Development of Hepatocellular Carcinoma After Seroclearance of Hepatitis B Surface Antigen

CLINICAL GASTROENTEROLOGY AND HEPATOLOGY 2009;7:889 893 Development of Hepatocellular Carcinoma After Seroclearance of Hepatitis B Surface Antigen MYRON JOHN TONG,*, MICHAEL ONG NGUYEN, LORI TERESE TONG, and LAWRENCE MITCHELL BLATT*, *The Pfleger Liver Institute and the Division of Digestive Diseases, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, California; The Liver Center, Huntington Medical Research Institutes, Pasadena, California; Alios Biopharma, South San Francisco, California See related article, Gehring AJ et al, on page 682 in Gastroenterology. BACKGROUND & AIMS: During the natural course of chronic hepatitis B virus infection, a small proportion of patients experience hepatitis B surface antigen (HBsAg) seroclearance. However, the long-term clinical outcomes of this process are not well established. METHODS: Thirty-five patients with chronic hepatitis B, followed between 1976 and 2008 at a community liver clinic, experienced HBsAg seroclearance. Ten patients were Caucasian and 25 were Asian. These patients continued to undergo surveillance for hepatocellular carcinoma that included test for -fetoprotein levels and abdominal ultrasound examinations. The median follow-up time was 185 months (range, 27 400 months). RESULTS: During the initial visit to the clinic, the median age of the patients was 41 years (range, 1.5 72 years). Eighteen patients (51.4%) were hepatitis B e antigen (HBeAg) positive 25 (71.4%) were hepatitis B virus DNA positive, and 13 (37.1%) had cirrhosis. At the time of HBsAg loss, the median age was 54 years (range, 13 77 years) and all were hepatitis B e antigen- as well as hepatitis B virus DNA negative. During the long-term follow-up, 4 patients with cirrhosis developed hepatocellular carcinoma (HCC), which was discovered by ultrasound examination. Factors associated with development of HCC were low baseline levels of albumin (P.04), family histories of HBsAg positivity (P.01) and HCC (P.04), and age of less than 50 years at the time of HBsAg clearance (P.03). CONCLUSIONS: HCC can still develop after HBsAg seroclearance. Thus, surveillance should be continued after HBsAg loss in the same manner as for HBsAg positive patients. Hepatitis B surface antigen (HBsAg) seroclearance is defined as the loss of HBsAg detectability in the blood of patients with chronic hepatitis B. In individuals with long standing chronic infection, disappearance of circulating HBsAg is an uncommon finding especially in areas of high endemicity for hepatitis B. In many countries in Asia where HBsAg carrier rates are up to 15% of the population, the annual HBsAg seroclearance rates were reported to be 0.1% to 1.2%. 1 3 In low endemic areas such as North America and Europe, the annual rates of HBsAg loss ranged from 0.1% to 2.1% per year. 4 8 There are few studies from the United States. In a report from Alaska, the annual HBsAg clearance rate in Eskimo HBsAg carriers was 0.5% per year. 9 The outcomes after HBsAg seroclearance are generally favorable. Recent studies have indicated that after HBsAg loss, there was significant improvement in serum alanine aminotransferase levels, and liver biopsy findings in these patients showed improvement in necroinflammation. 10 12 However, serum hepatitis B viral DNA (HBV DNA) was still detectable in 31% 71.4% of patients at the time of HBsAg seroclearance, which decreased to 21.4% and to 14.3% after 5 and 10 years of follow-up respectively. 13 15 Intrahepatic HBV DNA also was detected in up to 100% of patients after HBsAg loss, and included both integrated HBV DNA as well as covalently closed circular DNA in 37% 63.5% of patients. 10,12,16 Clinical complications after HBsAg seroclearance may occur. In reports from Asia, 1.6% 10.9% of patients with chronic hepatitis B progressed to cirrhosis, and 13.8% of patients with established cirrhosis experienced hepatic decompensation. 14,17,18 In both of the latter instances, progression of liver disease was associated with coexisting hepatitis C virus or hepatitis delta virus infection. 14,19 In addition, hepatocellular carcinoma (HCC) developed in 2.9% 20% of cirrhosis patients and in 1.9% of noncirrhotic patients who had HBsAg loss. 3,8 12,18 In this report, we describe the characteristics of 35 chronic hepatitis B patients who experienced HBsAg loss and the clinical sequelae which occurred during long-term follow-up. Methods Patients Since 1976, over 1200 patients with chronic hepatitis B have been evaluated in our liver clinic. The HBsAg positive patients were routinely followed every 3 12 months with liver tests including serum albumin, total bilirubin, aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, and platelet counts. Hepatitis B e antigen (HBeAg) and antibodies to hepatitis B e antigen (anti-hbe) were measured during the first clinic visit and every 3 5 years thereafter. The HBsAg was measured every 5 10 years if liver tests were persistently normal, HBeAg was negative, anti-hbe was positive, and if the HBV DNA was persistently negative. Through the years, Abbreviations used in this paper: Anti-HBc, antibody to hepatitis B core antigen; Anti-HBe, antibody to hepatitis B e antigen; Anti-HBs, antibody to hepatitis B surface antigen; Anti-HCV, antibody to hepatitis C virus; HBeAg, hepatitis B e antigen; HBsAg, hepatitis B surface antigen; HBV DNA, hepatitis B viral DNA; HCC, hepatocellular carcinoma; HCV RNA, hepatitis C viral RNA. 2009 by the AGA Institute 1542-3565/09/$36.00 doi:10.1016/j.cgh.2009.04.012

890 TONG ET AL CLINICAL GASTROENTEROLOGY AND HEPATOLOGY Vol. 7, No. 8 Table 1. Baseline Characteristics of 35 HBsAg Positive Patients Prior to HBsAg Seroclearance Age, y a 41 (2 72) Sex (male:female) 24:11 Race, n (%) Asian 25 (71.4) Caucasian 10 (28.6) Family history, n (%) HBsAg positivity 18 (51.4) Hepatocellular carcinoma 17 (48.6) HBeAg positive, n (%) 17 (48.6) Anti-HBe positive, n (%) 18 (51.4) HBV DNA positive, n (%) 25 (71.1) Albumin (g/dl) a 4.4 (2.7 5.1) Bilirubin (mg/dl) a 0.8 (0.2 2.9) Alkaline phosphatase (U/L) a 75 (26 245) Aspartate aminotransferase (U/L) a 43 (15 430) Alanine aminotransferase (U/L) a 67 (5 565) Platelet ( 10 9 /L) a 208 (70 366) -fetoprotein (ng/ml) a 3 (1.5 309) Anti-HCV positive, n (%) 2 (5.7) Cirrhosis, n (%) 13 (37.1) Months follow-up a 185 (27 400) a Median (range). the methodologies and the sensitivities for measurement of HBV DNA were different so that the results could not be compared. Therefore, the HBV DNA tests are reported herein as either positive or negative. In patients who presented to the clinic between 1976 1990, the HBV DNA was measured in sera stored at 70 C as previously described. 20 A family history of HBsAg positivity and presence of HCC in blood relatives were obtained at the first clinic visit. When queried about a history of alcohol consumption, the patients in this analysis revealed either none or only occasional alcohol use. Some patients who continued to attend our clinic were treated with antiviral therapy during the course of follow-up. All patients were observed for development of liver-related complications or progression to HCC. 21 Surveillance for HCC Abdominal ultrasound examinations and -fetoprotein tests were performed every 6 12 months in noncirrhotic patients, and every 3 6 months in patients with cirrhosis. If the -fetoprotein was elevated or a lesion was detected by abdominal ultrasound, then additional tests such as computerized tomography scan or magnetic resonance imaging were performed. Each of the HCC cases described herein had liver biopsy confirmation of malignancy. Statistical Analysis Categorical data were summarized by using frequencies and were analyzed by 2 methods for assessment of differences. All continuous data were descriptively summarized with means and standard deviations, and further analyzed for assessment of differences by analysis of variance (ANOVA) with post hoc pair-wise Student t tests. Due to the small sample size, continuous data were analyzed by both the parametric and nonparametric tests. The data reported herein are from parametric tests and these results were confirmed by nonparametric methodology in each instance. Analyses were conducted by using SAS software version 9.1 (SAS Institute Inc, Cary, NC). Statistical significance was defined as 2-sided P values.05. Results During a median follow-up period of 185 months (range, 27 400 months; mean 205.6 months 97 SD), 35 HBsAg positive patients had HBsAg seroclearance. Twenty-four (69%) were males and 25 (71%) were Asian (5 born in the USA). In the Asian patients, 17 (68%) had family members who were HBsAg positive and 10 (40%) had a family history of HCC. During the first visit to our clinic, the median age was 41 years (range, 1.5 72 yrs), 18 (51.4%) were HBeAg positive, and 25 (71.4%) were HBV DNA positive. The baseline laboratory test results are shown in Table 1. Two (5.7%) patients were coinfected with hepatitis C virus. Thirteen (37.1%) patients had cirrhosis. At the time of HBsAg clearance, the median age was 54 years (range, 13 77 years). Eighteen patients who were HBeAg positive during the first visit had all seroconverted to HBeAg negative. Thus, at the time of HBsAg loss, the 35 patients were anti-hbe positive, HBV DNA negative, and had persistently normal levels of serum alanine aminotransferase and -fetoprotein (Table 2). During the course of follow-up, 14 patients received antiviral therapy (7 with interferon alone, 5 with interferon plus lamivudine (4) or adefovir (1), and 1 each with lamivudine or adefovir. Prior to treatment, 11 of the 14 patients were HBeAg positive, and 3 were HBeAg negative with positive HBV DNA. Two of the latter patients also were positive for antibodies to hepatitis C virus and hepatitis C viral RNA, and both were treated with interferon, ribavirin, and lamivudine resulting in clearance of hepatitis C and HBsAg loss. Six of the patients who received antiviral therapy had cirrhosis. After HBsAg seroclearance, all 35 patients had persistently normal liver tests and none had seroreversion of HBsAg. Twenty-six patients subsequently developed anti-hbs. During further follow-up, 31 patients remained clinically stable. However, with continued routine surveillance for HCC, 4 patients had appearance of liver lesions on abdominal ultrasound examinations and each had liver biopsy confirmation of HCC (Table 3). The -fetoprotein was normal in 3 of the 4 patients who developed Table 2. Characteristics of 35 Patients at the Time of HBsAg Seroclearance Age, y a 54 (13 77) HBeAg positive, n (%) 0 (0) Anti-HBe positive, n (%) 35 (100) HBV DNA positive, n (%) 0 (0) Albumin (g/dl) a 4.6 (2.8 4.8) Bilirubin (mg/dl) a 0.7 (0.4 1.4) Alkaline phosphatase (U/L) a 64 (30 235) Aspartate aminotransferase (U/L) a 22 (13 34) Alanine aminotransferase (U/L) a 22 (9 59) Platelet ( 10 9 /L) a 211 (55 317) -fetoprotein (ng/ml) a 2.3 (1.2 5) Prior antiviral treatment, n (%) 14 (40) Interferon 7 Interferon lamivudine (4) or adefovir (1) 5 Lamivudine or adefovir 2 a Median (range).

August 2009 HEPATOCELLULAR CARCINOMA AFTER HBsAg CLEARANCE 891 Table 3. Characteristics of 4 Patients Who Developed Hepatocellular Carcinoma After HBsAg Seroclearance Patient A B C D Age at HCC diagnosis, y 55 67 69 73 Sex Male Male Male Male Race Asian Asian Caucasian Asian Family history HCC Brother Brother None Brother; father HBeAg/anti-HBe at HCC diagnosis / / / / HBV DNA at HCC diagnosis Negative Positive Negative Negative Anti-HBs AFP at HCC diagnosis (ng/ml) 2.8 4 342 3.5 Interval between ultrasound examination a 4 months 7 months 6 months 3 months HCC size (cm) 2.5 8 1.7 5.5 HCC treatment Resection TACE TACE TACE Recurrent HCC No No No Yes Clinical status Alive Dead Alive Alive AFP, alphafetoprotein; TACE, transcatheter arterial chemoembolization. a Time from the last negative ultrasound to the first positive ultrasound examination. HCC. The time interval between HBsAg seroclearance to HCC appearance in these 4 patients was 15, 16, 20, and 24 months. The median age at HCC detection was 69 years (range, 55 73 years). The 4 patients who developed HCC were male, all had cirrhosis, and 3 had family members with HCC. At the time of HCC discovery, the HBeAg was negative and anti-hbe was positive in all 4 patients. In 1 patient, the HBV DNA became detectable when the HCC appeared. In 3 patients, the tumors were treated with transcatheter arterial chemoembolization. A second HCC was subsequently detected in 1 of these latter patients 3 years later, which also was similarly treated. One other patient had hepatic resection of a left lobe HCC. Factors associated with HCC development by univariate analysis after HBsAg seroclearance were low baseline serum albumin (P.04), a family history of HBsAg positivity (P.01), a family history of HCC (P.04), and age less than 50 years at the time of HBsAg seroclearance (P.03) (Table 4). The data from 4 patients with HBsAg seroclearance who developed HCC are illustrated in Figure 1. Discussion HBsAg seroclearance is an unusual event. In a report of 245 HBsAg positive asymptomatic carriers in Taiwan, the mean annual rate of HBsAg loss was only 1.2% per year. 3 In this study, older age at presentation and sustained clinical remission of hepatitis were associated with HBsAg seroclearance. In another report from Korea, HBeAg negativity and undetectable HBV DNA at enrollment were associated with subsequent loss of HBsAg. 22 However, in a study from Europe, HBeAg positivity at entry was a significant predictive factor for HBsAg seroclearance. 8 Another report from Hong Kong indicated that patients with genotype B more often had HBsAg seroclearance than those with genotype C. 10 Another important factor which may influence HBsAg loss was previous treatment with interferon or oral antiviral agents. 8,23 Loss of HBsAg after interferon therapy in chronic hepatitis B patients was reported to be 3% 7.8%. 22,24 HBsAg seroclearance after treatment with lamivudine or adefovir was less than 1%. Prior to HBsAg clearance, 14 of 35 (40%) of our patients had received a course of antiviral therapy consisting of interferon alone, interferon plus lamivudine or adefovir, and lamivudine or adefovir alone. Eleven of the patients were HBeAg positive at baseline and seroconverted to HBeAg negative after antiviral therapy. Also, both patients who were coinfected with hepatitis C virus had a sustained virologic response with clearance of hepatitis C as well as HBsAg loss after treatment with interferon, ribavirin, and lamivudine. The remaining 21 of 35 (60%) of our patients who had HBsAg seroclearance did not receive antiviral therapy and therefore had spontaneous HBsAg loss. In the majority of instances, the outcomes after HBsAg seroclearance are favorable. The serum alanine aminotransferase values remained normal and antibody to hepatitis B core antigen titers gradually decreased. 11,15 Improvement in necroinflammation in liver biopsies after HBsAg loss was also noted. 10,11,12 However, the serum HBV DNA was detectable in 31% 98% of patients at the time of HBsAg seroclearance, 10,11,14,16 and remained so in up to 14% of patients even 10 years after HBsAg loss. 15 Also, intrahepatic HBV DNA was detected in liver tissue in the majority of patients and may be in the form of covalently closed circular DNA. 10,12,17 Table 4. Analysis of Factors Associated With Hepatocellular Carcinoma in Patients With HBsAg Seroclearance Factor HCC No HCC P value Number of patients 4 31 Family history.01 HBsAg Yes 3/4 13/31 No 1/4 18/31 Family history.04 HCC Yes 3/4 7/31 No 1/4 24/31 Age at HBsAg.03 seroconversion 50 years 14 0 50 years 4 17 Baseline albumin, gm/dl SD 3.75 0.88 4.31 0.45.04

892 TONG ET AL CLINICAL GASTROENTEROLOGY AND HEPATOLOGY Vol. 7, No. 8 Figure 1. (A) Patient A: a 55-year-old Asian male. (B) Patient B: a 67-yearold Asian male. (C) Patient C: a 69- year-old Caucasian male. (D) Patient D: a 73-year-old Asian male. AFP, alphafetoprotein; ALT, alanine aminotransferase. Clinical complications following HBsAg seroclearance are rare. In patients with HBsAg loss, progression from chronic hepatitis to cirrhosis was reported to be 1.6% 5.6% and hepatic decompensation occurred in 2.5% of patients with pre-existing cirrhosis. 3,14,19 However, many of these latter patients had superimposed hepatitis C virus or hepatitis delta virus infection which may have contributed to the progression of their liver disease even after HBsAg seroclearance. 14,20 None of our patients had liver-related complications after HBsAg seroclearance. The most serious clinical sequelae after HBsAg loss is development of HCC. The time interval between HBsAg seroclearance and HCC detection ranged from 9 months to 9 years. 8 14,18 In the latter reports, up to one third of patients developed HCC more than 5 years after HBsAg loss. The annual rates of HCC development in patients without and with cirrhosis were reported to be 0.21% and 0.45% to 6.2% respectively, and were higher in patients who were coinfected with hepatitis C or hepatitis delta virus. 8,10,12,14,18 Significant factors associated with development of HCC included a history of perinatal transmission, greater than 50 years duration of HBsAg positivity, age of greater than 50 years at the time of HBsAg seroclearance, and the presence of cirrhosis. 10,12 The mean age of our patients at HCC development was 66 years and all had cirrhosis at the time of the initial visit. Also, as recently reported, our patients who had HBsAg seroclearance after the age of 50 years were more apt to develop HCC than those who had HBsAg loss under the age of 50 years. 25 Reported herein, 3 of the Asian patients who developed HCC had a family history of HCC and this finding was an independent risk factor for HCC development in our patients after HBsAg loss. Thus, the 7 additional patients with HBsAg seroclearance who had family members with HCC are at high risk for developing HCC themselves and will require close surveillance for HCC. Further studies are needed to identify specific genetic factors which may predispose these patients for development of HCC. Because HBsAg seroclearance is an uncommon event, a large number of chronic hepatitis B patients must be observed for a long period of time. Thus, the majority of such reports are mainly from Asian countries where hepatitis B is endemic. A study from Europe of 32 patients with HBsAg loss required a multicenter study which involved at least 7 countries. 8 The patients reported herein are from a community-based liver clinic in Pasadena, California, which has been a referral source for over 1200 HBsAg positive patients for close to 3 decades. Our cohort of 35 patients who had HBsAg seroclearance consisted of both Asian and Caucasian patients. The Asian patients most likely contracted hepatitis B by perinatal transmission or via intrafamilial spread of hepatitis B virus since 68% of their family members also were HBsAg positive and 40% had family members with HCC. This finding is not only observed in Asian patients with HBsAg seroclearance but also in those who remain HBsAg positive. 26 In contrast, our Caucasian patients most likely contracted hepatitis B as adults either by sexual contact, blood transfusion, or by needle stick exposure. None of the Caucasian patients had family histories of HBsAg positivity nor had family members with HCC. In a recent report, the serum albumin levels were consistently lower throughout the course of follow-up in our HBsAg positive patients who later died from liver-related complications or who developed HCC. 20 A low baseline albumin level in our patients who progressed to HCC after HBsAg loss was an independent risk factor for HCC development, and was a reflection of the established liver damage which was already present when these patients first presented to our clinic. In summary, chronic hepatitis B patients who have HBsAg seroclearance still may develop HCC. Thus, surveillance for HCC must be continued indefinitely in patients who have established cirrhosis at the time of HBsAg loss, and especially in those with a family history of HCC, and those who had HBsAg clearance after the age of 50 years. Early detection of a small HCC may result in successful treatments which improve survival as demonstrated in our patients who developed HCC after HBsAg seroclearance.

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A comparative study of hepatitis B viral markers in the family members of Asian and non- Asian patients with hepatitis B surface antigen-positive hepatocellular carcinoma and with chronic hepatitis B infection. J Infect Dis 1979;140:506 512. Reprint requests Address requests for reprints to: Dr Myron J. Tong, Liver Center, 660 South Fair Oaks Avenue, Pasadena, California 91105. e-mail: myrontong@hmri.org; fax: (626) 397-5827. Acknowledgments The authors thank Carlos Hsien and Hai-En Sun for technical assistance during the preparation of this manuscript. Conflicts of interest The authors disclose no conflicts.