ORIGINAL ARTICLE
Occult Hepatitis B Virus Infection in Chinese Cryptogenic Intrahepatic Cholangiocarcinoma Patient Population YuQiong Li, MM,* Hui Wang, MM,* DaPeng Li, MM,w JingYi Hu, MM,z Hao Wang, MD,* DongXun Zhou, MD,* QiaoMei Li, MD,* XiaoLan Jiang, MM,* HuaBang Zhou, MD,* and HePing Hu, MD*
Background: There is no information available about occult hepatitis B virus (HBV) infection (OBI) in individuals with intrahepatic cholangiocarcinoma (ICC). Goals: To investigate the correlation between OBI and ICC. Study: A retrospective case-control study was conducted. The cases were 183 cryptogenic ICC patients (group I), and the controls were 549 healthy individuals (group II). The cases and controls were matched for age, sex, and inhabitancy. Adjusted odds ratios and 95% confidence intervals were calculated. Intrahepatic total HBV DNA in 63 paraffin-embedded samples was collected from patients in group I (n = 44), HBV-associated ICC patients (n = 3), and hepatic cavernous hemangioma patients with seronegative HBsAg (hepatitis B S antigen) (group III; n = 16). We determined the levels of serum and intrahepatic HBV DNA and compared the level of intrahepatic HBV DNA in 44 cryptogenic patients from group I with the level in the patients from group III. Results: Compared with group II, group I had a lower prevalence of anti-HBs (antibody against HBsAg) and a higher prevalence of anti-HBe (antibody against hepatitis B e antigen) and anti-HBc (antibody against hepatitis B c antigen). Multivariate analysis confirmed that anti-HBe and anti-HBc positivity were associated with ICC. The odds ratios and 95% confidence intervals for antiHBe and anti-HBc were 2.482 and 1.482-4.158, 4.556 and 2.9387.066, respectively. Compared with group III, cryptogenic ICC cases showed more frequent detection of intrahepatic total HBV DNA (63.64% vs. 18.75%, P = 0.002). Conclusions: OBI may represent an important risk factor for ICC. HBsAg seroclearance does not signify eradication of HBV and may not entirely prevent the development of ICC. Key Words: intrahepatic cholangiocarcinoma, occult hepatitis B virus, carcinogenesis
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ntrahepatic cholangiocarcinoma (ICC) is a malignant tumor that arises from the peripheral intrahepatic bile duct epithelium and is far less prevalent than hepatocellular
Received for publication June 12, 2013; accepted November 17, 2013. From the *Department of Hepatobiliary I, Eastern Hepatobiliary Surgery Hospital, Second Military Medical University; zSchool of Medicine, Jiao Tong University, Shanghai; and wDepartment of Gastroenterology, First Affiliated Hospital, Bengbu Medical College, Anhui, China. Y.L., H.W., and D.L. are the co-first authors. The authors declare that they have nothing to disclose. Reprints: HuaBang Zhou, MD, Department of Hepatobiliary I, Eastern Hepatobiliary Surgery Hospital, Second Military Medical University, 225 Changhai road, Shanghai 200438, China (e-mail: [email protected]). Copyright r 2013 by Lippincott Williams & Wilkins
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carcinoma (HCC). HCC accounts for >90% of all primary liver cancers in areas endemic for hepatitis B virus (HBV), whereas ICC accounts for approximately 5% of primary liver cancers.1 The etiology and carcinogenic mechanism of ICC remain unclear. Recent evidence suggests a possible etiological role for chronic HBV infection with detectable circulating hepatitis B surface antigens (HBsAg) in ICC development.2,3 China is an HBV-endemic area, and HBV is the predominant cause of HCC in this region. Data from our previous study also demonstrated that the incidence of HBV infection in ICC patients was significantly higher than that in individuals without cancer (48.6% vs. 6.6%) and that chronic HBV infection was the most important independent risk factor for ICC in the Chinese population.4 Several risk factors in addition to HBV infection have been reported as potential risk factors for ICC, such as liver fluke infestation,5 primary sclerosing cholangitis (PSC),6 hepatolithiasis, and chronic HCV infection7; however, nearly 40% of ICC cases in the Chinese population occur in the absence of known etiological factors.4 Detailed studies delineating the exact cause of ICC in this group of patients are currently lacking. During the course of chronic hepatitis B (CHB) infection, some patients may undergo HBsAg seroclearance and have undetectable circulating HBsAg.8,9 With the development of sensitive nucleic acid detection techniques, HBsAg-negative subjects with detectable HBV DNA in sera or liver tissues can be identified. Patients who have undetectable HBsAg and yet have detectable HBV DNA in the serum or liver are defined as having occult HBV infection (OBI).10–15 Although the clinical significance of this condition is not well known, several clinical observations show that OBI is an important clinical factor that can lead to: (1) reactivation of HBV infection in patients without HBsAg after chemotherapy, (2) HBV transmission from HBsAgnegative and anti-HBc-positive blood or organ donors, and (3) the development of cryptogenic cirrhosis.16–18 Recently, some researchers have also demonstrated that OBI patients with HBsAg seroclearance can still develop HCC8,9,16–18; OBI is thought to be an important risk factor for HCC development because it maintains the pro-oncogenic properties typical of the overt infection. The potential role of OBI as a risk factor for ICC development has not been addressed to date. In the present study, we therefore conducted a retrospective case-control study comprised of 183 cryptogenic ICC patients and 549 healthy individuals; in addition, we tested 63 intrahepatic HBV DNA samples (44 cryptogenic ICC patients, 3 HBV-associated ICC patients, and 16 hepatic cavernous hemangioma patients with seronegative HBsAg). Our goal was to investigate whether OBI was a risk factor for ICC development.
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MATERIALS AND METHODS Study Subjects From January 2003 to October 2007, 515 ICC patients underwent surgical dissection at the Eastern Hepatobiliary Surgery Hospital of the Second Military Medical University (Shanghai, China). The diagnosis of ICC was confirmed by pathology. Using our exclusion criteria (Fig. 1), we excluded 332 patients because of seropositive HBsAg (n = 247), hepatolithiasis (n = 39) (36 hepatolithiasis alone, 1 hepatolithiasis plus a history of chronic HBV infection, 1 hepatolithiasis plus hepatic schistosomiasis, and 1 hepatolithiasis plus HCV infection), PSC (n = 1), HCV infection alone (n = 1), hepatic schistosomiasis alone (n = 14), history of chronic HBV infection alone (n = 17), alcoholassociated cirrhosis (n = 9), and cryptogenetic cirrhosis (n = 4). The remaining 183 cryptogenic ICC patients (group I) were selected for statistical analysis. The healthy controls consisted of 549 healthy individuals (group II) who had visited the Eastern Hepatobiliary Surgery Hospital of the Second Military Medical University for a routine checkup. Exclusion criteria were as follows: seropositive HBsAg, history of chronic HBV infection, HCV infection, PSC, cirrhosis, hepatolithiasis, hepatic schistosomiasis, and cancer. Controls were matched to the ICC cases for sex and age (± 4 y) and were recruited during the same study period as the cases. To further clarify the correlation between OBI and ICC development, 3 patients with HBV-associated ICC
OBI and ICC
(2 males and 1 female) were selected as positive controls. Forty-four patients (26 males and 18 females) with cryptogenic ICC who were randomly selected from group I and 16 seronegative HBsAg patients (group III; 4 males and 12 females) with hepatic cavernous hemangioma who underwent hepatectomy during the same period in the same hospital were selected for liver total HBV DNA analysis. The project was approved by the local ethics committee.
Laboratory Tests of Virology Blood samples were tested for HBsAg, the antibody to hepatitis B surface antigen (anti-HBs), hepatitis B e antigen (HBeAg), the antibody to hepatitis B e antigen (anti-HBe), the antibody to hepatitis core antigen (anti-HBc), and antibodies against the hepatitis C virus (anti-HCV) using enzyme-linked immunosorbent assays (Abbott Laboratories, North Chicago, IL).
Isolation of Genomic DNA From FFPE Tissue Sections Total liver DNA was extracted from paraffin-embedded specimens using the QIAamp DNA FFPE Tissue Kit (Qiagen GmbH, Hilden, Germany) according to the manufacturer’s instructions. The total liver DNA was diluted with buffer ATE to a final concentration of 50 ng/mL per case for subsequent quantitative measurement of total HBV DNA.
Quantitative Measurement of Serum and Intrahepatic Total HBV DNA Serum HBV DNA levels were measured in 104 patients (56.83%). HBV DNA was extracted from 100 mL of serum using a diagnostic kit for the quantification of HBV DNA (Shanghai Kehua Laboratory System Co. Ltd., China) according to the manufacturer’s instructions with a final elution volume of 2 mL. The extracted DNA was then quantitatively measured by the same kit using the ABI7500 Fast Real-time PCR System (Applied Biosystems, Mortlake) according to the manufacturer’s instructions. This kit is a standard commercial assay for the detection of HBV DNA in human serum samples. To ensure the specificity of the test and to prevent diagnostic errors, the limit of detection was defined as 1000 IU/mL for serum or liver tissue.
Histologic Assessment Immunoperoxidase staining was performed using monoclonal antibodies against HBsAg (dilution, 1:100; Signet, Dedham, MA).
Liver Cirrhosis The diagnosis of cirrhosis was made according to histologic evaluation of the liver parenchyma. If histologic information was not available, clinically diagnosed liver cirrhosis was defined as follows: (i) platelet count 12 cm); (ii) esophageal or gastric varices; or (iii) overt complications of liver cirrhosis, including ascites, variceal bleeding, and hepatic encephalopathy.
Statistical Methods FIGURE 1. Flow chart for patient selection. HBV indicates hepatitis B virus; HCV, hepatitis C virus; PSC, primary sclerosing cholangitis. r
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Descriptive analyses of the variables were conducted using SPSS 16.0 (SPSS Inc., Chicago, IL). Univariate analyses were performed using the w2 test for categorical variables and the independent samples t test for discrete variables. www.jcge.com |
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P < 0.05 was considered statistically significant. For each risk factor, the odds ratios for ICC and their 95% confidence intervals were computed as estimates of the relative risk by unconditional logistic regression analysis using maximum likelihood estimates.
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TABLE 2. HBV Serological Antibody Profiles in Cryptogenic ICC Cases and Controls
n (%) Controls (n = 549)
Serological Tests
RESULTS Demographic and Baseline Features of Cases and Controls A total of 183 patients (108 males and 75 females; male-to-female ratio, 1.44:1) diagnosed with cryptogenic ICC were enrolled in the present study. The mean age was 54.74 ± 10.43 years (range, 21 to 78 y). Table 1 shows the distribution between controls and cryptogenic ICC cases according to age and sex. No significant differences in sex and mean age were found between the 2 groups, which suggest that the pairing was effective.
HBV Antibody Profiles in Cryptogenic ICC Patients The profiles for the HBV antibodies in each of the categories are summarized in Tables 2 and 3. HBeAg was negative in all of the cryptogenic ICC patients and controls. The serum from 26.23% and 31.69% of cryptogenic ICC patients and controls, respectively, was negative for all serological antibodies against HBV. The serum from 19.13% and 4.01% of cryptogenic ICC patients and controls, respectively, was positive for all serological antibodies of HBV. Anti-HBs alone, anti-HBe alone, and anti-HBc alone were detectable in 9.29%, 1.64%, and 10.38% of the cryptogenic ICC patients and in 48.09%, 0.18%, and 4.37% of the controls, respectively. Both anti-HBs and anti-HBe were detectable in 1.09% of the cryptogenic ICC patients and in 0.18% of the controls, both anti-HBs and anti-HBc were detectable in 16.39% of the cryptogenic ICC patients and in 7.29% of the controls, and both anti-HBe and anti-HBc were detectable in 15.85% of the cryptogenic ICC patients and in 4.19% of the controls. Compared with controls, cryptogenic ICC patients had a lower prevalence of anti-HBs (50.27% vs. 59.93%, P = 0.022), but they had a higher prevalence of anti-HBe (37.16% vs. 8.01%, P < 0.001) and anti-HBc (62.30% vs. 20.40%, P < 0.001) (Table 3).
Multiple Logistic Regression Analyses Using a logistic regression model adjusted for demographics (age and sex), we calculated adjusted odds ratios for the different HBV antibodies. This analysis showed that after adjustment, each of the HBV antibodies still remained statistically significant in the multivariate analyses (Table 4). Anti-HBs positive status was negatively
Negative for all antibodies Anti-HBs alone + Anti-HBe alone + Anti-HBc alone + Both anti-HBs + and anti-HBe + Both anti-HBs + and anti-HBc + Both anti-HBe + and anti-HBc + Positive for all antibodies
174 264 1 24 1
(31.69) (48.09) (0.18) (4.37) (0.18)
ICC Cases (n = 183) 48 17 3 19 2
(26.23) (9.29) (1.64) (10.38) (1.09)
40 (7.29)
30 (16.39)
23 (4.19)
29 (15.85)
22 (4.01)
35 (19.13)
Anti-HBc indicates antibody against hepatitis B core antigen; anti-HBe, antibody against hepatitis B e antigen; anti-HBs, antibody against hepatitis B surface antigen; HBV, hepatitis B virus; ICC, intrahepatic cholangiocarcinoma.
correlated with ICC development, which means that antiHBs is a protective factor against ICC development; antiHBe and anti-HBc positive status was positively correlated with ICC development, which indicates that both antibodies are independent risk factors (Table 4).
HBV DNA Level in Sera and HBsAg Protein Expression in Liver Tissue To determine whether HBV DNA was detectable in the serum of cryptogenic ICC patients, samples from 104 patients (56.83%) were tested for total HBV DNA. Only 1 patient (0.96%) had a level of HBV DNA above the limit of detection (7100 IU/mL; limit of detection: 1000 IU/mL), 45 patients with cryptogenic ICC from group I, and 16 patients with cavernous hemangioma of the liver (serum HBV DNA < 1000 IU/mL TABLE 3. Comparison of HBV Antibody Between ICC Cases and Controls
TABLE 1. Demographic Characteristics of Cases With ICC and Control Subjects
Variables Mean age (mean ± SD) (y) Sex Male Female
Cryptogenic ICC Cases (n = 183) 54.74 ± 10.43 108 75
ICC indicates intrahepatic cholangiocarcinoma.
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Control Cases (n = 549)
P
54.90 ± 10.36 0.86 324 225
1.00
n (%) Serological Tests
Controls (n = 549)
ICC Cases (n = 183)
P
Anti-HBs + Anti-HBe + Anti-HBc +
329 (59.93) 44 (8.01) 112 (20.40)
92 (50.27) 68 (37.16) 114 (62.30)
0.022 < 0.001 < 0.001
Anti-HBc indicates antibody against hepatitis B core antigen; anti-HBe, antibody against hepatitis B e antigen; anti-HBs, antibody against hepatitis B surface antigen; HBV, hepatitis B virus; ICC, intrahepatic cholangiocarcinoma.
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TABLE 4. Multiple Logistic Regression Analysis Examining the Association Between HBV Antibodies and ICC
Antibodies Against HBV Anti-HBs + Anti-HBe + Anti-HBc +
95% CI P
ORs
Lower
Upper
0.004 0.001 < 0.001
0.579 2.482 4.556
0.398 1.482 2.938
0.843 4.158 7.066
95% CI indicates 95% confidence interval; anti-HBc, antibody against hepatitis B core antigen; anti-HBe, antibody against hepatitis B e antigen; anti-HBs, antibody against hepatitis B surface antigen; HBV, hepatitis B virus; ICC, intrahepatic cholangiocarcinoma; ORs, odds ratios.
and seronegative for HBsAg) was assessed for intrahepatic total HBV DNA. All 3 of the HBV-associated ICC patients (100%) had detectable intrahepatic HBV DNA (Z1000 IU/ mL), which confirms the feasibility of this method. Compared with hepatic cavernous hemangioma patients, cryptogenic ICC patients were more frequently male (male/female: 26/18 vs. 4/ 12) and older (54.11 ± 11.72 vs. 48.38 ± 6.21), but no significant difference in age was found between the 2 groups (P = 0.068). Intrahepatic total HBV DNA was more frequently detected in ICC cases than in cavernous hemangioma liver patients (63.64% vs. 18.75%, P = 0.002). Compared with cryptogenic patients without anti-HBc (n = 16), cryptogenic ICC patients with anti-HBc (n = 28) demonstrated more frequent detection of intrahepatic total HBV DNA (71.43% vs. 50.00%), but there was no statistically significant difference between the 2 groups (P = 0.155).
FIGURE 2. Immunohistochemical staining of intrahepatic cholangiocarcinoma (ICC). Positive reactivity was observed in the cytoplasm of hepatic cells adjacent to ICC. r
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OBI and ICC
DISCUSSION The diagnosis of infection by HBV is based on the persistent presence of HBsAg in the blood. HBsAg-negative HBV infection or OBI is a recently recognized entity. The causal relationship between CHB infection and HCC or ICC is known.1–4 Furthermore, there have been a number of studies on the detection of tissue HBV DNA in HBsAgnegative HCC patients. A large amount of evidence supports OBI as an important risk factor for the development of HCC.10,13,19,20 However, the association between OBI and ICC is less defined. In this study, we not only found that OBI may represent an important risk factor for ICC development in the Chinese population but also provided circumstantial evidence to support the hypothesis that some cryptogenic ICC patients in the Chinese population had experienced HBsAg seroclearance. First, expression profiles of HBV antibodies were significantly different between cryptogenic ICC and healthy individuals, and the detection rate of anti-HBc in cryptogenic ICC patients was significantly higher than that in healthy individuals. Second, nearly one sixth of the cryptogenic ICC cases (15.39%) had positive reactivity of HBsAg in liver tissue. Third, more than half of the cryptogenic ICC cases (63.64%) had detectable HBV DNA in liver tissue. Finally, 18 patients had a clear history of CHB and HBsAg seroclearance of the 85 ICC patients with seronegative HBsAg cases excluded. The presence of anti-HBc indicates previous HBV infection and is a useful surrogate marker of OBI.21 In the present study, we found a significant difference in the distribution profiles of HBV antibodies between cryptogenic ICC patients and healthy individuals. The detection rate of anti-HBc in cryptogenic ICC patients was significantly higher than that in healthy individuals (62.30% vs. 20.40%), and anti-HBc was an independent risk factor for cryptogenic ICC development. The present results indicate that OBI may be an important risk factor for cryptogenic ICC development. Anti-HBc positivity does not always indicate OBI, and it is not a true marker of OBI. HBV DNA is the only reliable diagnostic maker of OBI.21 Although serum HBV DNA is a good marker for OBI, as OBI status is related to the persistence over time of viral genomes in hepatocytes, it is not sufficient for identifying all cases. Consequently, the analysis of liver DNA extracts for HBV DNA remains the gold standard for the diagnosis of OBI.21 In the present study, we detected serum total HBV DNA in 104 of the 183 cryptogenic ICC patients (56.83%) using a diagnostic kit for the quantification of HBV DNA (a standard commercial assay for the detection of HBV DNA in human serum samples); only 1 patient had levels of serum HBV DNA above the limit of detection. In contrast, we found that 28 of the 44 cryptogenic ICC cases (63.64%) had detectable HBV DNA in the liver tissue. The detection rate for intrahepatic total HBV DNA in cryptogenic ICC patients was significantly higher than that in hepatic cavernous hemangioma patients with seronegative HBsAg (63.64% vs. 18.75%). These results indicate that OBI may be an important risk factor for ICC development. A large amount of evidence suggests that the prevalence of OBI is higher among individuals with anti-HBc than among those who are anti-HBc-negative.22 In the present study, we also found that the prevalence of OBI was higher among cryptogenic ICC patients who were anti-HBc positive than among those who were anti-HBc-negative (74.43% vs. 50.00%). Our data further support the results of former studies. www.jcge.com |
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Seroclearance of HBsAg is a rare event in CHB infection, with an incidence of approximately 0.1% to 0.8% per year in patients acquiring CHB early in life (ie, Asian, African, and some Mediterranean patients) and 0.4% to 2% per year in patients acquiring CHB during adolescence and adulthood (ie, white patients).8 Although HBsAg seroclearance is usually associated with favorable virologic, biochemical, and histologic parameters, all patients who experience seroclearance still harbor HBV inside the liver.8 Moreover, several studies have shown that HCC can still develop after HBsAg seroclearance, especially for patients who experience HBsAg seroclearance after the age of 50 years.8,9,23,24 In the present study, we found that the expression profiles of HBV antibodies were significantly different between cryptogenic ICC patients and healthy individuals. In total, 28 of the 182 cryptogenic ICC cases (15.39%) had positive reactivity for HBsAg in liver tissue, and 28 of the 44 cryptogenic ICC cases (63.64%) had detectable intrahepatic HBV DNA. Eighteen patients had a clear history of CHB and HBsAg seroclearance of the 85 ICC patients with seronegative HBsAg excluded. These results strongly indicate that some cryptogenic ICC patients in the Chinese population had experienced HBsAg seroclearance and that ICC can still develop after HBsAg seroclearance. Finally, much evidence indicates that HBV-induced liver injury, including necroinflammation, fibrosis, and cirrhosis,25 is a possible mechanism underlying OBI-induced hepatocellular carcinogenesis.26 However, OBI-induced intrahepatic cholangiocarcinogenesis is not completely understood. Although HBV-induced liver injury is also a possible mechanism for OBI-induced intrahepatic cholangiocarcinogenesis, only 15 of the 183 cryptogenic ICC patients in the present study had liver necroinflammation and fibrosis on pathologic observation. Thus, we speculate that HBV-induced liver injury may not be an important mechanism for OBI-induced intrahepatic cholangiocarcinogenesis. Indeed, occult HBV may also contribute to hepatocyte transformation through direct mechanisms, including potential transforming activity of the hepatitis B X protein and truncated hepatitis B middle surface protein and chromosomal instability resulting from the integration of HBV DNA into the host chromosome.24 The association between occult HBV and HCC had been widely confirmed by sensitive and specific molecular techniques. According to our previous study, ICC patients with seronegative HBsAg have a significantly lower proportion of abnormal aminotransferase, histologic necroinflammation, fibrosis, and cirrhosis relative to ICC patients with seropositive HBsAg.4 These phenomena suggest that the direct mechanism mentioned above may be the main mechanism for OBIinduced ICC. We plan to further investigate this mechanism in a future study. In conclusion, these data suggest that OBI may represent an important risk factor for the development of ICC in the Chinese population. HBsAg seroclearance does not signify eradication of HBV and does not entirely prevent the development of ICC. Although HBV-induced liver injury is a possible mechanism underlying OBI-induced intrahepatic cholangiocarcinogenesis, it may not be the major mechanism for carcinogenesis.
3. 4.
5.
6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22.
23. 24.
25.
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lymphoma: a cohort study of parous women in Taiwan. Hepatology. 2011;53:1217–1225. Lee CH, Chang CJ, Lin YJ, et al. Viral hepatitis-associated intrahepatic cholangiocarcinoma shares common disease processes with hepatocellular carcinoma. Br J Cancer. 2009;100:1765–1770. Zhou H, Wang H, Zhou D, et al. Hepatitis B virus-associated intrahepatic cholangiocarcinoma and hepatocellular carcinoma may hold common disease process for carcinogenesis. Eur J Cancer. 2010;46:1056–1061. Hughes NR, Pairojkul C, Royce SG, et al. Liver flukeassociated and sporadic cholangiocarcinoma: an immunohistochemical study of bile duct, peribiliary gland and tumour cell phenotypes. J Clin Pathol. 2006;59:1073–1078. Chalasani N, Baluyut A, Ismail A, et al. Cholangiocarcinoma in patients with primary sclerosing cholangitis: a multicenter case-control study. Hepatology. 2000;31:7–11. Shaib YH, El-Serag HB, Davila JA, et al. Risk factors of intrahepatic cholangiocarcinoma in the United States: a casecontrol study. Gastroenterology. 2005;128:620–626. Yuen MF, Wong DK, Fung J, et al. HBsAg Seroclearance in chronic hepatitis B in Asian patients: replicative level and risk of hepatocellular carcinoma. Gastroenterology. 2008;135:1192–1199. Yuen MF, Wong DK, Sablon E, et al. HBsAg seroclearance in chronic hepatitis B in the Chinese: virological, histological, and clinical aspects. Hepatology. 2004;39:1694–1701. Raimondo G, Pollicino T, Cacciola I, et al. Occult hepatitis B virus infection. J Hepatol. 2007;46:160–170. Raimondo G, Pollicino T, Romano L, et al. A 2010 update on occult hepatitis B infection. Pathol Biol (Paris). 2010;58:254–257. Hollinger FB, Sood G. Occult hepatitis B virus infection: a covert operation. J Viral Hepat. 2010;17:1–15. Torbenson M, Thomas DL. Occult hepatitis B. Lancet Infect Dis. 2002;2:479–486. Raimondo G, Navarra G, Mondello S, et al. Occult hepatitis B virus in liver tissue of individuals without hepatic disease. J Hepatol. 2008;48:743–746. Cacciola I, Pollicino T, Squadrito G, et al. Occult hepatitis B virus infection in patients with chronic hepatitis C liver disease. N Engl J Med. 1999;341:22–26. Chemin I, Trepo C. Clinical impact of occult HBV infections. J Clin Virol. 2005;34(suppl 1):S15–S21. Hu KQ. Occult hepatitis B virus infection and its clinical implications. J Viral Hepat. 2002;9:243–257. Mulrooney-Cousins PM, Michalak TI. Persistent occult hepatitis B virus infection: experimental findings and clinical implications. World J Gastroenterol. 2007;13:5682–5686. Brechot C. Pathogenesis of hepatitis B virus-related hepatocellular carcinoma: old and new paradigms. Gastroenterology. 2004;127:S56–S61. Chemin I, Zoulim F. Hepatitis B virus induced hepatocellular carcinoma. Cancer Lett. 2009;286:52–59. Raimondo G, Allain JP, Brunetto MR, et al. Statements from the Taormina expert meeting on occult hepatitis B virus infection. J Hepatol. 2008;49:652–657. Nirei K, Kaneko M, Moriyama M, et al. The clinical features of chronic hepatitis C are not affected by the coexistence of hepatitis B virus DNA in patients negative for hepatitis B surface antigen. Intervirology. 2000;43:95–101. Ahn SH, Park YN, Park JY, et al. Long-term clinical and histological outcomes in patients with spontaneous hepatitis B surface antigen seroclearance. J Hepatol. 2005;42:188–194. Shiota G, Oyama K, Udagawa A, et al. Occult hepatitis B virus infection in HBs antigen-negative hepatocellular carcinoma in a Japanese population: involvement of HBx and p53. J Med Virol. 2000;62:151–158. De Mitri MS, Cassini R, Bernardi M. Hepatitis B virus-related hepatocarcinogenesis: molecular oncogenic potential of clear or occult infections. Eur J Cancer. 2010;46:2178–2186. Squadrito G, Pollicino T, Cacciola I, et al. Occult hepatitis B virus infection is associated with the development of hepatocellular carcinoma in chronic hepatitis C patients. Cancer. 2006;106:1326–1330. r
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Occult Hepatitis B Virus Infection in Chinese Cryptogenic Intrahepatic Cholangiocarcinoma Patient Population YuQiong Li, MM,* Hui Wang, MM,* DaPeng Li, MM,w JingYi Hu, MM,z Hao Wang, MD,* DongXun Zhou, MD,* QiaoMei Li, MD,* XiaoLan Jiang, MM,* HuaBang Zhou, MD,* and HePing Hu, MD*
Background: There is no information available about occult hepatitis B virus (HBV) infection (OBI) in individuals with intrahepatic cholangiocarcinoma (ICC). Goals: To investigate the correlation between OBI and ICC. Study: A retrospective case-control study was conducted. The cases were 183 cryptogenic ICC patients (group I), and the controls were 549 healthy individuals (group II). The cases and controls were matched for age, sex, and inhabitancy. Adjusted odds ratios and 95% confidence intervals were calculated. Intrahepatic total HBV DNA in 63 paraffin-embedded samples was collected from patients in group I (n = 44), HBV-associated ICC patients (n = 3), and hepatic cavernous hemangioma patients with seronegative HBsAg (hepatitis B S antigen) (group III; n = 16). We determined the levels of serum and intrahepatic HBV DNA and compared the level of intrahepatic HBV DNA in 44 cryptogenic patients from group I with the level in the patients from group III. Results: Compared with group II, group I had a lower prevalence of anti-HBs (antibody against HBsAg) and a higher prevalence of anti-HBe (antibody against hepatitis B e antigen) and anti-HBc (antibody against hepatitis B c antigen). Multivariate analysis confirmed that anti-HBe and anti-HBc positivity were associated with ICC. The odds ratios and 95% confidence intervals for antiHBe and anti-HBc were 2.482 and 1.482-4.158, 4.556 and 2.9387.066, respectively. Compared with group III, cryptogenic ICC cases showed more frequent detection of intrahepatic total HBV DNA (63.64% vs. 18.75%, P = 0.002). Conclusions: OBI may represent an important risk factor for ICC. HBsAg seroclearance does not signify eradication of HBV and may not entirely prevent the development of ICC. Key Words: intrahepatic cholangiocarcinoma, occult hepatitis B virus, carcinogenesis
(J Clin Gastroenterol 2014;48:878–882)
I
ntrahepatic cholangiocarcinoma (ICC) is a malignant tumor that arises from the peripheral intrahepatic bile duct epithelium and is far less prevalent than hepatocellular
Received for publication June 12, 2013; accepted November 17, 2013. From the *Department of Hepatobiliary I, Eastern Hepatobiliary Surgery Hospital, Second Military Medical University; zSchool of Medicine, Jiao Tong University, Shanghai; and wDepartment of Gastroenterology, First Affiliated Hospital, Bengbu Medical College, Anhui, China. Y.L., H.W., and D.L. are the co-first authors. The authors declare that they have nothing to disclose. Reprints: HuaBang Zhou, MD, Department of Hepatobiliary I, Eastern Hepatobiliary Surgery Hospital, Second Military Medical University, 225 Changhai road, Shanghai 200438, China (e-mail: [email protected]). Copyright r 2013 by Lippincott Williams & Wilkins
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carcinoma (HCC). HCC accounts for >90% of all primary liver cancers in areas endemic for hepatitis B virus (HBV), whereas ICC accounts for approximately 5% of primary liver cancers.1 The etiology and carcinogenic mechanism of ICC remain unclear. Recent evidence suggests a possible etiological role for chronic HBV infection with detectable circulating hepatitis B surface antigens (HBsAg) in ICC development.2,3 China is an HBV-endemic area, and HBV is the predominant cause of HCC in this region. Data from our previous study also demonstrated that the incidence of HBV infection in ICC patients was significantly higher than that in individuals without cancer (48.6% vs. 6.6%) and that chronic HBV infection was the most important independent risk factor for ICC in the Chinese population.4 Several risk factors in addition to HBV infection have been reported as potential risk factors for ICC, such as liver fluke infestation,5 primary sclerosing cholangitis (PSC),6 hepatolithiasis, and chronic HCV infection7; however, nearly 40% of ICC cases in the Chinese population occur in the absence of known etiological factors.4 Detailed studies delineating the exact cause of ICC in this group of patients are currently lacking. During the course of chronic hepatitis B (CHB) infection, some patients may undergo HBsAg seroclearance and have undetectable circulating HBsAg.8,9 With the development of sensitive nucleic acid detection techniques, HBsAg-negative subjects with detectable HBV DNA in sera or liver tissues can be identified. Patients who have undetectable HBsAg and yet have detectable HBV DNA in the serum or liver are defined as having occult HBV infection (OBI).10–15 Although the clinical significance of this condition is not well known, several clinical observations show that OBI is an important clinical factor that can lead to: (1) reactivation of HBV infection in patients without HBsAg after chemotherapy, (2) HBV transmission from HBsAgnegative and anti-HBc-positive blood or organ donors, and (3) the development of cryptogenic cirrhosis.16–18 Recently, some researchers have also demonstrated that OBI patients with HBsAg seroclearance can still develop HCC8,9,16–18; OBI is thought to be an important risk factor for HCC development because it maintains the pro-oncogenic properties typical of the overt infection. The potential role of OBI as a risk factor for ICC development has not been addressed to date. In the present study, we therefore conducted a retrospective case-control study comprised of 183 cryptogenic ICC patients and 549 healthy individuals; in addition, we tested 63 intrahepatic HBV DNA samples (44 cryptogenic ICC patients, 3 HBV-associated ICC patients, and 16 hepatic cavernous hemangioma patients with seronegative HBsAg). Our goal was to investigate whether OBI was a risk factor for ICC development.
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MATERIALS AND METHODS Study Subjects From January 2003 to October 2007, 515 ICC patients underwent surgical dissection at the Eastern Hepatobiliary Surgery Hospital of the Second Military Medical University (Shanghai, China). The diagnosis of ICC was confirmed by pathology. Using our exclusion criteria (Fig. 1), we excluded 332 patients because of seropositive HBsAg (n = 247), hepatolithiasis (n = 39) (36 hepatolithiasis alone, 1 hepatolithiasis plus a history of chronic HBV infection, 1 hepatolithiasis plus hepatic schistosomiasis, and 1 hepatolithiasis plus HCV infection), PSC (n = 1), HCV infection alone (n = 1), hepatic schistosomiasis alone (n = 14), history of chronic HBV infection alone (n = 17), alcoholassociated cirrhosis (n = 9), and cryptogenetic cirrhosis (n = 4). The remaining 183 cryptogenic ICC patients (group I) were selected for statistical analysis. The healthy controls consisted of 549 healthy individuals (group II) who had visited the Eastern Hepatobiliary Surgery Hospital of the Second Military Medical University for a routine checkup. Exclusion criteria were as follows: seropositive HBsAg, history of chronic HBV infection, HCV infection, PSC, cirrhosis, hepatolithiasis, hepatic schistosomiasis, and cancer. Controls were matched to the ICC cases for sex and age (± 4 y) and were recruited during the same study period as the cases. To further clarify the correlation between OBI and ICC development, 3 patients with HBV-associated ICC
OBI and ICC
(2 males and 1 female) were selected as positive controls. Forty-four patients (26 males and 18 females) with cryptogenic ICC who were randomly selected from group I and 16 seronegative HBsAg patients (group III; 4 males and 12 females) with hepatic cavernous hemangioma who underwent hepatectomy during the same period in the same hospital were selected for liver total HBV DNA analysis. The project was approved by the local ethics committee.
Laboratory Tests of Virology Blood samples were tested for HBsAg, the antibody to hepatitis B surface antigen (anti-HBs), hepatitis B e antigen (HBeAg), the antibody to hepatitis B e antigen (anti-HBe), the antibody to hepatitis core antigen (anti-HBc), and antibodies against the hepatitis C virus (anti-HCV) using enzyme-linked immunosorbent assays (Abbott Laboratories, North Chicago, IL).
Isolation of Genomic DNA From FFPE Tissue Sections Total liver DNA was extracted from paraffin-embedded specimens using the QIAamp DNA FFPE Tissue Kit (Qiagen GmbH, Hilden, Germany) according to the manufacturer’s instructions. The total liver DNA was diluted with buffer ATE to a final concentration of 50 ng/mL per case for subsequent quantitative measurement of total HBV DNA.
Quantitative Measurement of Serum and Intrahepatic Total HBV DNA Serum HBV DNA levels were measured in 104 patients (56.83%). HBV DNA was extracted from 100 mL of serum using a diagnostic kit for the quantification of HBV DNA (Shanghai Kehua Laboratory System Co. Ltd., China) according to the manufacturer’s instructions with a final elution volume of 2 mL. The extracted DNA was then quantitatively measured by the same kit using the ABI7500 Fast Real-time PCR System (Applied Biosystems, Mortlake) according to the manufacturer’s instructions. This kit is a standard commercial assay for the detection of HBV DNA in human serum samples. To ensure the specificity of the test and to prevent diagnostic errors, the limit of detection was defined as 1000 IU/mL for serum or liver tissue.
Histologic Assessment Immunoperoxidase staining was performed using monoclonal antibodies against HBsAg (dilution, 1:100; Signet, Dedham, MA).
Liver Cirrhosis The diagnosis of cirrhosis was made according to histologic evaluation of the liver parenchyma. If histologic information was not available, clinically diagnosed liver cirrhosis was defined as follows: (i) platelet count 12 cm); (ii) esophageal or gastric varices; or (iii) overt complications of liver cirrhosis, including ascites, variceal bleeding, and hepatic encephalopathy.
Statistical Methods FIGURE 1. Flow chart for patient selection. HBV indicates hepatitis B virus; HCV, hepatitis C virus; PSC, primary sclerosing cholangitis. r
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Descriptive analyses of the variables were conducted using SPSS 16.0 (SPSS Inc., Chicago, IL). Univariate analyses were performed using the w2 test for categorical variables and the independent samples t test for discrete variables. www.jcge.com |
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P < 0.05 was considered statistically significant. For each risk factor, the odds ratios for ICC and their 95% confidence intervals were computed as estimates of the relative risk by unconditional logistic regression analysis using maximum likelihood estimates.
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TABLE 2. HBV Serological Antibody Profiles in Cryptogenic ICC Cases and Controls
n (%) Controls (n = 549)
Serological Tests
RESULTS Demographic and Baseline Features of Cases and Controls A total of 183 patients (108 males and 75 females; male-to-female ratio, 1.44:1) diagnosed with cryptogenic ICC were enrolled in the present study. The mean age was 54.74 ± 10.43 years (range, 21 to 78 y). Table 1 shows the distribution between controls and cryptogenic ICC cases according to age and sex. No significant differences in sex and mean age were found between the 2 groups, which suggest that the pairing was effective.
HBV Antibody Profiles in Cryptogenic ICC Patients The profiles for the HBV antibodies in each of the categories are summarized in Tables 2 and 3. HBeAg was negative in all of the cryptogenic ICC patients and controls. The serum from 26.23% and 31.69% of cryptogenic ICC patients and controls, respectively, was negative for all serological antibodies against HBV. The serum from 19.13% and 4.01% of cryptogenic ICC patients and controls, respectively, was positive for all serological antibodies of HBV. Anti-HBs alone, anti-HBe alone, and anti-HBc alone were detectable in 9.29%, 1.64%, and 10.38% of the cryptogenic ICC patients and in 48.09%, 0.18%, and 4.37% of the controls, respectively. Both anti-HBs and anti-HBe were detectable in 1.09% of the cryptogenic ICC patients and in 0.18% of the controls, both anti-HBs and anti-HBc were detectable in 16.39% of the cryptogenic ICC patients and in 7.29% of the controls, and both anti-HBe and anti-HBc were detectable in 15.85% of the cryptogenic ICC patients and in 4.19% of the controls. Compared with controls, cryptogenic ICC patients had a lower prevalence of anti-HBs (50.27% vs. 59.93%, P = 0.022), but they had a higher prevalence of anti-HBe (37.16% vs. 8.01%, P < 0.001) and anti-HBc (62.30% vs. 20.40%, P < 0.001) (Table 3).
Multiple Logistic Regression Analyses Using a logistic regression model adjusted for demographics (age and sex), we calculated adjusted odds ratios for the different HBV antibodies. This analysis showed that after adjustment, each of the HBV antibodies still remained statistically significant in the multivariate analyses (Table 4). Anti-HBs positive status was negatively
Negative for all antibodies Anti-HBs alone + Anti-HBe alone + Anti-HBc alone + Both anti-HBs + and anti-HBe + Both anti-HBs + and anti-HBc + Both anti-HBe + and anti-HBc + Positive for all antibodies
174 264 1 24 1
(31.69) (48.09) (0.18) (4.37) (0.18)
ICC Cases (n = 183) 48 17 3 19 2
(26.23) (9.29) (1.64) (10.38) (1.09)
40 (7.29)
30 (16.39)
23 (4.19)
29 (15.85)
22 (4.01)
35 (19.13)
Anti-HBc indicates antibody against hepatitis B core antigen; anti-HBe, antibody against hepatitis B e antigen; anti-HBs, antibody against hepatitis B surface antigen; HBV, hepatitis B virus; ICC, intrahepatic cholangiocarcinoma.
correlated with ICC development, which means that antiHBs is a protective factor against ICC development; antiHBe and anti-HBc positive status was positively correlated with ICC development, which indicates that both antibodies are independent risk factors (Table 4).
HBV DNA Level in Sera and HBsAg Protein Expression in Liver Tissue To determine whether HBV DNA was detectable in the serum of cryptogenic ICC patients, samples from 104 patients (56.83%) were tested for total HBV DNA. Only 1 patient (0.96%) had a level of HBV DNA above the limit of detection (7100 IU/mL; limit of detection: 1000 IU/mL), 45 patients with cryptogenic ICC from group I, and 16 patients with cavernous hemangioma of the liver (serum HBV DNA < 1000 IU/mL TABLE 3. Comparison of HBV Antibody Between ICC Cases and Controls
TABLE 1. Demographic Characteristics of Cases With ICC and Control Subjects
Variables Mean age (mean ± SD) (y) Sex Male Female
Cryptogenic ICC Cases (n = 183) 54.74 ± 10.43 108 75
ICC indicates intrahepatic cholangiocarcinoma.
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Control Cases (n = 549)
P
54.90 ± 10.36 0.86 324 225
1.00
n (%) Serological Tests
Controls (n = 549)
ICC Cases (n = 183)
P
Anti-HBs + Anti-HBe + Anti-HBc +
329 (59.93) 44 (8.01) 112 (20.40)
92 (50.27) 68 (37.16) 114 (62.30)
0.022 < 0.001 < 0.001
Anti-HBc indicates antibody against hepatitis B core antigen; anti-HBe, antibody against hepatitis B e antigen; anti-HBs, antibody against hepatitis B surface antigen; HBV, hepatitis B virus; ICC, intrahepatic cholangiocarcinoma.
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TABLE 4. Multiple Logistic Regression Analysis Examining the Association Between HBV Antibodies and ICC
Antibodies Against HBV Anti-HBs + Anti-HBe + Anti-HBc +
95% CI P
ORs
Lower
Upper
0.004 0.001 < 0.001
0.579 2.482 4.556
0.398 1.482 2.938
0.843 4.158 7.066
95% CI indicates 95% confidence interval; anti-HBc, antibody against hepatitis B core antigen; anti-HBe, antibody against hepatitis B e antigen; anti-HBs, antibody against hepatitis B surface antigen; HBV, hepatitis B virus; ICC, intrahepatic cholangiocarcinoma; ORs, odds ratios.
and seronegative for HBsAg) was assessed for intrahepatic total HBV DNA. All 3 of the HBV-associated ICC patients (100%) had detectable intrahepatic HBV DNA (Z1000 IU/ mL), which confirms the feasibility of this method. Compared with hepatic cavernous hemangioma patients, cryptogenic ICC patients were more frequently male (male/female: 26/18 vs. 4/ 12) and older (54.11 ± 11.72 vs. 48.38 ± 6.21), but no significant difference in age was found between the 2 groups (P = 0.068). Intrahepatic total HBV DNA was more frequently detected in ICC cases than in cavernous hemangioma liver patients (63.64% vs. 18.75%, P = 0.002). Compared with cryptogenic patients without anti-HBc (n = 16), cryptogenic ICC patients with anti-HBc (n = 28) demonstrated more frequent detection of intrahepatic total HBV DNA (71.43% vs. 50.00%), but there was no statistically significant difference between the 2 groups (P = 0.155).
FIGURE 2. Immunohistochemical staining of intrahepatic cholangiocarcinoma (ICC). Positive reactivity was observed in the cytoplasm of hepatic cells adjacent to ICC. r
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OBI and ICC
DISCUSSION The diagnosis of infection by HBV is based on the persistent presence of HBsAg in the blood. HBsAg-negative HBV infection or OBI is a recently recognized entity. The causal relationship between CHB infection and HCC or ICC is known.1–4 Furthermore, there have been a number of studies on the detection of tissue HBV DNA in HBsAgnegative HCC patients. A large amount of evidence supports OBI as an important risk factor for the development of HCC.10,13,19,20 However, the association between OBI and ICC is less defined. In this study, we not only found that OBI may represent an important risk factor for ICC development in the Chinese population but also provided circumstantial evidence to support the hypothesis that some cryptogenic ICC patients in the Chinese population had experienced HBsAg seroclearance. First, expression profiles of HBV antibodies were significantly different between cryptogenic ICC and healthy individuals, and the detection rate of anti-HBc in cryptogenic ICC patients was significantly higher than that in healthy individuals. Second, nearly one sixth of the cryptogenic ICC cases (15.39%) had positive reactivity of HBsAg in liver tissue. Third, more than half of the cryptogenic ICC cases (63.64%) had detectable HBV DNA in liver tissue. Finally, 18 patients had a clear history of CHB and HBsAg seroclearance of the 85 ICC patients with seronegative HBsAg cases excluded. The presence of anti-HBc indicates previous HBV infection and is a useful surrogate marker of OBI.21 In the present study, we found a significant difference in the distribution profiles of HBV antibodies between cryptogenic ICC patients and healthy individuals. The detection rate of anti-HBc in cryptogenic ICC patients was significantly higher than that in healthy individuals (62.30% vs. 20.40%), and anti-HBc was an independent risk factor for cryptogenic ICC development. The present results indicate that OBI may be an important risk factor for cryptogenic ICC development. Anti-HBc positivity does not always indicate OBI, and it is not a true marker of OBI. HBV DNA is the only reliable diagnostic maker of OBI.21 Although serum HBV DNA is a good marker for OBI, as OBI status is related to the persistence over time of viral genomes in hepatocytes, it is not sufficient for identifying all cases. Consequently, the analysis of liver DNA extracts for HBV DNA remains the gold standard for the diagnosis of OBI.21 In the present study, we detected serum total HBV DNA in 104 of the 183 cryptogenic ICC patients (56.83%) using a diagnostic kit for the quantification of HBV DNA (a standard commercial assay for the detection of HBV DNA in human serum samples); only 1 patient had levels of serum HBV DNA above the limit of detection. In contrast, we found that 28 of the 44 cryptogenic ICC cases (63.64%) had detectable HBV DNA in the liver tissue. The detection rate for intrahepatic total HBV DNA in cryptogenic ICC patients was significantly higher than that in hepatic cavernous hemangioma patients with seronegative HBsAg (63.64% vs. 18.75%). These results indicate that OBI may be an important risk factor for ICC development. A large amount of evidence suggests that the prevalence of OBI is higher among individuals with anti-HBc than among those who are anti-HBc-negative.22 In the present study, we also found that the prevalence of OBI was higher among cryptogenic ICC patients who were anti-HBc positive than among those who were anti-HBc-negative (74.43% vs. 50.00%). Our data further support the results of former studies. www.jcge.com |
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Seroclearance of HBsAg is a rare event in CHB infection, with an incidence of approximately 0.1% to 0.8% per year in patients acquiring CHB early in life (ie, Asian, African, and some Mediterranean patients) and 0.4% to 2% per year in patients acquiring CHB during adolescence and adulthood (ie, white patients).8 Although HBsAg seroclearance is usually associated with favorable virologic, biochemical, and histologic parameters, all patients who experience seroclearance still harbor HBV inside the liver.8 Moreover, several studies have shown that HCC can still develop after HBsAg seroclearance, especially for patients who experience HBsAg seroclearance after the age of 50 years.8,9,23,24 In the present study, we found that the expression profiles of HBV antibodies were significantly different between cryptogenic ICC patients and healthy individuals. In total, 28 of the 182 cryptogenic ICC cases (15.39%) had positive reactivity for HBsAg in liver tissue, and 28 of the 44 cryptogenic ICC cases (63.64%) had detectable intrahepatic HBV DNA. Eighteen patients had a clear history of CHB and HBsAg seroclearance of the 85 ICC patients with seronegative HBsAg excluded. These results strongly indicate that some cryptogenic ICC patients in the Chinese population had experienced HBsAg seroclearance and that ICC can still develop after HBsAg seroclearance. Finally, much evidence indicates that HBV-induced liver injury, including necroinflammation, fibrosis, and cirrhosis,25 is a possible mechanism underlying OBI-induced hepatocellular carcinogenesis.26 However, OBI-induced intrahepatic cholangiocarcinogenesis is not completely understood. Although HBV-induced liver injury is also a possible mechanism for OBI-induced intrahepatic cholangiocarcinogenesis, only 15 of the 183 cryptogenic ICC patients in the present study had liver necroinflammation and fibrosis on pathologic observation. Thus, we speculate that HBV-induced liver injury may not be an important mechanism for OBI-induced intrahepatic cholangiocarcinogenesis. Indeed, occult HBV may also contribute to hepatocyte transformation through direct mechanisms, including potential transforming activity of the hepatitis B X protein and truncated hepatitis B middle surface protein and chromosomal instability resulting from the integration of HBV DNA into the host chromosome.24 The association between occult HBV and HCC had been widely confirmed by sensitive and specific molecular techniques. According to our previous study, ICC patients with seronegative HBsAg have a significantly lower proportion of abnormal aminotransferase, histologic necroinflammation, fibrosis, and cirrhosis relative to ICC patients with seropositive HBsAg.4 These phenomena suggest that the direct mechanism mentioned above may be the main mechanism for OBIinduced ICC. We plan to further investigate this mechanism in a future study. In conclusion, these data suggest that OBI may represent an important risk factor for the development of ICC in the Chinese population. HBsAg seroclearance does not signify eradication of HBV and does not entirely prevent the development of ICC. Although HBV-induced liver injury is a possible mechanism underlying OBI-induced intrahepatic cholangiocarcinogenesis, it may not be the major mechanism for carcinogenesis.
3. 4.
5.
6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22.
23. 24.
25.
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lymphoma: a cohort study of parous women in Taiwan. Hepatology. 2011;53:1217–1225. Lee CH, Chang CJ, Lin YJ, et al. Viral hepatitis-associated intrahepatic cholangiocarcinoma shares common disease processes with hepatocellular carcinoma. Br J Cancer. 2009;100:1765–1770. Zhou H, Wang H, Zhou D, et al. Hepatitis B virus-associated intrahepatic cholangiocarcinoma and hepatocellular carcinoma may hold common disease process for carcinogenesis. Eur J Cancer. 2010;46:1056–1061. Hughes NR, Pairojkul C, Royce SG, et al. Liver flukeassociated and sporadic cholangiocarcinoma: an immunohistochemical study of bile duct, peribiliary gland and tumour cell phenotypes. J Clin Pathol. 2006;59:1073–1078. Chalasani N, Baluyut A, Ismail A, et al. Cholangiocarcinoma in patients with primary sclerosing cholangitis: a multicenter case-control study. Hepatology. 2000;31:7–11. Shaib YH, El-Serag HB, Davila JA, et al. Risk factors of intrahepatic cholangiocarcinoma in the United States: a casecontrol study. Gastroenterology. 2005;128:620–626. Yuen MF, Wong DK, Fung J, et al. HBsAg Seroclearance in chronic hepatitis B in Asian patients: replicative level and risk of hepatocellular carcinoma. Gastroenterology. 2008;135:1192–1199. Yuen MF, Wong DK, Sablon E, et al. HBsAg seroclearance in chronic hepatitis B in the Chinese: virological, histological, and clinical aspects. Hepatology. 2004;39:1694–1701. Raimondo G, Pollicino T, Cacciola I, et al. Occult hepatitis B virus infection. J Hepatol. 2007;46:160–170. Raimondo G, Pollicino T, Romano L, et al. A 2010 update on occult hepatitis B infection. Pathol Biol (Paris). 2010;58:254–257. Hollinger FB, Sood G. Occult hepatitis B virus infection: a covert operation. J Viral Hepat. 2010;17:1–15. Torbenson M, Thomas DL. Occult hepatitis B. Lancet Infect Dis. 2002;2:479–486. Raimondo G, Navarra G, Mondello S, et al. Occult hepatitis B virus in liver tissue of individuals without hepatic disease. J Hepatol. 2008;48:743–746. Cacciola I, Pollicino T, Squadrito G, et al. Occult hepatitis B virus infection in patients with chronic hepatitis C liver disease. N Engl J Med. 1999;341:22–26. Chemin I, Trepo C. Clinical impact of occult HBV infections. J Clin Virol. 2005;34(suppl 1):S15–S21. Hu KQ. Occult hepatitis B virus infection and its clinical implications. J Viral Hepat. 2002;9:243–257. Mulrooney-Cousins PM, Michalak TI. Persistent occult hepatitis B virus infection: experimental findings and clinical implications. World J Gastroenterol. 2007;13:5682–5686. Brechot C. Pathogenesis of hepatitis B virus-related hepatocellular carcinoma: old and new paradigms. Gastroenterology. 2004;127:S56–S61. Chemin I, Zoulim F. Hepatitis B virus induced hepatocellular carcinoma. Cancer Lett. 2009;286:52–59. Raimondo G, Allain JP, Brunetto MR, et al. Statements from the Taormina expert meeting on occult hepatitis B virus infection. J Hepatol. 2008;49:652–657. Nirei K, Kaneko M, Moriyama M, et al. The clinical features of chronic hepatitis C are not affected by the coexistence of hepatitis B virus DNA in patients negative for hepatitis B surface antigen. Intervirology. 2000;43:95–101. Ahn SH, Park YN, Park JY, et al. Long-term clinical and histological outcomes in patients with spontaneous hepatitis B surface antigen seroclearance. J Hepatol. 2005;42:188–194. Shiota G, Oyama K, Udagawa A, et al. Occult hepatitis B virus infection in HBs antigen-negative hepatocellular carcinoma in a Japanese population: involvement of HBx and p53. J Med Virol. 2000;62:151–158. De Mitri MS, Cassini R, Bernardi M. Hepatitis B virus-related hepatocarcinogenesis: molecular oncogenic potential of clear or occult infections. Eur J Cancer. 2010;46:2178–2186. Squadrito G, Pollicino T, Cacciola I, et al. Occult hepatitis B virus infection is associated with the development of hepatocellular carcinoma in chronic hepatitis C patients. Cancer. 2006;106:1326–1330. r
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