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hepatitis G virus infection in Chinese patients with hepatocellular carcinoma
Hepatology Research 16 (2000) 91 – 97 www.elsevier.com/locate/ihepcom
GB virus C/hepatitis G virus infection in Chinese patients with hepatocellular carcinoma Lei Xuezhong a, Shigeko Naitoh b, Deng Xuewen b, Lianshan Zhao a, Yoshihiro Akahane b,* a Department of Infectious Diseases, The First Uni6ersity Hospital of West China Uni6ersity of Medical Sciences, Chengdu, China b The First Department of Internal Medicine, Yamanashi Medical Uni6ersity, Yamanashi-Ken, Japan
Received 31 July 1998; received in revised form 17 December 1998; accepted 6 January 1999
Abstract Although hepatitis B and C viruses are closely related to hepatocellular carcinoma (HCC), the association between the newly discovered GB virus C (GBV-C)/hepatitis G virus (HGV) and HCC is not yet known. Sera from 124 patients with HCC in China were tested for GBV-C/HGV RNA by the reverse-transcription polymerase chain reaction with subsequent hybridization using GBV-C/HGV probes located at the 3%-untranslated region of the reported genomes. GBV-C/HGV RNA was detected in 33 (27%) of them, including 18 of the 59 (31%) patients with HBsAg, 12 of the 22 (55%) with both HBsAg and anti-HCV, and three of the 33 (9%) negative for either HBsAg or anti-HCV; it was not detected in any of the 10 with anti-HCV alone. There were no differences in clinical backgrounds between the patients with HBsAg and/or anti-HCV who possessed and who did not possess GBV-C/ HGV RNA in serum. These results indicate that, although GBV-C/HGV is highly prevalent in Chinese patients with HCC, the role of GBV-C/HGV in hepatocarcinogenesis would be minimal in the development of non-B, non-C HCC. © 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: GB virus C; Hepatitis G virus; Hepatitis B virus; Hepatitis C virus; Hepatocellular carcinoma; Epidemiology
* Corresponding author. Tel.: +81-552-73-9584; fax: +81-552-73-6748. 1386-6346/00/$ - see front matter © 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S 1 3 8 6 - 6 3 4 6 ( 9 9 ) 0 0 0 0 7 - 8
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L. Xuezhong et al. / Hepatology Research 16 (2000) 91–97
1. Introduction Hepatocellular carcinoma (HCC) is common in China, and its mortality rate is reported to be 20.00 – 24.32 deaths per 100 000 [1]. HCC is currently an important health problem in China. An intimate relationship between HCC and chronic hepatitis B virus (HBV) infection has long been proved, and a close association of HCC with chronic hepatitis C virus (HCV) infection is strongly suggested ever since this virus was identified [2]. Additionally, repeated exposures to the aflatoxin B1 and mycotoxin are other known etiologic factors for the development of HCC [3]. However, substantial numbers of patients with HCC do not have any of these known etiologic factors. The involvement of other factors, including non-B, non-C virus in the development of HCC has not been identified, as yet. A flavivirus-like RNA virus, with a genome different from hepatitis A–E viruses, has been identified recently in two independent laboratories from serum of non-A to E hepatitis patients, and was designated GB virus-C (GBV-C) or hepatitis G virus (HGV) [4,5]. It has been proved that the virus has a global distribution and is transmitted by blood-borne horizontal infection [6–8]. In order to determine the possible role of GBV-C/HGV infection in the development of HCC and its possible coordination with HBV and/or HCV in hepatocarcinogenesis, we examined GBVC/HGV RNA in 124 Chinese patients with HCC, including 33 with a non-B, non-C cause.
2. Materials and methods
2.1. Studied subjects One hundred and twenty-four patients with a diagnosis of HCC at admission to the First University Hospital of West China University of Medical Sciences, Chengdu, Southwest China, during August 1995 through March 1997 were enrolled in the study. They aged 43.6 910.1 years and included 101 males and 23 females. The diagnosis of HCC was based on a combination of clinical features, demonstration of one or more mass-lesions in the liver on hepatic imaging, and a higher concentration of serum a-fetoprotein; those patients with a serum a-fetoprotein higher than 500 ng/ml had hepatic masses. The diagnosis was confirmed further in 39 patients by histopathological evidence of HCC in liver tissue specimens obtained at fine needle biopsy or surgical operation. The underlying liver disease, chronic hepatitis (CH) or liver cirrhosis(LC), was diagnosed by clinical symptoms as well as biochemical and ultrasonographic findings, and confirmed further by biopsy of non-cancerous portions of liver tissue in some indefinite cases. Blood samples from the patients were obtained with great care for avoiding contamination, and separated sera were stored at − 20°C until they were tested for makers of hepatitis virus infections. The study was approved by the ethics committee of the hospital, and all patients gave informed consent.
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2.2. Serological tests Hepatitis B surface antigen (HBsAg) was determined by passive hemagglutination using commercial kits (Mycell, Institute of Immunology, Tokyo, Japan). Antibody to HCV (anti-HCV) was determined by a second-generation enzyme immunoassay (EIA-II, Ortho Diagnostic Systems, Tokyo, Japan).
2.3. Detection of GBV-C/HGV RNA Total RNA was extracted from 50 ml of serum with the guanidine isothiocyanate – phenol/chloroform procedures (SMITEST EX-R&D: Sumitomo Metal Industries, Tokyo, Japan), and then subjected to the reverse-transcription polymerase chain reaction (PCR) with use of SMITEST GBV-C RNA (3%UTR) Detection Kit (Sumitomo Metal Industries). In essence, the procedures of reverse transcription and amplification cycles using the specific rTth DNA polymerase (Perkin-Elmer, CT) and dinitrophenyl-labeled GBV-C/HGV-specific primers derived from the 3%-untranslated region were as follows: 70°C for 3 min, 60°C for 30 min, and 94°C for 1 min; then five cycles of PCR (93°C for 15 s, 62°C for 30 s in each cycle); followed by 35 cycles of PCR (90°C for 15 s, 62°C for 30 s in each cycle); finally, the extension reaction at 60°C for 4 min and kept at 10°C thereafter (9600-R Gene Amp PCR System: Perkin-Elmer). Amplified products were analyzed by hybridization and chemical-coloring in wells of a microplate coated with GBV-C/HGV probe derived from the 3%-untranslated region. The absorbance was determined at 450 nm and readings over 0.500 were considered positive. The sensitivity of this assay was 10 copies of GBV-C/HGV RNA per reaction, corresponding to about 200 copies per ml of the initial serum.
2.4. Statistical analysis The frequency was compared between groups with the two-tailed Mantel–Haenszel x 2 or two-tailed Fisher’s exact test, and group means were compared with the
Table 1 Demographic features, underlying liver disease and markers of hepatitis virus infections in HCC patients in China Underlying diseasea
CH LC Total a b
N
Age (years)b
Male/female
HBsAg
Anti-HCV
Both
Neither
N (%)
N (%)
N (%)
N (%)
3 (9%) 19 (21%)
8 (23%) 25 (28%)
22 (18%)
33 (27%)
35 89
44.1 9 10.6 43.49 10.0
26/9 75/14
23 (66%) 36 (40%)
124
43.6 9 10.1
101/23
59 (48%)
CH, chronic hepatitis; LC, liver cirrhosis. Mean9 S.D. age is shown.
1 (3%) 9 (10%) 10 (8%)
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94
Table 2 GBV-C RNA in HCC patients of various etiologies Etiology HBV HCV HBV plus HCV Non-B, non-C Total a b
N (%) 59 10 22 33 124
(48%) (8%) (18%) (18%)
Age (years)
Male
42.1910.2 44.3 97.8 44.4 9 11.9 45.5 9 9.4
45 9 18 30
43 6910.1
(76%) (90%) (82%) (91%)
101 (82%)
GBV-C RNA 18 (31%)a 0 12 (55%)b 3 (9%) 33 (27%)
Different from non-B, non-C etiology (PB0.05). Different from non-B, non-C etiology (PB0.01).
Student’s t-test. A P-value of less than 0.05 was considered to indicate statistical significance.
3. Results Demographic features, underlying liver disease and markers of HBV and HCV are shown for the 124 patients with HCC in Table 1. Of them, 35 were non-cirrhotic and 96 were cirrhotic in their non-neoplastic regions of the liver. Twenty-three of the 59 HBsAg-positive cases, one of the 10 anti-HCV positive cases, eight of the 33 cases negative for both HBsAg and anti-HCV, and three of the 22 cases positive for both HBsAg and anti-HCV had non-cirrhotic livers. Of the 124 patients with HCC, 33 were negative for both HBsAg and anti-HCV, 10 were positive for anti-HCV only, 59 were positive for HBsAg only, and 22 were positive for both anti-HCV and HBsAg. In total, GBV-C/HGV RNA was detected in 33 of the 124 (27%) patients with HCC (Table 2). Of these 33 patients, 18 (55%) were also positive for HBsAg, none were positive for anti-HCV only, 12 (36.4%) were positive for both HBsAg and anti-HCV, and only three (9%) were positive for GBV-C/HGV RNA alone. Interestingly, among the 10 cases with HCV infection only, none were found to be positive for GBV-C/HGV RNA, while in the HBsAg-positive cases, 18 (31%) were positive for GBV-C/HGV RNA. Based on these data, GBV-C/HGV RNA was highly prevalent among Chinese HCC patients who were co-infected with HBV (PB 0.05), with the highest prevalence in the patients with both HBsAg and anti-HCV (PB 0.01). Table 3 compares underlying liver disease, demographic features, and markers of HBV and HCV infections between the HCC patients with and without GBV-C/HGV RNA. No significant differences were observed in age, sex and underlying diseases between the cases with and without GBV-C/HGV RNA.
4. Discussion Since the identification of GBV-C/HGV in 1995, several lines of evidence suggested
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this flavivirus-like RNA virus caused acute and chronic infections, and induced non-A to E fulminant hepatitis, while the pathological consequences of chronic GBV-C/HGV infection remain to be seen [4,5,9,10]. It is yet to be determined if GBV-C/HGV is hepatocarcinogenic by itself and can coordinate with co-infecting HBV and HCV toward the development of HCC. In the present study, GBV-C/HGV RNA was detected in 33 of the 124 (27%) patients with HCC. It was detected in 30 of the 91 (33%) patients with HBsAg, anti-HCV or both, at a much higher prevalence than that in only three of the 33 (9%) patients of a non-B, non-C etiology (PB 0.01). Such a low prevalence of GBV-C/HGV RNA in the patients with non-B, non-C HCC is consistent with the results of similar studies performed in the HCC patients from Japan (3.8%), Europe (6.7%) and America (9.5%) [11 –13]. Similar results can also be found in recently reported studies which unanimously indicate that GBV-C/HGV would not be a major etiologic agent for advanced non-B, non-C chronic liver diseases including HCC [14]. Interestingly, we found a significantly greater prevalence of GBV-C/HGV infection in the HCC patients with HBV infection (35%) and in those with both HBV and HCV infections (55%), but not in those with HCV infection (0%). This implies that in China, GBV-C/HGV may have transmission modes in common with HBV, which seem to be different from those in Japan where HCV infection is the major etiologic agent of HCC. While most Chinese HBsAg carriers have contracted HBV infection during the perinatal period or in early childhood, super-infection with GBV-C/HGV may occur later in life. It has been recently proved, however, that GBV-C/HGV can be transmitted perinatally; the high prevalence of GBV-C/HGV in Chinese HCC patients might reflect frequent vertical transmission of HBV in China [15]. In addition, just like the transmission of HCV, blood transfusions have Table 3 Demographic features, underlying liver disease and etiologies of the patients with HCC who possessed and who did not possess GBV-C RNA in serum Features
Age (years)a Male Underlying diseaseb CH LC Etiology HBV HCV HBV plus HCV Non-B, non-C a
Positive (n =33)
Negative (n = 91)
44.79 12.6 28 (85%)
43.1 99.1 73 (80%)
NS NS
7 (22%) 26 (79%)
28 (31%) 63 (69%)
NS NS
18 (55%) 0 12 (36%) 3 (9%)
41 10 10 30
PB0.05 NS PB0.05 PB0.05
Mean9 S.D. age is shown. CH, chronic hepatitis; LC, liver cirrhosis. c Not significant. b
Differencesc
GBV-C RNA
(45%) (11%) (11%) (33%)
96
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been proved as an important route of GBV-C transmission, even though the present study did not produce sufficient evidence to indicate routes of transmission for GBV-C/HGV common with those for HCV in China [16–18]. Because many hepatitis viruses share the same modes of transmission, multiple viral infection may occur in a given patient. Previous studies have also suggested a reciprocal inverse relation among different hepatitis viruses. In the present study, we took advantage of extremely common chronic HBV and HCV infections in China to investigate the presence and possible significance of GBV-C/HGV co-infection in HCC patients with HBV and HCV infection. Although the co-infection with GBV-C/HGV was observed in 30 of the 91 (33%) HCC patients with HBV and/or HCV infection, there were no differences in the clinical features between the patients with and without GBV-C/HGV co-infection, as is reported for chronic hepatitis patients with HBV or HCV infection in Taiwan, where viral hepatitis is also endemic [19]. These observations suggest that the influence of GBV-C/HGV on the clinical course of HCC would be minimal. Acknowledgements The authors thank Professor Hiroshi Suzuki, the Dean of Yamanashi Medical University and the President of Miyakawa Memorial Research Foundation, for valuable help and encouragement. This study was supported, in part, by the Ministry of Health and Welfare of Japan. We also thank the staff in the Viral Hepatitis Research Unit, West China University of Medical Sciences for their great efforts in recruiting blood samples. References [1] Yu SZ. Primary prevention of hepatocellular carcinoma. J Gastroenterol Hepatol 1995;10:674 – 82. [2] Choo QL, Kuo G, Weiner AJ, Overby LR, Bradley DW, Houghton M. Isolation of a cDNA clone derived from a blood-borne non-A, non-B viral hepatitis genome. Science 1989;244:359 – 62. [3] Yen FS, Yu MC, Mo CC, et al. Hepatitis B virus, aflatoxins, and hepatocellular carcinoma in Southern Guangxi, China. Cancer Res 1989;49:2506 – 9. [4] Simons JN, Pilot-Matias TJ, Leary TP, et al. Identification of two flavivirus-like genomes in the GB hepatitis agent. Proc Natl Acad Sci USA 1995;92:3401 – 5. [5] Linnen J, Wages J, Zhang-Keck ZY, et al. Molecular cloning and disease association of hepatitis G virus: a transfusion-transmissible agent. Science 1996;271:505 – 8. [6] Alter HJ, Nakatsuji Y, Melpolder J, et al. The incidence of transfusion-associated hepatitis G virus infection and its relation to liver disease. New Engl J Med 1997;336:747 – 54. [7] Nakatsuji Y, Shih JW, Tanaka E, et al. Prevalence and disease association of hepatitis G virus infection in Japan. J Viral Hepat 1996;3:307 – 16. [8] Sarrazin C, Herrmann G, Roth WK, Lee JH, Marx S, Zeuzem S. Prevalence and clinical and histological manifestation of hepatitis G/GBV-C infections in patients with elevated aminotransferases of unknown etiology. J Hepatol 1997;27:276 – 83. [9] Yoshiba M, Okamoto H, Mishiro S. Detection of the GBV-C hepatitis virus genome in serum from patients with fulminant hepatitis of unknown aetiology. Lancet 1995;346:1131 – 2. [10] Heringlake S, Osterkamp S, Trautwein C, et al. Association between fulminant hepatic failure and a strain of GBV virus C. Lancet 1996;348:1626– 9.
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[11] Kanda T, Yokosuka O, Imazeki F, et al. GB virus-C RNA in Japanese patients with hepatocellular carcinoma and cirrhosis. J Hepatol 1997;27:464 – 9. [12] Lightfoot K, Skelton M, Kew MC, et al. Does hepatitis GB virus-C infection cause hepatocellular carcinoma in black Africans? Hepatology 1997;26:740 – 2. [13] Shibayama T, Hayashi S, Saeki S, et al. Infection with GB virus C in the patients with primary hepatocellular carcinoma in Japan. Hepatol Res 1997;8:37 – 43. [14] Tagger A, Donato F, Ribero ML, et al. A case-control study on GB virus C/hepatitis G virus infection and hepatocellular carcinoma. Hepatology 1997;26:1653 – 7. [15] Feucht HH, Zollner B, Polywka S, Laufs R. Vertical transmission of hepatitis G (letter). Lancet 1996;347:615–6. [16] Sugai Y, Nakayama H, Fukuda M, et al. Infection with GB virus C in patients with chronic liver disease. J Med Virol 1997;51:175–81. [17] Masuko K, Mitsui T, Iwano K, et al. Infection with hepatitis GB virus C in patients on maintenance hemodialysis. New Engl J Med 1996;334:1485– 90. [18] Wang Y, Chen HS, Fan MH, et al. Infection with GB virus C and hepatitis C virus in hemodialysis patients and blood donors in Beijing. J Med Virol 1997;52:26 – 30. [19] Kao JH, Chen PJ, Lai MY, et al. GB virus-C/hepatitis G virus infection in an area endemic for viral hepatitis, chronic liver disease, and liver cancer. Gastroenterology 1997;112:1265– 70.
.
GB virus C/hepatitis G virus infection in Chinese patients with hepatocellular carcinoma Lei Xuezhong a, Shigeko Naitoh b, Deng Xuewen b, Lianshan Zhao a, Yoshihiro Akahane b,* a Department of Infectious Diseases, The First Uni6ersity Hospital of West China Uni6ersity of Medical Sciences, Chengdu, China b The First Department of Internal Medicine, Yamanashi Medical Uni6ersity, Yamanashi-Ken, Japan
Received 31 July 1998; received in revised form 17 December 1998; accepted 6 January 1999
Abstract Although hepatitis B and C viruses are closely related to hepatocellular carcinoma (HCC), the association between the newly discovered GB virus C (GBV-C)/hepatitis G virus (HGV) and HCC is not yet known. Sera from 124 patients with HCC in China were tested for GBV-C/HGV RNA by the reverse-transcription polymerase chain reaction with subsequent hybridization using GBV-C/HGV probes located at the 3%-untranslated region of the reported genomes. GBV-C/HGV RNA was detected in 33 (27%) of them, including 18 of the 59 (31%) patients with HBsAg, 12 of the 22 (55%) with both HBsAg and anti-HCV, and three of the 33 (9%) negative for either HBsAg or anti-HCV; it was not detected in any of the 10 with anti-HCV alone. There were no differences in clinical backgrounds between the patients with HBsAg and/or anti-HCV who possessed and who did not possess GBV-C/ HGV RNA in serum. These results indicate that, although GBV-C/HGV is highly prevalent in Chinese patients with HCC, the role of GBV-C/HGV in hepatocarcinogenesis would be minimal in the development of non-B, non-C HCC. © 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: GB virus C; Hepatitis G virus; Hepatitis B virus; Hepatitis C virus; Hepatocellular carcinoma; Epidemiology
* Corresponding author. Tel.: +81-552-73-9584; fax: +81-552-73-6748. 1386-6346/00/$ - see front matter © 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S 1 3 8 6 - 6 3 4 6 ( 9 9 ) 0 0 0 0 7 - 8
92
L. Xuezhong et al. / Hepatology Research 16 (2000) 91–97
1. Introduction Hepatocellular carcinoma (HCC) is common in China, and its mortality rate is reported to be 20.00 – 24.32 deaths per 100 000 [1]. HCC is currently an important health problem in China. An intimate relationship between HCC and chronic hepatitis B virus (HBV) infection has long been proved, and a close association of HCC with chronic hepatitis C virus (HCV) infection is strongly suggested ever since this virus was identified [2]. Additionally, repeated exposures to the aflatoxin B1 and mycotoxin are other known etiologic factors for the development of HCC [3]. However, substantial numbers of patients with HCC do not have any of these known etiologic factors. The involvement of other factors, including non-B, non-C virus in the development of HCC has not been identified, as yet. A flavivirus-like RNA virus, with a genome different from hepatitis A–E viruses, has been identified recently in two independent laboratories from serum of non-A to E hepatitis patients, and was designated GB virus-C (GBV-C) or hepatitis G virus (HGV) [4,5]. It has been proved that the virus has a global distribution and is transmitted by blood-borne horizontal infection [6–8]. In order to determine the possible role of GBV-C/HGV infection in the development of HCC and its possible coordination with HBV and/or HCV in hepatocarcinogenesis, we examined GBVC/HGV RNA in 124 Chinese patients with HCC, including 33 with a non-B, non-C cause.
2. Materials and methods
2.1. Studied subjects One hundred and twenty-four patients with a diagnosis of HCC at admission to the First University Hospital of West China University of Medical Sciences, Chengdu, Southwest China, during August 1995 through March 1997 were enrolled in the study. They aged 43.6 910.1 years and included 101 males and 23 females. The diagnosis of HCC was based on a combination of clinical features, demonstration of one or more mass-lesions in the liver on hepatic imaging, and a higher concentration of serum a-fetoprotein; those patients with a serum a-fetoprotein higher than 500 ng/ml had hepatic masses. The diagnosis was confirmed further in 39 patients by histopathological evidence of HCC in liver tissue specimens obtained at fine needle biopsy or surgical operation. The underlying liver disease, chronic hepatitis (CH) or liver cirrhosis(LC), was diagnosed by clinical symptoms as well as biochemical and ultrasonographic findings, and confirmed further by biopsy of non-cancerous portions of liver tissue in some indefinite cases. Blood samples from the patients were obtained with great care for avoiding contamination, and separated sera were stored at − 20°C until they were tested for makers of hepatitis virus infections. The study was approved by the ethics committee of the hospital, and all patients gave informed consent.
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2.2. Serological tests Hepatitis B surface antigen (HBsAg) was determined by passive hemagglutination using commercial kits (Mycell, Institute of Immunology, Tokyo, Japan). Antibody to HCV (anti-HCV) was determined by a second-generation enzyme immunoassay (EIA-II, Ortho Diagnostic Systems, Tokyo, Japan).
2.3. Detection of GBV-C/HGV RNA Total RNA was extracted from 50 ml of serum with the guanidine isothiocyanate – phenol/chloroform procedures (SMITEST EX-R&D: Sumitomo Metal Industries, Tokyo, Japan), and then subjected to the reverse-transcription polymerase chain reaction (PCR) with use of SMITEST GBV-C RNA (3%UTR) Detection Kit (Sumitomo Metal Industries). In essence, the procedures of reverse transcription and amplification cycles using the specific rTth DNA polymerase (Perkin-Elmer, CT) and dinitrophenyl-labeled GBV-C/HGV-specific primers derived from the 3%-untranslated region were as follows: 70°C for 3 min, 60°C for 30 min, and 94°C for 1 min; then five cycles of PCR (93°C for 15 s, 62°C for 30 s in each cycle); followed by 35 cycles of PCR (90°C for 15 s, 62°C for 30 s in each cycle); finally, the extension reaction at 60°C for 4 min and kept at 10°C thereafter (9600-R Gene Amp PCR System: Perkin-Elmer). Amplified products were analyzed by hybridization and chemical-coloring in wells of a microplate coated with GBV-C/HGV probe derived from the 3%-untranslated region. The absorbance was determined at 450 nm and readings over 0.500 were considered positive. The sensitivity of this assay was 10 copies of GBV-C/HGV RNA per reaction, corresponding to about 200 copies per ml of the initial serum.
2.4. Statistical analysis The frequency was compared between groups with the two-tailed Mantel–Haenszel x 2 or two-tailed Fisher’s exact test, and group means were compared with the
Table 1 Demographic features, underlying liver disease and markers of hepatitis virus infections in HCC patients in China Underlying diseasea
CH LC Total a b
N
Age (years)b
Male/female
HBsAg
Anti-HCV
Both
Neither
N (%)
N (%)
N (%)
N (%)
3 (9%) 19 (21%)
8 (23%) 25 (28%)
22 (18%)
33 (27%)
35 89
44.1 9 10.6 43.49 10.0
26/9 75/14
23 (66%) 36 (40%)
124
43.6 9 10.1
101/23
59 (48%)
CH, chronic hepatitis; LC, liver cirrhosis. Mean9 S.D. age is shown.
1 (3%) 9 (10%) 10 (8%)
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94
Table 2 GBV-C RNA in HCC patients of various etiologies Etiology HBV HCV HBV plus HCV Non-B, non-C Total a b
N (%) 59 10 22 33 124
(48%) (8%) (18%) (18%)
Age (years)
Male
42.1910.2 44.3 97.8 44.4 9 11.9 45.5 9 9.4
45 9 18 30
43 6910.1
(76%) (90%) (82%) (91%)
101 (82%)
GBV-C RNA 18 (31%)a 0 12 (55%)b 3 (9%) 33 (27%)
Different from non-B, non-C etiology (PB0.05). Different from non-B, non-C etiology (PB0.01).
Student’s t-test. A P-value of less than 0.05 was considered to indicate statistical significance.
3. Results Demographic features, underlying liver disease and markers of HBV and HCV are shown for the 124 patients with HCC in Table 1. Of them, 35 were non-cirrhotic and 96 were cirrhotic in their non-neoplastic regions of the liver. Twenty-three of the 59 HBsAg-positive cases, one of the 10 anti-HCV positive cases, eight of the 33 cases negative for both HBsAg and anti-HCV, and three of the 22 cases positive for both HBsAg and anti-HCV had non-cirrhotic livers. Of the 124 patients with HCC, 33 were negative for both HBsAg and anti-HCV, 10 were positive for anti-HCV only, 59 were positive for HBsAg only, and 22 were positive for both anti-HCV and HBsAg. In total, GBV-C/HGV RNA was detected in 33 of the 124 (27%) patients with HCC (Table 2). Of these 33 patients, 18 (55%) were also positive for HBsAg, none were positive for anti-HCV only, 12 (36.4%) were positive for both HBsAg and anti-HCV, and only three (9%) were positive for GBV-C/HGV RNA alone. Interestingly, among the 10 cases with HCV infection only, none were found to be positive for GBV-C/HGV RNA, while in the HBsAg-positive cases, 18 (31%) were positive for GBV-C/HGV RNA. Based on these data, GBV-C/HGV RNA was highly prevalent among Chinese HCC patients who were co-infected with HBV (PB 0.05), with the highest prevalence in the patients with both HBsAg and anti-HCV (PB 0.01). Table 3 compares underlying liver disease, demographic features, and markers of HBV and HCV infections between the HCC patients with and without GBV-C/HGV RNA. No significant differences were observed in age, sex and underlying diseases between the cases with and without GBV-C/HGV RNA.
4. Discussion Since the identification of GBV-C/HGV in 1995, several lines of evidence suggested
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95
this flavivirus-like RNA virus caused acute and chronic infections, and induced non-A to E fulminant hepatitis, while the pathological consequences of chronic GBV-C/HGV infection remain to be seen [4,5,9,10]. It is yet to be determined if GBV-C/HGV is hepatocarcinogenic by itself and can coordinate with co-infecting HBV and HCV toward the development of HCC. In the present study, GBV-C/HGV RNA was detected in 33 of the 124 (27%) patients with HCC. It was detected in 30 of the 91 (33%) patients with HBsAg, anti-HCV or both, at a much higher prevalence than that in only three of the 33 (9%) patients of a non-B, non-C etiology (PB 0.01). Such a low prevalence of GBV-C/HGV RNA in the patients with non-B, non-C HCC is consistent with the results of similar studies performed in the HCC patients from Japan (3.8%), Europe (6.7%) and America (9.5%) [11 –13]. Similar results can also be found in recently reported studies which unanimously indicate that GBV-C/HGV would not be a major etiologic agent for advanced non-B, non-C chronic liver diseases including HCC [14]. Interestingly, we found a significantly greater prevalence of GBV-C/HGV infection in the HCC patients with HBV infection (35%) and in those with both HBV and HCV infections (55%), but not in those with HCV infection (0%). This implies that in China, GBV-C/HGV may have transmission modes in common with HBV, which seem to be different from those in Japan where HCV infection is the major etiologic agent of HCC. While most Chinese HBsAg carriers have contracted HBV infection during the perinatal period or in early childhood, super-infection with GBV-C/HGV may occur later in life. It has been recently proved, however, that GBV-C/HGV can be transmitted perinatally; the high prevalence of GBV-C/HGV in Chinese HCC patients might reflect frequent vertical transmission of HBV in China [15]. In addition, just like the transmission of HCV, blood transfusions have Table 3 Demographic features, underlying liver disease and etiologies of the patients with HCC who possessed and who did not possess GBV-C RNA in serum Features
Age (years)a Male Underlying diseaseb CH LC Etiology HBV HCV HBV plus HCV Non-B, non-C a
Positive (n =33)
Negative (n = 91)
44.79 12.6 28 (85%)
43.1 99.1 73 (80%)
NS NS
7 (22%) 26 (79%)
28 (31%) 63 (69%)
NS NS
18 (55%) 0 12 (36%) 3 (9%)
41 10 10 30
PB0.05 NS PB0.05 PB0.05
Mean9 S.D. age is shown. CH, chronic hepatitis; LC, liver cirrhosis. c Not significant. b
Differencesc
GBV-C RNA
(45%) (11%) (11%) (33%)
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been proved as an important route of GBV-C transmission, even though the present study did not produce sufficient evidence to indicate routes of transmission for GBV-C/HGV common with those for HCV in China [16–18]. Because many hepatitis viruses share the same modes of transmission, multiple viral infection may occur in a given patient. Previous studies have also suggested a reciprocal inverse relation among different hepatitis viruses. In the present study, we took advantage of extremely common chronic HBV and HCV infections in China to investigate the presence and possible significance of GBV-C/HGV co-infection in HCC patients with HBV and HCV infection. Although the co-infection with GBV-C/HGV was observed in 30 of the 91 (33%) HCC patients with HBV and/or HCV infection, there were no differences in the clinical features between the patients with and without GBV-C/HGV co-infection, as is reported for chronic hepatitis patients with HBV or HCV infection in Taiwan, where viral hepatitis is also endemic [19]. These observations suggest that the influence of GBV-C/HGV on the clinical course of HCC would be minimal. Acknowledgements The authors thank Professor Hiroshi Suzuki, the Dean of Yamanashi Medical University and the President of Miyakawa Memorial Research Foundation, for valuable help and encouragement. This study was supported, in part, by the Ministry of Health and Welfare of Japan. We also thank the staff in the Viral Hepatitis Research Unit, West China University of Medical Sciences for their great efforts in recruiting blood samples. References [1] Yu SZ. Primary prevention of hepatocellular carcinoma. J Gastroenterol Hepatol 1995;10:674 – 82. [2] Choo QL, Kuo G, Weiner AJ, Overby LR, Bradley DW, Houghton M. Isolation of a cDNA clone derived from a blood-borne non-A, non-B viral hepatitis genome. Science 1989;244:359 – 62. [3] Yen FS, Yu MC, Mo CC, et al. Hepatitis B virus, aflatoxins, and hepatocellular carcinoma in Southern Guangxi, China. Cancer Res 1989;49:2506 – 9. [4] Simons JN, Pilot-Matias TJ, Leary TP, et al. Identification of two flavivirus-like genomes in the GB hepatitis agent. Proc Natl Acad Sci USA 1995;92:3401 – 5. [5] Linnen J, Wages J, Zhang-Keck ZY, et al. Molecular cloning and disease association of hepatitis G virus: a transfusion-transmissible agent. Science 1996;271:505 – 8. [6] Alter HJ, Nakatsuji Y, Melpolder J, et al. The incidence of transfusion-associated hepatitis G virus infection and its relation to liver disease. New Engl J Med 1997;336:747 – 54. [7] Nakatsuji Y, Shih JW, Tanaka E, et al. Prevalence and disease association of hepatitis G virus infection in Japan. J Viral Hepat 1996;3:307 – 16. [8] Sarrazin C, Herrmann G, Roth WK, Lee JH, Marx S, Zeuzem S. Prevalence and clinical and histological manifestation of hepatitis G/GBV-C infections in patients with elevated aminotransferases of unknown etiology. J Hepatol 1997;27:276 – 83. [9] Yoshiba M, Okamoto H, Mishiro S. Detection of the GBV-C hepatitis virus genome in serum from patients with fulminant hepatitis of unknown aetiology. Lancet 1995;346:1131 – 2. [10] Heringlake S, Osterkamp S, Trautwein C, et al. Association between fulminant hepatic failure and a strain of GBV virus C. Lancet 1996;348:1626– 9.
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