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Hepatitis B virus transmission and hepatocarcinogenesis: a 9 year retrospective cohort of 13 676 relatives with hepatocellular carcinoma
Journal of Hepatology 40 (2004) 653–659 www.elsevier.com/locate/jhep
Hepatitis B virus transmission and hepatocarcinogenesis: a 9 year retrospective cohort of 13 676 relatives with hepatocellular carcinoma Chien-Hung Chen1, Yang Yuan Chen2, Gran-Hum Chen3, Sien-Sing Yang4, Huang-Shang Tang5, Hsien Hong Lin6, Deng-Yn Lin7, Sing Kai Lo14, Jeng-Ming Du8, Ting-Tsung Chang9, Shinn-Cherng Chen10, Li-Ying Liao11, Chung-Huang Kuo12, Kwo-Chuan Lin2, Dar-In Tai7,12,*, Chi-Sin Changchien12, Wen-Yu Chang10, Jin-Chuan Sheu1, Ding-Shinn Chen1, Yun-Fan Liaw7, Juei-Low Sung13 1
Department of Internal Medicine, National Taiwan University Hospital and National Taiwan University College of Medicine, Taipei, Taiwan, ROC 2 Department of Internal Medicine, Changhua Christian Hospital, Changhua, Taiwan, ROC 3 Department of Internal Medicine, Taichung Veterans General Hospital, Taichung, Taiwan, ROC 4 Liver Unit, Clinical Research Center, Cathay General Hospital, Taipei, Taiwan, ROC 5 Department of Internal Medicine, Tri-service General Hospital, National Defence Medical Center, Taipei, Taiwan, ROC 6 Department of Internal Medicine, Buddhist Tzu Chi General Hospital, Hualien, Taiwan, ROC 7 Liver Research Unit, Chang Gung Memorial Hospital and Chang Gung University College of Medicine, 199 Tun-Hwa North Road, Taipei 105, Taiwan, ROC 8 Department of Medical Information Management, Chang Gung Memorial Hospital, Taipei, Taiwan, ROC 9 Department of Internal Medicine, National Cheng Kung University Hospital, Tainan, Taiwan, ROC 10 Hepatobiliary Division, Department of Internal Medicine, Kaohsiung Medical University Hospital, Kaohsiung, Taiwan, ROC 11 Department of Gastroenterology, Taipei Municipal Jen-Ai Hospital, Taipei, Taiwan, ROC 12 Liver Unit, Kaohsiung Chang Gung Memorial Hospital, Kaohsiung, Taiwan, ROC 13 Sun Yat-Sen Cancer Center Hospital, Taipei, Taiwan, ROC 14 Institute for International Health, Faculty of Medicine, University of Sydney, Sydney, NSW, Australia
Background/Aims: Familial clustering of hepatitis B virus (HBV) infection is related to perinatal transmission, and is the main cause of familial-type hepatocellular carcinoma (HCC). The route of HBV transmission differs between the children and siblings of patients with HCC. This study examined the differences in HBV carrier rates and HCC-related mortality between two generations in HCC families. Methods: From 1992 to 1997, relatives of individuals with HCC were screened prospectively with ultrasonography, alpha-fetoprotein, liver biochemistry tests and viral markers. Total HCC-related deaths during a 9-year period were compared between the generations of index patients and their children. Results: The study included a total of 13 676 relatives in two generations. More HCC-related deaths occurred in the index patient generation than in the child generation. Furthermore, children of female index patients had higher rates of liver cancer related mortality than children of male index patients. The same was true when the analysis was limited to male HBV carriers. The prevalence of HBsAg in the offspring of HBsAg positive mothers was 66% in the child generation and 72% in the index patient generation. These high prevalences indicated high maternal HBV replication status. Conclusions: Perinatal transmission and maternal viral load are important risk factors in hepatocarcinogenesis. q 2003 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved. Keywords: Maternal – fetal exchange; Taiwan; Survival analysis; Hepatitis B surface antigen; Family
Received 10 May 2003; received in revised form 22 October 2003; accepted 4 December 2003 * Corresponding author. Tel.: þ 886-3-328-1200; fax: þ886-3-327-2236. E-mail address: [email protected] (D.I. Tai). Abbreviations: HBsAg, hepatitis B surface antigen; HBV, hepatitis B virus; HCC, hepatocellular carcinoma; MP, HBsAg positive mother; MN, HBsAg negative mother; FP, HBsAg positive father; FN, HBsAg negative father. 0168-8278/$30.00 q 2003 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.jhep.2003.12.002
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1. Introduction Viral hepatitis, alcohol, smoking, familial tendency and aflatoxin are well-known risk factors for hepatocellular carcinoma (HCC) [1 – 3]. Several cohort studies have found that HBsAg carriers are at highest risk (relative risk ranges from 7 to 98) of developing HCC, while other factors appear to have a lower risk (relative risk ranges from one to four) [1 –3]. Nevertheless, a low risk of HCC in asymptomatic HBsAg carriers was observed in another study [4]. The reason for this discrepancy attracted our attention. One clue is that approximately 70% of the HBsAg positive patients with HCC acquired their HBV infection perinatally [5 – 7]. Because familial aggregation is a characteristic of HCC, a nationwide survey of relatives of patients with HCC was done between 1992 and 1997. The original purposes of the survey were to study viral infection status and have an early detection of HCC in HCC families [8 – 11]. The survey included the parents of the index patients, the index patients themselves, and their children. Given the same transmission route (perinatal transmission) as the index patients, the index patient generation had a high prevalence of HBsAg (approximately 60%) [8 –10]. In contrast, the prevalence of HBsAg was approximately 30% in the child generation [8 –10]. This difference may result from a difference in transmission route. The index patients were predominantly male, and the prevalence of HBsAg in their spouse is assumed to approximate that in the general population (15 – 20%) [12,13]. Therefore, the risk of perinatal transmission should be lower in the child generation than in the index patient generation. However, the prevalence of HBsAg in the child generation (30%) was still significantly higher than that in the general population. This phenomenon may be caused by high horizontal transmission owing to high paternal (mostly HCC) HBsAg prevalence (. 60%) [14]. Therefore, this study uses the index patient and child generations to assess the role of perinatal transmission of hepatitis B virus (HBV) in hepatocarcinogenesis. This retrospective cohort study examined the difference in HCCrelated death rates between these two generations of HCC with different HBV transmission routes, and determined the underlying factors for HBsAg carriers to progress to severe liver disease, such as HCC.
original hospital. All eligible relatives, with or without symptoms of liver disease, were accepted. The study focuses on the generations of the index patients and their children, while the latter group was further sub-divided into the children of male and female index patients. Since hepatitis C virus infection also is an important risk factor in hepatocarcinogenesis [1], patients positive for hepatitis C virus antibodies were excluded from this analysis. The prevalence of HBsAg in the parent generation was examined to determine the perinatal transmission rate in the index patient generation.
2.2. Survey The study began by identifying the relationship between the HCC patients and their first-degree relatives. General information such as national citizen identification number, address, sex, race, and education level was recorded. Total number of siblings and children in a family was recorded after the second year. Information regarding numbers of siblings and children was then added after the second year. Blood samples were obtained from the relatives and sent for liver biochemical, HBsAg, and alpha-fetoprotein (AFP) tests (radio-immunoassay or enzyme-linked immunosorbent assay; Abbott Laboratories, Abbott Park, IL, or General Biologicals Corp, Science Based Industrial Park, Hsin Chu, Taiwan). Since a test for hepatitis C virus antibodies (Abbott HCV EIA II; Abbott Laboratories) became commercially available in 1993, it was also included in the survey. In addition, liver ultrasonography was performed at each visit. This study was approved by the Ethics Committee of each hospital and also by the Department of Health, Taiwan. All participants provided informed consent.
2.3. Search for fatalities This study used the national citizen identification numbers of the relatives of patients with HCC to search the mortality data bank established by the Statistics Office, Department of Health, Taiwan. The mortality data bank stored death certificate data, which included patient demographic data such as the time, place, and cause of death; and the name of the official who issued the document. Cause of death was classified using the International Classifications of Diseases, Injuries and Causes of Death (ICD-9, World Health Organization, 1977) [15].
2.4. Correlation of HBsAg status between parents and offspring
2. Patients and methods
The correlation of HBsAg status between parents and index patient generation, as well as between index case and their children, were examined. Relatives with known maternal HBsAg status were divided into the HBsAg positive mother (MP) group and the HBsAg negative mother (MN) group. Cases without maternal data but with known paternal HBsAg status also were divided into an HBsAg positive father (FP) group and an HBsAg negative father (FN) group. The prevalence of HBsAg in each group was determined in the index patient and child generations. Prevalence of maternal HBsAg among total HBsAg carriers in each generation also was estimated.
2.1. Patients
2.5. Statistical analysis
The present survey was conducted over a 6-year period. Relatives of patients with HCC, including the generations of parents, siblings, and children older than 15 were recruited. Eleven major teaching hospitals in Taiwan participated in the survey. Patients in whom HCC was diagnosed served as index cases. Relatives of the index cases were invited to participate in the survey at Outpatient Clinics, by mail, radio broadcasts, newspapers, or television. Relatives of patients with HCC diagnosed at other hospitals also were included, provided pertinent clinical information was available from the
The x2 -test, Fisher’s exact test, and Student’s t-test were used, when appropriate. Additionally, the Mantel–Cox procedure was applied to compare survival functions among different groups. Moreover, the sharpened Bonferroni procedure [16] was used to adjust for individual alpha levels, and thus keeping the overall alpha level at 0.05 when performing multiple tests. Stepwise logistic regression was conducted to identify independent risk factors of HCC. All statistical analyses were performed using BMDP statistical software (BMDP statistical software, Inc, Los Angeles, CA).
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Table 1 General information in relatives of patients with HCC stratified by familial generation and viral status Familial generation
No. of subjects
Age, years
No. of malesa
No. HBsAg positive
Total Mortalities from HCC
13 676 117
39.6 ^ 10.3 46.3 ^ 9.9
7164 (52.4) 103 (88.0)
5515 (40.3) 102 (87.5)
Total siblings Mortalities from HCC
4181 79
44.4 ^ 12.0* 46.9 ^ 10.3
2150 (51.4) 69 (87.3)
2190* (52.4) 69 (87.3)
Total children Mortalities from HCC
9495 38
37.5 ^ 8.7* 45.0 ^ 9.1
5014 (52.8) 34 (89.5)
3325* (35.0) 33 (86.8)
Children of male index patients Mortalities from HCC
6730 15
36.7 ^ 8.7* 45.3 ^ 9.1
3655 (54.3) 15 (100)
2194* (32.6) 14 (93.3)
Children of female index patients Mortalities from HCC
2765 23
39.5 ^ 8.3* 44.6 ^ 9.3
1359 (49.2) 19 (82.6)
1131* (40.9) 19 (82.6)
a
*Significant difference after sharpened Bonferroni adjustment. Numbers in parentheses are percentages of the relatives in each generation group.
3. Results 3.1. General information This study analyzed a total of 13 676 relatives of patients with HCC in the index patient and child generations. The sample included 4181 siblings and 9495 children. Table 1 lists the general information and prevalence of different viral infection groups in each generation. The mean age in the index patient generation (44.4 years) exceeded that of the child generation (37.7 years). However, the difference in mean age between the two generations was only 7 years, because these two groups were generally enrolled from different index patients. The prevalence of HBsAg was higher for the index patient generation (52.4%) than the child generation (35.0%). For the child generation, children of male index HCC patients (36.7 years) were slightly younger than children of female index HCC patients (39.5 years). Moreover, HBsAg was more prevalent in children of female index patients with HCC (40.9%) than in children of male index patients with HCC (32.6%). 3.1.1. Maternal role on HBV infection in index patient generation We correlate HBsAg status between parents of index
patients and the index patient generation and found that 308 (54.2%) of 568 mothers of index HCC cases were HBsAg carriers (Table 2). Among the relatives of the index patient generation, 71.6% (421/588) of the MP group and 43.7% (311/712) of the MN group were HBsAg carriers. The HBsAg prevalence is much higher in MP group than MN group ðP , 0:001Þ: The high prevalence of HBsAg in the MP group of the index patient generation suggests a high viral load in the mothers of this group [17]. Forty-four percent (311/712) of the MN group of the index patient generation were HBsAg carriers. This proportion appears too high to result from horizontal transmission alone. The prevalence of HBsAg is known to decline progressively with increasing age [11,13,18]. This study examined the prevalence of maternal HBsAg and found that the prevalence of HBsAg was 62% in mothers born after 1939, compared to just 41% in mothers born before 1939. Therefore, some the HBsAg negative mothers were likely to be HBsAg carriers during their pregnancies. For mothers unable to participate in this study, only 93 (22.2%) of the 419 fathers of index HCC cases were HBsAg carriers. This is similar to the 15 –20% prevalence in the general population [12,13]. The index patient generation had a high HBsAg carrier rate (76.2 and 64.0% in FP and FN groups, respectively) regardless of paternal HBsAg status.
Table 2 Correlation of HBsAg status between parents and their offspring in the index patient generation HBsAg positive cases/total cases (%) Relation to index patient
Index patients’ fathers
Index patients’ mothers
Parents
93/419 (22.2)
308/568 (54.2)
Index patients’ fathers
Index patients’ mothers
Siblings *P , 0:001:
HBsAg (þ) FP group 32/42 (76.2)
HBsAg (2) FN group 130/203 (64%)
HBsAg (þ ) MP group *421/588 (71.6)
HBsAg (2) MN group *311/712 (43.7)
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Notably, mortality from liver disease prevented some mothers from participating in this study. These findings further indicate a strong maternal rather than paternal role in transmitting HBV infection. The high prevalence of HBsAg in index patient generation and the little influence by paternal HBsAg status agrees with previous reports concerning the high prevalence of maternal HBsAg (65 –86%) [5 –7] in patients with HCC. 3.1.2. Maternal role on HBV infection in children of female index patient We correlate HBsAg status between female index patients and their children. HBsAg data were collected from 811 indexed patients, of whom 160 (19.7%) were women. Notably, 91 of the 160 (56.9%) female index patients were positive for HBsAg (Table 3). This rate approximates that in a recent Taiwanese report [14]. The prevalence of HBsAg was 66.3% in the MP group of the child generation. This rate is quite high, also suggesting a high maternal viral load [17]. On the other hand, the prevalence of HBsAg is just 18.0% in the MN group of the child generation, approximating the prevalence in the general population. For all HBsAg carriers in children of female index patients, the prevalence of maternal HBsAg is 79.8% (138/173). This finding supports a high perinatal transmission rate in children of female index patients. 3.1.3. High horizontal transmission of HBV in children of male index patients We correlate HBsAg status between male index patients and their children. Out of 651 male index patients 427 (64.7%) were HBsAg carriers. The prevalence of HBsAg was 35.3% (340/963) in the FP group, compared to 24.9% (75/301) in the FN group of the children’s generation. The higher HBsAg prevalence in the FP group compared to the FN group ðP , 0:001Þ suggests a high horizontal transmission in the child generation. 3.1.4. Estimation of maternal HBsAg prevalence in children of male index patients This study excluded spouses, thus we were unable to know the maternal prevalence of HBsAg in children of male index patients. However, the carrier rate (24.9%) observed in the FN group of the children’s generation approximated that of the general population (15 –20%) [12,13]. The prevalence of maternal HBsAg in HBsAg carriers from the FN group of the child generation is also expected to be close to that in the general population (35 –50%) [7,19]. Since the higher prevalence of HBsAg in the FP group of the child generation was related to higher paternal transmission, maternal HBsAg prevalence may also approximate that in the general population. Based on these considerations, 8.7 – 12.5% (24.9% £ 0.35 to 24.9% £ 0.5) of children of male index patients were expected to be related to perinatal transmission. For all the HBsAg carriers in this group, the prevalence of maternal HBsAg
is estimated to be approximately 26.5 – 37.9% [(963 þ 301) £ (8.7 to 12.5%)/(340 þ 75)]. Maternal HBsAg was much less prevalent in this group than in the index patient group (65 – 86%) or in children with female index patients (79.8%). 3.2. Mortality from HCC During the 9-year period from 1992 to 2000, 117 relatives died of HCC. Cumulative deaths from HCC were significantly higher in the index patient generation than in the child generation ðP ¼ 0:004Þ (Fig. 1A). When the analysis was confined to relatives of male HBsAg carriers, cumulative mortality from HCC remained higher in the index patient generation than in the child generation ðP ¼ 0:02Þ (Fig. 1B). The relatives in the child generation were further divided into two groups based on the gender of the index patients. Cumulative deaths from HCC were higher in the children of female index patients than in those of male index patients ðP ¼ 0:003Þ (Fig. 2A). When comparing male HBsAg carriers only, we also found that the children of female index patients had higher mortality from HCC than did those of male index patients ðP ¼ 0:054Þ (Fig. 2B). 3.2.1. Factors associated with HCC development Stepwise logistic regression found familial generation, male gender, age, HBsAg carrier status and gender of index cases to be significantly associated with HCC development (Table 4).
4. Discussion The survival analysis revealed higher mortality from HCC in the index patient generation than in the child generation. When the study population was confined to male HBsAg carriers—focusing on the two major risk factors in hepatocarcinogenesis—the mortality rate remained higher in the index patient generation than in the child generation. As age had been taken into consideration in the survival analysis, the difference between two generations was therefore mainly attributable to perinatal transmission. HBV infection via perinatal transmission, rather than familial or genetic factors, is important in hepatocarcinogenesis. The subgroups in the child generation further showed the importance of perinatal transmission in hepatocarcinogenesis. Liver cancer related mortality was higher in children of female index patients than in those of male index patients. Moreover, the pattern remained similar when the sample was limited only to male HBsAg carriers, despite the P-value being marginally non-significant. Multivariate analysis by stepwise logistic regression confirms that HBsAg carriers, index patient generation and male gender are independent risk factors associated with
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Fig. 1. (A) Mortality from HCC was higher in the index patient generation than in the child generation (Mantel–Cox test P 5 0:004). (B) When the study relatives were limited to male HBsAg carriers, mortality from HCC remained higher in the index patient generation than in the child generation (Mantel– Cox test P 5 0:02). [This figure appears in colour on the web.]
Fig. 2. (A) In the child generation alone, the children of female index patients displayed higher mortality from HCC than the children of male index patients (Mantel–Cox test P 5 0:003). (B) When the child generation was limited to HBsAg positive men, the children of female index patients again displayed higher mortality than those of male index patients (Mantel–Cox test P 5 0:054). [This figure appears in colour on the web.]
HCC development. Meanwhile, male index and young age are associated with reduced likelihood of developing HCC. Why perinatal transmission determines the outcome of chronic HBV infection is an important question. One possible answer to this question is that perinatal transmission induces a high tolerance for HBV [20]. High perinatal transmission also means a high vertical transmission rate, which could deliver a higher dose of HBV DNA during the early stage of life than delivery via
horizontal transmission. This high dose of HBV DNA then may increase tolerance to HBV [17]. Persistent HBV infection generally causes repeated hepatic necrotic-inflammatory activities and regeneration, which is one of the main causes of hepatocarcinogenesis [21 – 23]. Perinatal transmission apparently is not the only determinant of the occurrence of hepatocellular carcinoma. Approximately 35– 50% of HBsAg carriers in the general population are also infected with HBV through perinatal
Table 3 Correlation of HBsAg status between index patients and their children HBsAg positive cases/total case (%) Relation to index case
Male index patients
Female index patients
Index cases
427/651 (65.6)
91/160 (56.9)
Fathers (male index patients)
Mothers (female index patients)
Children *,†P , 0:001:
HBsAg (þ ) FP group *340/963 (35.3)
HBsAg (2) FN group *75/301 (24.9)
HBsAg (þ) MP group 138/208 (66.3)
†
HBsAg (2) MN group 35/194 (18)
†
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transmission [7,19], but HCC is rare in asymptomatic HBsAg carriers [4]. The dose of HBV DNA transmitted from a mother to her offspring could be another risk factor for hepatocarcinogenesis. Several notable pieces of evidence exist regarding HBV transmission between mother and infant. First, in cases of delivery with threatened abortion, more HBV could pass through the impaired placenta barrier, possibly explaining the higher vaccination failure rate in newborn babies compared to other infants [24 –26]. Second, in 773 samples of paired sera from mothers and offspring, the outcome of perinatal HBV infection depended on maternal HBV DNA level [17]. The authors found that 78.8% of the children of 416 hepatitis B e antigen-positive mothers became chronic HBsAg carriers, compared to just 1.3% of the children of 269 mothers with antibodies to hepatitis B e antigen. Logistic regression analysis identified maternal HBV DNA as a strong independent predictor of persistent HBV infection. Because families with HCC have a long HBV replication period [10], their offsprings receive a larger dose of HBV DNA and thus develop stronger tolerance to HBV [20]. Third, observation of the HCC relatives participating in this study showed that hepatitis B e antigen and HBV DNA frequently clear earlier in younger siblings than in older siblings [27]. This observation implies that maternal HBV replication was more active when older offsprings were born, inducing strong tolerance to HBV in older siblings. This finding suggests that vertical transmission and a dynamic interaction with maternal HBV significantly influence the outcomes of offspring. This study also found a HBsAg prevalence of 72% in the index patient and 66% in the child generation of MP groups (Tables 2 and 3). The high prevalence of HBsAg among the offspring indicates a high maternal viral load [17]. Moreover, the high infectivity could result from prolonged viral replication phase [10], more virulent HBV strain [28], or younger maternal age [27,29]. This study concludes that HBV perinatal transmission with a high maternal viral load is important in familial-type HCC.
Table 4 Factors significantly associated with HCC as identified using stepwise logistic regression Step
Variable entered
Odds ratio
95% CI* for OR†
P-value
1 2 3 4 5
HBsAg (þ ) Age Male gender Index patients’ generation Male index patients
10.9 0.949 6.99 3.0 0.591
(6.26, 18.9) (0.933, 0.905) (4.05, 12.0) (1.99, 4.52) (0.353, 0.898)
,0.001 ,0.001 ,0.001 ,0.001 0.014
*Confidence interval; †Odds ratio.
Acknowledgements This study was supported by grants from the Bureau of Health Promotion and Protection, Department of Health (DOH-81) and the National Science Council (NSC89-2315B-182A-003), Republic of China (Taiwan). The authors are very grateful to the Office of Statistics, Department of Health, Republic of China (Taiwan) for providing national mortality data.
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Hepatitis B virus transmission and hepatocarcinogenesis: a 9 year retrospective cohort of 13 676 relatives with hepatocellular carcinoma Chien-Hung Chen1, Yang Yuan Chen2, Gran-Hum Chen3, Sien-Sing Yang4, Huang-Shang Tang5, Hsien Hong Lin6, Deng-Yn Lin7, Sing Kai Lo14, Jeng-Ming Du8, Ting-Tsung Chang9, Shinn-Cherng Chen10, Li-Ying Liao11, Chung-Huang Kuo12, Kwo-Chuan Lin2, Dar-In Tai7,12,*, Chi-Sin Changchien12, Wen-Yu Chang10, Jin-Chuan Sheu1, Ding-Shinn Chen1, Yun-Fan Liaw7, Juei-Low Sung13 1
Department of Internal Medicine, National Taiwan University Hospital and National Taiwan University College of Medicine, Taipei, Taiwan, ROC 2 Department of Internal Medicine, Changhua Christian Hospital, Changhua, Taiwan, ROC 3 Department of Internal Medicine, Taichung Veterans General Hospital, Taichung, Taiwan, ROC 4 Liver Unit, Clinical Research Center, Cathay General Hospital, Taipei, Taiwan, ROC 5 Department of Internal Medicine, Tri-service General Hospital, National Defence Medical Center, Taipei, Taiwan, ROC 6 Department of Internal Medicine, Buddhist Tzu Chi General Hospital, Hualien, Taiwan, ROC 7 Liver Research Unit, Chang Gung Memorial Hospital and Chang Gung University College of Medicine, 199 Tun-Hwa North Road, Taipei 105, Taiwan, ROC 8 Department of Medical Information Management, Chang Gung Memorial Hospital, Taipei, Taiwan, ROC 9 Department of Internal Medicine, National Cheng Kung University Hospital, Tainan, Taiwan, ROC 10 Hepatobiliary Division, Department of Internal Medicine, Kaohsiung Medical University Hospital, Kaohsiung, Taiwan, ROC 11 Department of Gastroenterology, Taipei Municipal Jen-Ai Hospital, Taipei, Taiwan, ROC 12 Liver Unit, Kaohsiung Chang Gung Memorial Hospital, Kaohsiung, Taiwan, ROC 13 Sun Yat-Sen Cancer Center Hospital, Taipei, Taiwan, ROC 14 Institute for International Health, Faculty of Medicine, University of Sydney, Sydney, NSW, Australia
Background/Aims: Familial clustering of hepatitis B virus (HBV) infection is related to perinatal transmission, and is the main cause of familial-type hepatocellular carcinoma (HCC). The route of HBV transmission differs between the children and siblings of patients with HCC. This study examined the differences in HBV carrier rates and HCC-related mortality between two generations in HCC families. Methods: From 1992 to 1997, relatives of individuals with HCC were screened prospectively with ultrasonography, alpha-fetoprotein, liver biochemistry tests and viral markers. Total HCC-related deaths during a 9-year period were compared between the generations of index patients and their children. Results: The study included a total of 13 676 relatives in two generations. More HCC-related deaths occurred in the index patient generation than in the child generation. Furthermore, children of female index patients had higher rates of liver cancer related mortality than children of male index patients. The same was true when the analysis was limited to male HBV carriers. The prevalence of HBsAg in the offspring of HBsAg positive mothers was 66% in the child generation and 72% in the index patient generation. These high prevalences indicated high maternal HBV replication status. Conclusions: Perinatal transmission and maternal viral load are important risk factors in hepatocarcinogenesis. q 2003 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved. Keywords: Maternal – fetal exchange; Taiwan; Survival analysis; Hepatitis B surface antigen; Family
Received 10 May 2003; received in revised form 22 October 2003; accepted 4 December 2003 * Corresponding author. Tel.: þ 886-3-328-1200; fax: þ886-3-327-2236. E-mail address: [email protected] (D.I. Tai). Abbreviations: HBsAg, hepatitis B surface antigen; HBV, hepatitis B virus; HCC, hepatocellular carcinoma; MP, HBsAg positive mother; MN, HBsAg negative mother; FP, HBsAg positive father; FN, HBsAg negative father. 0168-8278/$30.00 q 2003 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.jhep.2003.12.002
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1. Introduction Viral hepatitis, alcohol, smoking, familial tendency and aflatoxin are well-known risk factors for hepatocellular carcinoma (HCC) [1 – 3]. Several cohort studies have found that HBsAg carriers are at highest risk (relative risk ranges from 7 to 98) of developing HCC, while other factors appear to have a lower risk (relative risk ranges from one to four) [1 –3]. Nevertheless, a low risk of HCC in asymptomatic HBsAg carriers was observed in another study [4]. The reason for this discrepancy attracted our attention. One clue is that approximately 70% of the HBsAg positive patients with HCC acquired their HBV infection perinatally [5 – 7]. Because familial aggregation is a characteristic of HCC, a nationwide survey of relatives of patients with HCC was done between 1992 and 1997. The original purposes of the survey were to study viral infection status and have an early detection of HCC in HCC families [8 – 11]. The survey included the parents of the index patients, the index patients themselves, and their children. Given the same transmission route (perinatal transmission) as the index patients, the index patient generation had a high prevalence of HBsAg (approximately 60%) [8 –10]. In contrast, the prevalence of HBsAg was approximately 30% in the child generation [8 –10]. This difference may result from a difference in transmission route. The index patients were predominantly male, and the prevalence of HBsAg in their spouse is assumed to approximate that in the general population (15 – 20%) [12,13]. Therefore, the risk of perinatal transmission should be lower in the child generation than in the index patient generation. However, the prevalence of HBsAg in the child generation (30%) was still significantly higher than that in the general population. This phenomenon may be caused by high horizontal transmission owing to high paternal (mostly HCC) HBsAg prevalence (. 60%) [14]. Therefore, this study uses the index patient and child generations to assess the role of perinatal transmission of hepatitis B virus (HBV) in hepatocarcinogenesis. This retrospective cohort study examined the difference in HCCrelated death rates between these two generations of HCC with different HBV transmission routes, and determined the underlying factors for HBsAg carriers to progress to severe liver disease, such as HCC.
original hospital. All eligible relatives, with or without symptoms of liver disease, were accepted. The study focuses on the generations of the index patients and their children, while the latter group was further sub-divided into the children of male and female index patients. Since hepatitis C virus infection also is an important risk factor in hepatocarcinogenesis [1], patients positive for hepatitis C virus antibodies were excluded from this analysis. The prevalence of HBsAg in the parent generation was examined to determine the perinatal transmission rate in the index patient generation.
2.2. Survey The study began by identifying the relationship between the HCC patients and their first-degree relatives. General information such as national citizen identification number, address, sex, race, and education level was recorded. Total number of siblings and children in a family was recorded after the second year. Information regarding numbers of siblings and children was then added after the second year. Blood samples were obtained from the relatives and sent for liver biochemical, HBsAg, and alpha-fetoprotein (AFP) tests (radio-immunoassay or enzyme-linked immunosorbent assay; Abbott Laboratories, Abbott Park, IL, or General Biologicals Corp, Science Based Industrial Park, Hsin Chu, Taiwan). Since a test for hepatitis C virus antibodies (Abbott HCV EIA II; Abbott Laboratories) became commercially available in 1993, it was also included in the survey. In addition, liver ultrasonography was performed at each visit. This study was approved by the Ethics Committee of each hospital and also by the Department of Health, Taiwan. All participants provided informed consent.
2.3. Search for fatalities This study used the national citizen identification numbers of the relatives of patients with HCC to search the mortality data bank established by the Statistics Office, Department of Health, Taiwan. The mortality data bank stored death certificate data, which included patient demographic data such as the time, place, and cause of death; and the name of the official who issued the document. Cause of death was classified using the International Classifications of Diseases, Injuries and Causes of Death (ICD-9, World Health Organization, 1977) [15].
2.4. Correlation of HBsAg status between parents and offspring
2. Patients and methods
The correlation of HBsAg status between parents and index patient generation, as well as between index case and their children, were examined. Relatives with known maternal HBsAg status were divided into the HBsAg positive mother (MP) group and the HBsAg negative mother (MN) group. Cases without maternal data but with known paternal HBsAg status also were divided into an HBsAg positive father (FP) group and an HBsAg negative father (FN) group. The prevalence of HBsAg in each group was determined in the index patient and child generations. Prevalence of maternal HBsAg among total HBsAg carriers in each generation also was estimated.
2.1. Patients
2.5. Statistical analysis
The present survey was conducted over a 6-year period. Relatives of patients with HCC, including the generations of parents, siblings, and children older than 15 were recruited. Eleven major teaching hospitals in Taiwan participated in the survey. Patients in whom HCC was diagnosed served as index cases. Relatives of the index cases were invited to participate in the survey at Outpatient Clinics, by mail, radio broadcasts, newspapers, or television. Relatives of patients with HCC diagnosed at other hospitals also were included, provided pertinent clinical information was available from the
The x2 -test, Fisher’s exact test, and Student’s t-test were used, when appropriate. Additionally, the Mantel–Cox procedure was applied to compare survival functions among different groups. Moreover, the sharpened Bonferroni procedure [16] was used to adjust for individual alpha levels, and thus keeping the overall alpha level at 0.05 when performing multiple tests. Stepwise logistic regression was conducted to identify independent risk factors of HCC. All statistical analyses were performed using BMDP statistical software (BMDP statistical software, Inc, Los Angeles, CA).
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Table 1 General information in relatives of patients with HCC stratified by familial generation and viral status Familial generation
No. of subjects
Age, years
No. of malesa
No. HBsAg positive
Total Mortalities from HCC
13 676 117
39.6 ^ 10.3 46.3 ^ 9.9
7164 (52.4) 103 (88.0)
5515 (40.3) 102 (87.5)
Total siblings Mortalities from HCC
4181 79
44.4 ^ 12.0* 46.9 ^ 10.3
2150 (51.4) 69 (87.3)
2190* (52.4) 69 (87.3)
Total children Mortalities from HCC
9495 38
37.5 ^ 8.7* 45.0 ^ 9.1
5014 (52.8) 34 (89.5)
3325* (35.0) 33 (86.8)
Children of male index patients Mortalities from HCC
6730 15
36.7 ^ 8.7* 45.3 ^ 9.1
3655 (54.3) 15 (100)
2194* (32.6) 14 (93.3)
Children of female index patients Mortalities from HCC
2765 23
39.5 ^ 8.3* 44.6 ^ 9.3
1359 (49.2) 19 (82.6)
1131* (40.9) 19 (82.6)
a
*Significant difference after sharpened Bonferroni adjustment. Numbers in parentheses are percentages of the relatives in each generation group.
3. Results 3.1. General information This study analyzed a total of 13 676 relatives of patients with HCC in the index patient and child generations. The sample included 4181 siblings and 9495 children. Table 1 lists the general information and prevalence of different viral infection groups in each generation. The mean age in the index patient generation (44.4 years) exceeded that of the child generation (37.7 years). However, the difference in mean age between the two generations was only 7 years, because these two groups were generally enrolled from different index patients. The prevalence of HBsAg was higher for the index patient generation (52.4%) than the child generation (35.0%). For the child generation, children of male index HCC patients (36.7 years) were slightly younger than children of female index HCC patients (39.5 years). Moreover, HBsAg was more prevalent in children of female index patients with HCC (40.9%) than in children of male index patients with HCC (32.6%). 3.1.1. Maternal role on HBV infection in index patient generation We correlate HBsAg status between parents of index
patients and the index patient generation and found that 308 (54.2%) of 568 mothers of index HCC cases were HBsAg carriers (Table 2). Among the relatives of the index patient generation, 71.6% (421/588) of the MP group and 43.7% (311/712) of the MN group were HBsAg carriers. The HBsAg prevalence is much higher in MP group than MN group ðP , 0:001Þ: The high prevalence of HBsAg in the MP group of the index patient generation suggests a high viral load in the mothers of this group [17]. Forty-four percent (311/712) of the MN group of the index patient generation were HBsAg carriers. This proportion appears too high to result from horizontal transmission alone. The prevalence of HBsAg is known to decline progressively with increasing age [11,13,18]. This study examined the prevalence of maternal HBsAg and found that the prevalence of HBsAg was 62% in mothers born after 1939, compared to just 41% in mothers born before 1939. Therefore, some the HBsAg negative mothers were likely to be HBsAg carriers during their pregnancies. For mothers unable to participate in this study, only 93 (22.2%) of the 419 fathers of index HCC cases were HBsAg carriers. This is similar to the 15 –20% prevalence in the general population [12,13]. The index patient generation had a high HBsAg carrier rate (76.2 and 64.0% in FP and FN groups, respectively) regardless of paternal HBsAg status.
Table 2 Correlation of HBsAg status between parents and their offspring in the index patient generation HBsAg positive cases/total cases (%) Relation to index patient
Index patients’ fathers
Index patients’ mothers
Parents
93/419 (22.2)
308/568 (54.2)
Index patients’ fathers
Index patients’ mothers
Siblings *P , 0:001:
HBsAg (þ) FP group 32/42 (76.2)
HBsAg (2) FN group 130/203 (64%)
HBsAg (þ ) MP group *421/588 (71.6)
HBsAg (2) MN group *311/712 (43.7)
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Notably, mortality from liver disease prevented some mothers from participating in this study. These findings further indicate a strong maternal rather than paternal role in transmitting HBV infection. The high prevalence of HBsAg in index patient generation and the little influence by paternal HBsAg status agrees with previous reports concerning the high prevalence of maternal HBsAg (65 –86%) [5 –7] in patients with HCC. 3.1.2. Maternal role on HBV infection in children of female index patient We correlate HBsAg status between female index patients and their children. HBsAg data were collected from 811 indexed patients, of whom 160 (19.7%) were women. Notably, 91 of the 160 (56.9%) female index patients were positive for HBsAg (Table 3). This rate approximates that in a recent Taiwanese report [14]. The prevalence of HBsAg was 66.3% in the MP group of the child generation. This rate is quite high, also suggesting a high maternal viral load [17]. On the other hand, the prevalence of HBsAg is just 18.0% in the MN group of the child generation, approximating the prevalence in the general population. For all HBsAg carriers in children of female index patients, the prevalence of maternal HBsAg is 79.8% (138/173). This finding supports a high perinatal transmission rate in children of female index patients. 3.1.3. High horizontal transmission of HBV in children of male index patients We correlate HBsAg status between male index patients and their children. Out of 651 male index patients 427 (64.7%) were HBsAg carriers. The prevalence of HBsAg was 35.3% (340/963) in the FP group, compared to 24.9% (75/301) in the FN group of the children’s generation. The higher HBsAg prevalence in the FP group compared to the FN group ðP , 0:001Þ suggests a high horizontal transmission in the child generation. 3.1.4. Estimation of maternal HBsAg prevalence in children of male index patients This study excluded spouses, thus we were unable to know the maternal prevalence of HBsAg in children of male index patients. However, the carrier rate (24.9%) observed in the FN group of the children’s generation approximated that of the general population (15 –20%) [12,13]. The prevalence of maternal HBsAg in HBsAg carriers from the FN group of the child generation is also expected to be close to that in the general population (35 –50%) [7,19]. Since the higher prevalence of HBsAg in the FP group of the child generation was related to higher paternal transmission, maternal HBsAg prevalence may also approximate that in the general population. Based on these considerations, 8.7 – 12.5% (24.9% £ 0.35 to 24.9% £ 0.5) of children of male index patients were expected to be related to perinatal transmission. For all the HBsAg carriers in this group, the prevalence of maternal HBsAg
is estimated to be approximately 26.5 – 37.9% [(963 þ 301) £ (8.7 to 12.5%)/(340 þ 75)]. Maternal HBsAg was much less prevalent in this group than in the index patient group (65 – 86%) or in children with female index patients (79.8%). 3.2. Mortality from HCC During the 9-year period from 1992 to 2000, 117 relatives died of HCC. Cumulative deaths from HCC were significantly higher in the index patient generation than in the child generation ðP ¼ 0:004Þ (Fig. 1A). When the analysis was confined to relatives of male HBsAg carriers, cumulative mortality from HCC remained higher in the index patient generation than in the child generation ðP ¼ 0:02Þ (Fig. 1B). The relatives in the child generation were further divided into two groups based on the gender of the index patients. Cumulative deaths from HCC were higher in the children of female index patients than in those of male index patients ðP ¼ 0:003Þ (Fig. 2A). When comparing male HBsAg carriers only, we also found that the children of female index patients had higher mortality from HCC than did those of male index patients ðP ¼ 0:054Þ (Fig. 2B). 3.2.1. Factors associated with HCC development Stepwise logistic regression found familial generation, male gender, age, HBsAg carrier status and gender of index cases to be significantly associated with HCC development (Table 4).
4. Discussion The survival analysis revealed higher mortality from HCC in the index patient generation than in the child generation. When the study population was confined to male HBsAg carriers—focusing on the two major risk factors in hepatocarcinogenesis—the mortality rate remained higher in the index patient generation than in the child generation. As age had been taken into consideration in the survival analysis, the difference between two generations was therefore mainly attributable to perinatal transmission. HBV infection via perinatal transmission, rather than familial or genetic factors, is important in hepatocarcinogenesis. The subgroups in the child generation further showed the importance of perinatal transmission in hepatocarcinogenesis. Liver cancer related mortality was higher in children of female index patients than in those of male index patients. Moreover, the pattern remained similar when the sample was limited only to male HBsAg carriers, despite the P-value being marginally non-significant. Multivariate analysis by stepwise logistic regression confirms that HBsAg carriers, index patient generation and male gender are independent risk factors associated with
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Fig. 1. (A) Mortality from HCC was higher in the index patient generation than in the child generation (Mantel–Cox test P 5 0:004). (B) When the study relatives were limited to male HBsAg carriers, mortality from HCC remained higher in the index patient generation than in the child generation (Mantel– Cox test P 5 0:02). [This figure appears in colour on the web.]
Fig. 2. (A) In the child generation alone, the children of female index patients displayed higher mortality from HCC than the children of male index patients (Mantel–Cox test P 5 0:003). (B) When the child generation was limited to HBsAg positive men, the children of female index patients again displayed higher mortality than those of male index patients (Mantel–Cox test P 5 0:054). [This figure appears in colour on the web.]
HCC development. Meanwhile, male index and young age are associated with reduced likelihood of developing HCC. Why perinatal transmission determines the outcome of chronic HBV infection is an important question. One possible answer to this question is that perinatal transmission induces a high tolerance for HBV [20]. High perinatal transmission also means a high vertical transmission rate, which could deliver a higher dose of HBV DNA during the early stage of life than delivery via
horizontal transmission. This high dose of HBV DNA then may increase tolerance to HBV [17]. Persistent HBV infection generally causes repeated hepatic necrotic-inflammatory activities and regeneration, which is one of the main causes of hepatocarcinogenesis [21 – 23]. Perinatal transmission apparently is not the only determinant of the occurrence of hepatocellular carcinoma. Approximately 35– 50% of HBsAg carriers in the general population are also infected with HBV through perinatal
Table 3 Correlation of HBsAg status between index patients and their children HBsAg positive cases/total case (%) Relation to index case
Male index patients
Female index patients
Index cases
427/651 (65.6)
91/160 (56.9)
Fathers (male index patients)
Mothers (female index patients)
Children *,†P , 0:001:
HBsAg (þ ) FP group *340/963 (35.3)
HBsAg (2) FN group *75/301 (24.9)
HBsAg (þ) MP group 138/208 (66.3)
†
HBsAg (2) MN group 35/194 (18)
†
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transmission [7,19], but HCC is rare in asymptomatic HBsAg carriers [4]. The dose of HBV DNA transmitted from a mother to her offspring could be another risk factor for hepatocarcinogenesis. Several notable pieces of evidence exist regarding HBV transmission between mother and infant. First, in cases of delivery with threatened abortion, more HBV could pass through the impaired placenta barrier, possibly explaining the higher vaccination failure rate in newborn babies compared to other infants [24 –26]. Second, in 773 samples of paired sera from mothers and offspring, the outcome of perinatal HBV infection depended on maternal HBV DNA level [17]. The authors found that 78.8% of the children of 416 hepatitis B e antigen-positive mothers became chronic HBsAg carriers, compared to just 1.3% of the children of 269 mothers with antibodies to hepatitis B e antigen. Logistic regression analysis identified maternal HBV DNA as a strong independent predictor of persistent HBV infection. Because families with HCC have a long HBV replication period [10], their offsprings receive a larger dose of HBV DNA and thus develop stronger tolerance to HBV [20]. Third, observation of the HCC relatives participating in this study showed that hepatitis B e antigen and HBV DNA frequently clear earlier in younger siblings than in older siblings [27]. This observation implies that maternal HBV replication was more active when older offsprings were born, inducing strong tolerance to HBV in older siblings. This finding suggests that vertical transmission and a dynamic interaction with maternal HBV significantly influence the outcomes of offspring. This study also found a HBsAg prevalence of 72% in the index patient and 66% in the child generation of MP groups (Tables 2 and 3). The high prevalence of HBsAg among the offspring indicates a high maternal viral load [17]. Moreover, the high infectivity could result from prolonged viral replication phase [10], more virulent HBV strain [28], or younger maternal age [27,29]. This study concludes that HBV perinatal transmission with a high maternal viral load is important in familial-type HCC.
Table 4 Factors significantly associated with HCC as identified using stepwise logistic regression Step
Variable entered
Odds ratio
95% CI* for OR†
P-value
1 2 3 4 5
HBsAg (þ ) Age Male gender Index patients’ generation Male index patients
10.9 0.949 6.99 3.0 0.591
(6.26, 18.9) (0.933, 0.905) (4.05, 12.0) (1.99, 4.52) (0.353, 0.898)
,0.001 ,0.001 ,0.001 ,0.001 0.014
*Confidence interval; †Odds ratio.
Acknowledgements This study was supported by grants from the Bureau of Health Promotion and Protection, Department of Health (DOH-81) and the National Science Council (NSC89-2315B-182A-003), Republic of China (Taiwan). The authors are very grateful to the Office of Statistics, Department of Health, Republic of China (Taiwan) for providing national mortality data.
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