBV reactivation in anti-HBe positive patients increased the chance of cirrhosis development.16 The present study also showed that 23% and 4.4% of our patients with HBeAgnegative hepatitis progressed to cirrhosis and HCC, respectively, with a significantly higher cumulative incidence of cirrhosis than patients with sustained remission (Fig. 2). Because data on HBV genotype and precore/core promotor variant were not available in the vast majority of the patients, the role of HBV genotypes in the clinical course of patients after HBeAg seroconversion is unknown. Similar to general HBV carriers in Taiwan, however, our patients with active hepatitis after HBeAg seroconversion also were genotype B predominant (85%, unpublished data). In conclusion, the present study suggests that spontaneous HBeAg seroconversion confers favorable long-term outcomes in the majority of patients. However, HBeAg reversion or HBeAg-negative hepatitis may develop in some patients who subsequently show an increased risk of cirrhosis or HCC, as compared with patients with sustained remission. Although the long-term outcomes after spontaneous HBeAg seroconversion are much better than HBeAg seropositive patients, surveillance of HCC is still required even in patients with sustained remission. Acknowledgment: The authors thank L.H. Lu and C.Y. Chen for their excellent technical assistance and S.C. Chen and S.C.Chu for their secretarial assistance.

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